Haschek and Rousseaux's Handbook of Toxicologic Pathology, Volume 3: Environmental Toxicologic Pathology and Major Toxicant Classes [4 ed.] 0443161534, 9780443161537

Table of contents :
Cover
Haschek and Rousseaux’s Handbook of Toxicologic Pathology
Copyright
Dedication
Contents
Contributors
About the Editors
Editors
Associate Editors
Illustrations Editor
Preface
PART 1 Toxicologic Pathology in Environmental and Food Protection
1
. Environmental Toxicologic Pathology and Human Health
1 Introduction
2 History of Carcinogenic Testing in Animal Species
3 Principles of Evaluations for Carcinogenic Potential
4 Examples of Environmental Pollutants
4.1 Workplace Exposure
4.2 General Environmental Contaminants
4.3 Air Pollutants (also see Respiratory Tract, Vol 5, Chap 4)
4.4 Water Pollutants
4.5 Ground and Soil Contamination
4.6 Radiofrequency Radiation (also see Radiation and Other Physical Agents, Vol 3, Chap 14)
4.7 Microplastics and Nanoplastics
5 The Role of Lifestyle and the Environment on Human Health
6 Methods of Toxicity and Carcinogenicity Testing
6.1 Fish Models
6.2 Transgenic Mouse Models
7 Current Considerations for Environmental Toxicity and Carcinogenicity Testing
7.1 Mechanism of Action versus Mode of Action
7.2 Human Relevancy
7.3 Alternative Testing Strategies
8 New Directions for Environmental Toxicity and Carcinogenicity Testing
8.1 Safe and Sustainable Alternatives
References
2
. Food and Toxicologic Pathology
1 Introduction
1.2 Overview
2 Chemicals Intentionally Added to Food
2.1 Preservatives
2.2 Food Coloring
2.3 Flavor Enhancers
Natural Flavoring Substances
Artificial Flavoring Substances
2.4 Emulsifiers, Stabilizers, and Thickeners
Emulsifiers
Stabilizers
Thickeners
2.5 Functional Foods
2.6 Medicated Feed
2.7 Dietary Supplements
3 Contamination of Food
3.1 Environmental Contaminants
3.2 Food Packaging and Food Processing Contaminants/Food Contact Substances
Food Packaging
Food Processing
Food Contact Substances
3.3 Natural Toxins as Food Contaminants
Algal Compounds in Food
Phycotoxins in Food
Cyanotoxins
Marine Algal Toxins
Domoic Acid and Amnesic Shellfish Poisoning
Mycotoxins
Bacterial Toxins
4 Compounds with Toxic Properties Naturally Present in Certain Foods
4.1 Cyanogenic Glycosides
4.2 Glucosinolates Brassica sp.
5 Novel Foods
5.1 Genetically Modified Food
5.2 Novel Food Colors—Anthocyanins
5.3 Novel Preservatives—Amygdalin
5.4 Novel Emulsifiers—Yeast
5.5 Novel Sweeteners—Stevia
5.6 Novel Proteins—Cell-Based Meats
5.7 Novel Oils—Olestra
5.8 Novel Carbohydrates—PrecticX
5.9 Recombinant Bovine Somatotropin
5.10 Cannabis—Cannabidiol
5.11 Nanomaterials
5.12 Probiotics and Prebiotics—Intelligent Labs Probiotics with Prebiotics
6 Adverse Reactions to Food Constituent
6.1 Food Allergies
6.2 Allergy-like Food Poisoning
6.3 Adverse Reactions to Gluten and Gluten-Related Disorders
Celiac Disease
“Leaky Gut”
Nonceliac Gluten Sensitivity
Wheat Allergy
6.4 Exposure of a Susceptible Population
6.5 Direct Chemical Toxicity
6.6 Nonallergic Food Hypersensitivity and Intolerance
6.7 Food Color and Food Allergy
7 Mechanism of Action of Clinical Disorders Related to Food
7.1 Gut Microbiota and Adverse Reactions to Food
Microbiome and Cytochrome P450
Microbiome and Immunity
Microbiome and Food Additives
7.2 Neurotransmission
Excitatory Amino Acids
Excitotoxicity of Glutamate
Glutamate, GABA, and Glutamate Receptors
Monosodium Glutamate
Domoic Acid
Gulf War Illness
Serotonin
Psilocybin—Mushrooms
Other Hallucinogens that May Contaminate Food
Cannabinoids
7.3 Channel Blockers
Saxitoxin
Tetrodotoxin
7.4 Endocrine Modifiers
Goitrogens
Phytoestrogens
Estrogenic Mycotoxins
8 Safety Assessment of Food
8.1 Risk/Safety Assessment in Food
8.2 Food Additives
Acceptable Daily Intake
Food Colors
8.3 Food Contaminants
Residues
Maximum Residue Limit
Withdrawal Periods
Antimicrobial Resistance: More than Residues
Clenbuterol: Withdrawal because of Residues
rBST: Minimal Residues, Withdrawn because of Animal Welfare Issues
Functional Foods
Genetically Modified Plants and Organisms
9 Regulation of Food
9.1 History of Food-Related Disease
9.2 History of Food Regulation
9.3 Food Regulations Around the World
Regulation and Approval of Foods Meant for Human Consumption
United States
Food and Drug Administration
USDA
Environmental Protection Agency
Center for Disease Control
State and Local Regulatory Systems
Canada
Europe
Japan
Other Countries
China
Brazil
Australia and New Zealand
United Kingdom
South Africa
Feed for Animal Consumption
United States
Ingredients and the Approval Process
Production, Storage, and Distribution of Safe Animal Feed Ingredients and Mixed Feed
Reporting Tools of Animal Food Hazards and Unsafe Animal Food
Feed Standards
Regulation of Pet Food and Its Labeling
Other Countries
Canada
European Union
Australia and New Zealand
Japan
China
South Africa
10 Challenges and Future Developments in Food Safety
11 Conclusions
Glossary
References
3
. Nutritional Toxicologic Pathology
1 Introduction
2 Caloric Excess and Obesity
3 Caloric Restriction
4 Macronutrients and Micronutrients
4.1 Introduction
4.2 Macronutrients
Proteins
Deficiency
Excess
Amino Acids
Deficiency
Excess
Carbohydrates
Deficiency
Excess
Fiber
Lipids
Deficiency
Excess
4.3 Micronutrients (for General Background References See Table 3.1)
Vitamins (see Table 3.2)
Vitamin A (All Trans-Retinol)
Deficiency
Excess
Vitamin D (Calcitrol)
Deficiency
Excess
Vitamin E
Deficiency
Excess
Vitamin K
Deficiency
Excess
Vitamin C (Ascorbic Acid)
Deficiency
Excess
The B Vitamins
Thiamine (Vitamin B1)
Deficiency
Excess
Riboflavin (Vitamin B2)
Deficiency
Excess
Niacin (Nicotinic Acid, Vitamin B3)
Deficiency
Excess
Pyridoxine (Vitamin B6)
Deficiency
Excess
Biotin (Vitamin B7)
Deficiency
Excess
Folic Acid (Folate, Vitamin B9, Folacin)
Deficiency
Excess
Cobalamin (Vitamin B12)
Deficiency
Excess
Choline
Deficiency
Excess
Minerals
Major Minerals
Calcium
Deficiency
Excess
Phosphorus
Deficiency
Excess
Magnesium
Deficiency
Excess
Sodium/Potassium
Deficiency
Excess
Sulfur
Deficiency
Excess
Trace Minerals
Chromium
Deficiency
Excess
Cobalt
Deficiency
Excess
Copper
Deficiency
Excess
Fluorine
Deficiency
Excess
Iodine
Deficiency
Excess
Iron
Deficiency
Excess
Manganese
Deficiency
Excess
Molybdenum
Deficiency
Excess
Selenium
Deficiency
Excess
Zinc
Deficiency
Excess
5 Dietary Contaminants (Also See Issues In Laboratory Animal Science that Impact Toxicologic Pathology, Vol 1, Chap 29)
5.1 Analyses for Contaminants
5.2 Pesticides
5.3 Mycotoxins, Heavy Metals, Phytoestrogens, and Other Contaminants
References
PART 2 Selected Toxicant Classes in the Environment
4
. Herbal Remedies
1 Introduction
1.1 Nomenclature
2 Apothecary to Pharmacy
3 Evidence for Herbal Remedy Efficacy
3.1 Empirical Evidence – Traditional Knowledge (Botanical)
3.2 Experimental Evidence – Controlled (Single Active Compound)
4 The Active Pharmaceutical ingredient(s)
4.1 Influencing Factors on the Concentration of the API(s) in the Plant
4.2 Dose and Response
4.3 Contaminants
4.4 Adulterants
4.5 A Comparison Between Properties of Herbal Remedies and Conventional Drugs
4.6 Acceptability of Herbal Remedies
5 Quality, Efficacy and Safety
6 Quality
6.1 The Active Pharmaceutical Ingredient
6.2 Quality Control
6.3 Manufacturing Processes and Controls
7 Efficacy and Effectiveness
7.1 Traditional Knowledge of Efficacy
7.2 Experimental Evidence
7.3 Randomized Clinical Trials Using Herbal Remedies
8 Safety
8.1 Safety, Side Effects and Toxicity
8.2 Adverse Reactions
8.3 Interactions
Herb–Drug Interaction
8.4 Herb-Herb Interaction
8.5 Direct Toxicity
8.6 Indirect Toxicity
8.7 Hypersensitivity – Idiopathic Allergic Reactions
9 Toxicology of Herbal Remedies
9.1 Lethality
9.2 Genotoxicity and Carcinogenesis
9.3 Herbal Toxicokinetics
9.4 Microbiome
9.5 Herbal Pharmacodynamics
9.6 Organ Toxicity
Hepatotoxicity
Renal Toxicity
Cardiotoxicity
Neurotoxicity
Dermal Toxicity
Primary Irritant Dermatitis
Allergic Contact Dermatitis
Photosensitization Dermatitis
10 Toxicologic Pathology of Select Herbal Remedies
10.1 Aloe vera – Aloe barbadensis
Whole Leaf Extract
Latex
Gel
10.2 Cannabis – Cannabis sativa and Cannabis indica
10.3 Chamomile – Chamomilla recutita
10.4 Coffee – Coffea arabica and C. Canephora
Pharmacokinetics
Human Health
Animal Studies
10.5 Cocoa – Theobroma cacao
10.6 Echinacea – Echinacea purpurea
10.7 Ephedra – Ephedra sinica
10.8 Garlic – Allium sativum
10.9 Ginkgo Biloba – Ginkgo biloba
10.10 Ginger – Zingiberis rhizome
10.11 Ginseng – Panax ginseng
10.12 Goldenseal – Hydrastis canadensis
10.13 Green Tea – Camellia sinensis
10.14 Indole-3-Carbinol – Brassica sp. Glucosinolates
10.15 Kava kava – Piper methysticum
10.16 Milk Thistle – Silybum marianum
10.17 Mint Mentha sp. [Contains Pulegone]
10.18 Rattlepods, Yellow Burrweed, and Groundsel—Crotalaria, Amsinckia, and Senecio Containing [Contains Riddelliine a Pyrrolizid ...
10.19 Saw Palmetto – Serenoa repens
10.20 Senna – Senna alexandrina
10.21 St. John's Wort – Hypericum perforatum
10.22 Tobacco – Nicotiana tabacum
10.23 Turmeric Oleoresin – Curcuma longa
11 International Regulatory Overview
11.1 Select List of Countries and Their Regulatory Requirements
Australia
Canada
China
European Union
India
Japan
Korea
Malaysia
Philippines
United States of America
12 Discussion
13 Summary
References
5 Phycotoxins
1 INTRODUCTION
1.1 Harmful Algal Blooms
1.2 Aquatic Hypoxia
1.3 Some Important Marine and Freshwater Toxins
1.4 The Need for Greater Access to and Reliance on Diagnostic Expertise and Instrumentation
1.5 A Future with Fewer Harmful Algal Blooms and Phycotoxin Poisonings
1.6 Rationale for the Subsequent Discussions of Phycotoxins
2 SAXITOXINS
2.1 Source/Occurrence
2.2 Toxicology
2.3 Clinical Signs and Pathology
2.4 Human Exposure and Disease
2.5 Diagnosis, Treatment, and Control
3 CYCLIC IMINES
3.1 Source/Occurrence
3.2 Toxicology
3.3 Clinical Signs and Pathology
3.4 Human Exposure and Disease
3.5 Diagnosis, Treatment, and Control
4 DOMOIC ACID
4.1 Occurrence and Species Susceptibility
4.2 Toxicology
4.3 Clinical Signs and Pathology
4.4 Human Exposure and Disease
4.5 Diagnosis, Treatment, and Control
5 BREVETOXINS
5.1 Source/Occurrence
5.2 Toxicology
5.3 Clinical Signs
5.4 Gross and Histologic Findings
5.5 Human Exposure and Disease
5.6 Diagnosis, Treatment, and Control
6 CIGUATOXINS
6.1 Source/Occurrence
6.2 Toxicology
6.3 Maitotoxins
6.4 Clinical Signs and Pathology
6.5 Human Exposure and Disease
6.6 Diagnosis, Treatment, and Control
7 OKADAIC ACID AND DINOPHYSISTOXINS
7.1 Source/Occurrence
7.2 Toxicology
7.3 Clinical Signs and Pathology
7.4 Human Exposure and Disease
7.5 Diagnosis, Treatment, and Control
8 AZASPIRACID TOXINS
8.1 Source/Occurrence
8.2 Toxicology
8.3 Clinical Signs and Pathology
8.4 Human Exposure and Disease
8.5 Diagnosis, Treatment, and Control
9 CYLINDROSPERMOPSINS
9.1 Source/Occurrence
9.2 Toxicology
9.3 Clinical Signs and Pathology
9.4 Human Exposure and Disease
9.5 Diagnosis, Treatment, and Control
10 MICROCYSTINS AND NODULARINS
10.1 Source/Occurrence
10.2 Toxicology
10.3 Clinical Signs and Pathology
10.4 Human Exposure and Disease
10.5 Diagnosis, Treatment, and Control
11 ANATOXINS
11.1 Source/Occurrence
11.2 Toxicology
11.3 Clinical Signs and Pathology
11.4 Human Exposure and Disease
11.5 Diagnosis, Treatment, and Control
12 GUANITOXIN [FORMERLY ANATOXIN-A(S)]
12.1 Source/Occurrence
12.2 Toxicology
12.3 Clinical Signs and Pathology
12.4 Human Exposure and Disease
12.5 Diagnosis and Treatment
13 LYNGBYATOXINS AND APLYSIATOXINS
13.1 Source/Occurrence
13.2 Toxicology
13.3 Clinical Signs and Pathology
13.4 Human Exposure and Disease
13.5 Diagnosis, Treatment, and Control
14 β-METHYLAMINOALANINE
14.1 Introduction
14.2 Sources/Occurrences/Exposures
14.3 Toxicology
14.4 Animal Studies
14.5 Mechanism of Action
14.6 Human Exposure and Disease
14.7 Analytical Methods for Detection and Quantification
14.8 Conclusion
15 EMERGING PHYCOTOXINS
15.1 Vacuolar Myelinopathy and Aetokthonotoxin
15.2 Palytoxins
15.3 Yessotoxins
16 CONCLUSIONS AND FUTURE NEEDS
REFERENCES
6
. Mycotoxins
1 Introduction
2 Aflatoxins
2.1 Source/Occurrence
2.2 Toxicology
Toxin
Species Susceptibility
Biodistribution, Metabolism, and Excretion
Mechanism of Action
2.3 Manifestations of Toxicity in Animals
Overview
Laboratory Animals
Poultry
Livestock
Other Species
Liver Cancer in Laboratory Animals
2.4 Human Risk and Disease
2.5 Diagnosis, Treatment, and Control
3 Ochratoxins
3.1 Source/Occurrence
3.2 Toxicology
Species Susceptibility
Biodistribution, Metabolism, and Excretion
Mechanism of Action
3.3 Manifestations of Toxicity in Animals
Laboratory Animals
Swine
Poultry
3.4 Human Risk and Disease
3.5 Diagnosis, Treatment, and Prevention
4 Patulin
4.1 Source/Occurrence
4.2 Toxicology
Toxin
Biodistribution, Metabolism, and Excretion
Mechanism of Action
4.3 Manifestations of Toxicity in Animals
4.4 Human Exposure and Disease
4.5 Diagnosis, Treatment, and Control
5 Trichothecene Mycotoxins
5.1 Sources/Occurrence
5.2 Toxicology
Toxins
Biodistribution, Metabolism, and Excretion
Mechanism of Action
Toxicity and Species Susceptibility
5.3 Manifestations of Toxicity in Animals
General
Deoxynivalenol
T-2 Toxin and Diacetoxyscirpenol
Overview
Swine
Ruminants
Poultry
Macrocyclic Trichothecenes (Stachybotryotoxicosis)
5.4 Human Risk and Disease
Introduction
Deoxynivalenol
T-2 Toxin and Diacetoxyscirpenol
Macrocyclic Trichothecenes
5.5 Diagnosis, Treatment, and Prevention
General
Livestock
Chemical Warfare Considerations for T-2 Toxin
6 Zearalenone
6.1 Source/Occurrence
6.2 Toxicology
Toxins
Mechanism of Action
Species Susceptibility
Biodistribution, Metabolism, and Excretion
6.3 Manifestations of Toxicity in Animals
Laboratory Animals
Swine
Cattle and Sheep
6.4 Human Risk and Disease
6.5 Diagnosis, Treatment, and Prevention
7 Fumonisins
7.1 Source/Occurrence/Exposure
7.2 Toxicology and Mode of Action (MOA)
Toxins
Species Susceptibility
Biodistribution, Metabolism, and Excretion
Mode of Action
7.3 Manifestations of Toxicity in Animals
Overview
Equidae
Swine
Laboratory Animals
Nonhuman Primates
7.4 Human Risk and Disease
7.5 Diagnosis, Treatment, and Prevention
7.6 Regulations and Guidances
8 Ergot Alkaloids
8.1 Introduction
8.2 Source/Occurrence
Claviceps spp.
Epichloe spp.
8.3 Toxicology
Toxins
Biodistribution, Metabolism, and Excretion
Mechanism of Action
Species Susceptibility
8.4 Manifestations of Toxicity in Animals (Reviewed by EFSA, 2005; Gupta et al., 2018a)
Laboratory Animals
Food Animals and Horses (Reviewed by Blodgett, 2001; Strickland et al., 2011, Klotz, 2015: Coufal-Majeewski et al., 2016; G ...
Gangrenous Syndrome
Hyperthermic Syndrome (Summer Fescue Toxicosis, Summer Slump, Systemic Hyperthermia)
Fat Necrosis
Reproductive Failure
Neurologic Syndrome
8.5 Human Risk and Disease
8.6 Pharmaceutical Use
8.7 Diagnosis, Treatment and Prevention
9 Emerging Mycotoxins
9.1 Introduction
9.2 Alternaria Toxins
Source/Occurrence
Toxicology
Toxins
Biodistribution, Metabolism, and Excretion
Mechanism of Action
Manifestations of Toxicity in Animals
Human Exposure and Disease
9.3 Aspergillus and Penicillium Toxins
Cyclopiazonic Acid
Source/Occurrence
Toxicology, Toxicokinetics and Mechanism of Action
Manifestations of Toxicity in Animals
Human Exposure and Disease
Sterigmatocystin (Zingales et al., 2020)
Source/Occurrence
Toxicology, Toxicokinetics, and Mechanism of Action
Manifestations of Toxicity in Animals
Human Exposure and Disease
9.4 Tremorgenic Mycotoxins
Source/Occurrence
Toxicology, Toxicokinetics and Mechanism of Action
Manifestations of Toxicity in Animals
Human Exposure and Disease
9.5 Fusarium Toxins
Beauvericin and Enniatins
Source/Occurrence
Toxicology, Toxicokinetics and Mechanism of Action
Manifestations of Toxicity in Animals
Human Exposure and Disease
Moniliformin
Butenolide
Culmorin, Fusaproliferins and Fusaric Acid
9.6 Diagnosis, Treatment, and Control
10 Summary/Conclusion
Acknowledgments
References
7
. Poisonous Plants
1 Introduction
2 Selected Hepatotoxic Plants
2.1 Dehydropyrrolizidine Alkaloid–Containing Plants
Chemical Structure and Diversity
Plant Sources
Toxicology
Clinical Signs and Pathology
Animal Species Susceptibility
Human Exposure and Disease
2.2 Saponin-Containing Plants
2.3 Plants Containing Fungal Hepatotoxins
Lupinosis
Sporidesmin
Plant/Mycotoxin-Related Liver Diseases and Syndromes
2.4 Alsike Clover
2.5 Lantana
2.6 Cocklebur (Xanthium strumarium) and Other Potent Hepatotoxic Plants
3 Selected Neurotoxic Plants
3.1 Plant-Induced Storage Diseases (Swainsonine/Calestegine/Castanospermine)
Swainsonine or Locoweed Intoxication
Toxin and Toxicity
Clinical Disease
Pathology
Reproductive Effects
Swainsonine in Human Health and Medicine
Calystegines
Castanospermine
3.2 Ryegrass Toxicity
Perennial Ryegrass Staggers
Annual Ryegrass Toxicity
3.3 Larkspur
3.4 Centaurea spp.
3.5 Nitro-Toxins
3.6 Hemlocks
3.7 Lupines
3.8 Death Camas
4 Selected Myotoxic Plants
4.1 Cardioactive Glycoside-Containing Plants
4.2 Rayless Goldenrod and White Snakeroot
4.3 Other Myotoxic Plants
Thermopsis spp.
Cassia or Senna spp.
Erythroxylum coca
Seleniferous Plants
5 Selected Teratogenic Plants
5.1 Lupine
5.2 Veratrum californicum
5.3 Poison Hemlock
6 Selected Nephrotoxic Plants
6.1 Oak
6.2 Lily and Grapes
6.3 Oxalate-Containing Plants
6.4 Amaranthus spp.
6.5 Calcinogenic Glycoside-Containing Plants
7 Other Toxic Plants
7.1 Pine Needles
7.2 Cyanogenic Plants
7.3 Nitrate-Accumulating Plants
7.4 Photosensitizing Plants
Primary Photosensitization
Hypericism
Fagopyrism
Furocoumarins
Drugs and Other Toxicants
Hepatogenous Photosensitization (Secondary Photosensitization)
Photosensitization Sequelae
7.5 Bracken Fern
Acute Hemorrhagic Disease and Enzootic Hematuria
Bright Blindness
Bracken Staggers
Human Poisoning
Treatment and Prevention
7.6 Ricinus spp.
8 Additional Resources
References
8
. Animal Toxins
1 Introduction
2 Sources of Exposure
2.1 Poisoning
Ingestion
Dermal or Mucosal Contact
Inhalation
2.2 Envenomation
Bites
Stings
2.3 Deliberate Administration
Aggression and Defense
Therapeutic Applications
3 Zootoxin Classification
3.1 Zootoxin Classification by Source
3.2 Zootoxin Classification by Molecular Structure
3.3 Zootoxin Classification by Function
Coagulotoxins
Necrotoxins
Neurotoxins
3.4 Zootoxin Classification by Mechanism of Action
Cell and Tissue Destruction
Phospholipase A2
Cardiotoxins
Metalloproteinases
Sphingomyelinase D
Circulatory Disturbances
Vascular Permeability Enhancement
Vascular Tone Modulation
Vascular Wall Damage
Hemostasis Abnormalities
Coagulotoxic Zootoxins
Coagulotoxic Mechanisms
Inflammation Induction
Innate Immune Responses to Zootoxins
Adaptive Immune Responses to Zootoxins
Neurotransmission Derangement
Altered Neurotransmitter Levels
Altered Neurotransmitter Availability
Exogenous signaling molecules
Altered synthesis and packaging
Altered Neurotransmitter Release
Inhibition of Na+ channels
Inhibition of K+ channels
Inhibition of Ca2+ channels
Modified Presynaptic Receptor Activity
Disruption of Axon Terminal Membranes
Altered Acetylcholinesterase Activity
Aberrant Postsynaptic Receptor Activation
Altered Action Potential Propagation
Altered Myofiber Integrity and Function
4 Clinical Presentations and Pathologic Manifestations of Zootoxin-Mediated Diseases
4.1 Blood Vessels and Blood Components
4.2 Epithelium (Cutaneous and Mucosal Surfaces)
4.3 Kidney
4.4 Liver
4.5 Lung
4.6 Muscle (Cardiac and Skeletal)
4.7 Neuromuscular Junction and Other Peripheral Synapses
4.8 Systemic (Multi-Organ) Failure
5 Diagnosis and Treatment of Zootoxin-Mediated Diseases
5.1 Diagnosis
History and Physical Examination
Field Tests
Molecular Procedures
Pathology Procedures
Clinical Pathology
Histopathology
5.2 Treatment
First Aid for Acute Exposures
Supportive (Nonspecific) Care for Acute Exposures
Curative (Specific) Therapies and Their Complications
Antivenom Therapy
Adverse Reactions to Antivenom
Care for Chronic Complications
Prophylactic Measures
6 Regulatory Guidance Regarding Zootoxins
6.1 Sources and Major Indications of Medicinal Zootoxins
6.2 Practices for Developing Zootoxin-Based Medical Products
6.3 Practices for Developing Antivenom Products
7 Summary
Glossary
Acknowledgments
References
9
. Bacterial Toxins
1 Introduction
2 Exotoxins
2.1 Sources of Exposure
Infection
Ingestion
Inhalation
Therapeutic Products
2.2 Toxicology
Exotoxin Classification by Function
Cytolysins
Bacterial Colonization Factors
Exotoxin Classification by Mechanism of Action
Type I Exotoxins—Superantigens Hijacking the Immune Response
Type II Exotoxins—Membrane-Damaging Toxins
Type III Exotoxins—Intracellular Effector Enzymes
Exotoxin Classification by Target Organ Spectrum
Enterotoxins
Hemolysins
Leukocidins
Myotoxins
Neurotoxins
3 Endotoxins
3.1 Sources of Exposure
Infection
Ingestion
Inhalation
Therapeutic Products
3.2 Toxicology
Structure and Functional Attributes of Endotoxin
Endotoxin-Mediated Cell Signaling
Pathogenesis of Endotoxin-Induced Immune Dysfunction
4 Clinical Presentations and Pathologic Manifestations of Bacterial Toxin-Mediated Diseases
4.1 Enteric Effects (Intestine)
4.2 Fascial Effects (Connective Tissue)
4.3 Hepatic Effects (Hepatocytes and Hepatic Immune Cells)
4.4 Microvascular Effects (Blood Vessels)
4.5 Myotoxic Effects (Cardiac and Skeletal Muscle)
4.6 Neurotoxic Effects (Brain and Terminal Nerve Synapses)
4.7 Pneumotoxic Effects (Lung)
4.8 Systemic Effects (Sepsis and Toxic Shock Syndrome)
4.9 Miscellaneous Effects Attributed to Bacterial Toxins
5 Diagnosis and Treatment of Bacterial Toxin–Mediated Diseases
5.1 Diagnosis
Imaging, Microbiological, and Molecular Procedures
Pathology Procedures
5.2 Treatment
Curative Therapies
Prophylactic Measures
6 Regulatory Guidance Regarding Bacterial Toxins
6.1 Food and Beverage Production and Water Treatment
6.2 Manufacturing Biomedical Products
6.3 Safety Assessment of Immunotoxins
Regulatory Guidance for Immunotoxin Safety Assessment
Toxic Effects Associated with Immunotoxin Administration
7 Summary
Glossary
Acknowledgments
References
10
. Metals
1 Introduction
2 Antimony
2.1 Sources and Exposure
2.2 Toxicology
2.3 Manifestations of Toxicosis
2.4 Diagnosis and Treatment
3 Arsenic
3.1 Inorganic Arsenic
Sources and Exposure
Toxicology
Manifestations of Toxicosis
3.2 Organic Arsenic
Sources and Exposure
Toxicology
Manifestations of Toxicosis
3.3 Arsine
3.4 Diagnosis and Treatment
3.5 Chemical Warfare Considerations
4 Beryllium
4.1 Sources and Exposure
4.2 Toxicology
4.3 Manifestations of Toxicosis
4.4 Diagnosis and Treatment
5 Bismuth
5.1 Sources and Exposure
5.2 Toxicology
5.3 Manifestations of Toxicosis
5.4 Diagnosis and Treatment
6 Cadmium
6.1 Sources and Exposure
6.2 Toxicology
6.3 Manifestations of Toxicosis
6.4 Diagnosis and Treatment
7 Chromium
7.1 Sources and Exposure
7.2 Toxicology
7.3 Manifestations of Toxicosis
7.4 Diagnosis and Treatment
8 Lead
8.1 Sources and Exposure
8.2 Toxicology
8.3 Manifestations of Toxicosis in Animals
8.4 Human Exposure and Disease
8.5 Diagnosis and Treatment
9 Mercury
9.1 Sources and Exposure
9.2 Toxicology
9.3 Elemental Mercury
9.4 Inorganic Mercury
9.5 Organic Mercury
9.6 Diagnosis and Treatment
10 Plutonium (See Also Volume 3, Chap 14, Radiation and Other Physical Agents)
10.1 Sources and Exposure
10.2 Toxicology
10.3 Manifestations of Toxicosis
10.4 Diagnosis and Treatment
11 Thallium
11.1 Sources and Exposure
11.2 Toxicology
11.3 Manifestations of Toxicosis
11.4 Diagnosis and Treatment
12 Uranium (See also Volume 3, Chap 14, Radiation and Other Physical Agents)
12.1 Sources and Exposure
12.2 Toxicology
12.3 Manifestation of Toxicosis
12.4 Diagnosis and Treatment
13 Summary and Conclusions
References
11
. Agrochemicals
1 Introduction
2 Herbicides
2.1 Introduction
2.2 Inhibition of Cell Division and Growth
Auxin Mimics
Chlorophenoxy Herbicides
Toxicology, Clinical Signs, and Pathology
Human Risk
Microtubule Organization Inhibitors
Carbamates
Toxicology, Clinical Signs, and Pathology
2.3 Activation of Reactive Oxygen Species
Pyridiniums: Paraquat and Diquat
Pyridiniums
Development and Use
Toxicology, Clinical Signs, and Pathology
Phosphonic Acids
Glufosinate
Toxicology, Clinical Signs, and Pathology
Human Risk
Ureas and Thioureas
Toxicology, Clinical Signs, and Pathology
Human Risk
Triazines
Toxicology, Clinical Signs, and Pathology
Hydroxyphenylpyruvate Dioxygenase Inhibitors
2.4 Inhibition of Cellular Metabolism
Inhibition of Enolpyruvyl Shikimate Phosphate Synthase
Glyphosate
Toxicology, Clinical Signs, and Pathology
Human Risk
Thiocarbamates
3 Fungicides
3.1 Introduction
3.2 Triazole-Containing Azole Fungicides (Conazoles)/DMI-Fungicides (Demethylation Inhibitors)/C14-Demethylase Inhibitors
Tebuconazole
Prothioconazole
Mefentrifluconazole
3.3 Succinate Dehydrogenase Inhibitor Fungicides
3.4 Strobilurins or Quinol Oxidation Site of Complex III Inhibitor Fungicides
4 Insecticides
4.1 Introduction
4.2 Organophosphates and Carbamates
Toxicology
Clinical Signs and Pathology
Human Risk
4.3 Organochlorines
Development and Use
Toxicology
Clinical Signs and Pathology
Human Risk
4.4 Pyrethrins and Pyrethroids
Development and Use
Toxicology
Clinical Signs and Pathology
Human Risk
4.5 New Insecticides
Neonicotinoids
Phenylpyrazoles
Macrocyclic Lactone Endectocides (Mectins)
Diamide Insecticides
Cyromazine
Diafenthiuron
Fenoxycarb
Lufenuron
Pymetrozine
Spiropidion
5 Rodenticides
5.1 Introduction
5.2 Anticoagulant Rodenticides
Development and Use
Toxicology
Metabolism
Clinical Signs and Pathology
Human Risk
5.3 Cholecalciferol
Development and Use
Toxicology
Clinical Signs and Pathology
Human Risk
Medical Data
Other Species
5.4 Inorganic Compounds: Metal Phosphides
Aluminum Phosphide/Zinc Phosphide
Development and Use
Toxicology
Absorption, Distribution, Excretion, and Metabolism
Clinical Signs
Other Species (Clinical Signs and Pathology)
Human Risk
5.5 Alphachloralose
Development and Use
Toxicology
Clinical Signs and Pathology
Human Risk
Risk to Environment/Other Species
5.6 Bromethalin
Development and Use
Toxicology
Clinical Signs
Other Species (Clinical Signs and Pathology)
5.7 Corn Cob
Development and Use
Toxicology
Clinical Signs and Pathology
Human Risk
Risk to Other Species
5.8 Strychnine
Toxicology
Clinical Signs
Risk to Other Species
6 Conclusions
References
12
. New Frontiers in Endocrine Disruptor Research∗
1 Introduction
2 Environmental Chemicals Can Disrupt Endocrine Signaling
2.1 History of Endocrine Disruptor Research
2.2 Types of Chemicals With Endocrine-Disrupting Activity
2.3 Routes of Exposure to Endocrine Disruptors
2.4 Regulatory Approaches to Endocrine Disruption
3 Mechanisms of Endocrine Disruption
4 Examples of Disruption of Endocrine Pathways by Some Environmental Contaminants and Emerging Endocrine Disruptors
4.1 Phthalates Disrupt Several Endocrine Pathways
4.2 Emerging Endocrine Disruptors: Glyphosate
4.3 Emerging Endocrine Disruptors: General Anesthetics as Endocrine Disruptors
5 Epigenetic Effects of EDCs
6 From Reactive to Proactive Endocrine Disruptor Analysis
7 Emerging Models in EDC Research
7.1 Zebrafish Model
7.2 CRISPR Screening
8 Omics Technologies to Evaluate Endocrine Disruption
8.1 Transcriptomics and Proteomics
8.2 Lipidomics and Metabolomics
8.3 Microbiome
8.4 Exposomics
9 New Frontiers in Bioinformatics and Integrative and Functional Enrichment Omics Approaches
9.1 Integrative Correlation Analyses
9.2 Integrative MultiOmics Pathway Resolution
10 Machine Learning and EDCs
10.1 How Machine Learning Works
10.2 Examples of Current Deep Learning Programs for Toxicology
11 Conclusions
Abbreviations
References
13
. Nanoparticulates
1 Background
1.1 Definitions
1.2 Historical Perspective
1.3 Development of Nanotechnology
1.4 Current and Future Nanotechnology Applications
1.5 Human Exposures
2 Experimental Toxicologic Pathology of NPs
2.1 Enhanced Toxicity of Nanoscale Particulates
Surface Area
Solubility
Quantum Chemistry
Size
2.2 Visualizing NPs in Tissue
Factors which Limit the Ability to Identify NPs in Tissue Sections
Labeled NPs
High-Resolution FESEM
Enhanced Darkfield Microscopy
2.3 Cytopathology
Cytoplasmic Membrane Damage
Mitotic Spindle Interactions
Overview of the Mitotic Spindle
Centrosomal Interactions
Microtubule Interactions
Chromosomal Interactions
Additional Cytopathologic Interactions
2.4 Target Organ and Tissue Toxicity
Pulmonary Pathology
Neurotoxicity/Neuropathology of NPs
Cardiovascular Pathology
Lymphatic Pathology
2.5 Human Relevance of Experimental Studies in Animals
3 Future Trends in Nanopathology and Nanotoxicology
4 Conclusions
Acknowledgments
References
14
. Radiation and Other Physical Agents
1 Introduction
1.1 Sources and Occurrence
Ionizing Radiation
External Radiation
Internally Deposited Radionuclides
Ultraviolet Radiation
Hyperthermia
1.2 Impact of Household Technologies and Nonionizing Radiation (see also Environmental Toxicologic Pathology and Human Health, ...
Part I Ionizing Radiation
2 Nature and Action of Ionizing Radiation
2.1 Radiation Biophysics
2.2 External Radiation and Internal Emitters
External Radiation
Internal Emitters
Definition
Emissions
Chemical Form
Metabolism
Chemical Class
Routes of Entry
Ingestion
Inhalation
Dose Localization
Health Effects
2.3 Chernobyl and Fukushima: Retrospective Overview of Pathology Associated with Ionizing Radiation Exposure
3 Mechanisms of Ionizing Radiation Injury
3.1 Interaction of Ionizing Radiation with Biological Materials
3.2 Subcellular and Cellular Effects of Ionizing Radiation
3.3 Cell and Tissue Radiosensitivity to Ionizing Radiation
3.4 General Tissue and Organ Effects of Ionizing Radiation
3.5 Molecular Mediators
3.6 Acute Radiation Syndromes and Combined Injury
3.7 Ionizing Radiation Carcinogenesis
4 Response to Injury Induced by Ionizing Radiation
4.1 Vascular and Connective Tissue Effects of Ionizing Radiation
4.2 Hematopoietic and Lymphoid Systems
General Reaction to Ionizing Radiation Injury – Hematopoietic System
Lymphoid Tissues
Radiation Leukemogenesis
4.3 Alimentary System
General Reaction to Ionizing Radiation Injury
Salivary Tissue
Small Intestine
Esophagus
Liver
4.4 Nervous System
General Reaction to Ionizing Radiation Injury
Peripheral/Cranial Nerves
Spinal Cord
Brain
4.5 Endocrine System
Thyroid Gland
Parathyroid Gland
Pancreas
Adrenal Gland
4.6 Special Senses
General Reaction to Ionizing Radiation Injury
Ear
Eye
Lens
Retina
Other Ocular Structures
4.7 Respiratory System
General Reaction to Ionizing Radiation Injury
Upper Respiratory Tract
Lung
Neoplasia
4.8 Musculoskeletal System
General Reaction to Ionizing Radiation Injury
Bone
Skeletal Muscle
4.9 Cardiovascular System
General Reaction to Ionizing Radiation Injury
Coronary Arteries
Pericardium
Heart
4.10 Urinary System
General Reaction to Ionizing Radiation Injury
Kidney
Ureter
Urinary Bladder
4.11 Fetal Effects
4.12 Reproductive Tract
Testes
Ovary
4.13 Integumentary System
General Reaction to Ionizing Radiation Injury
Part II Ultraviolet Radiation
5 Nature and Action of Ultraviolet Radiation
6 Mechanisms of Ultraviolet Radiation Injury
7 Response to Injury Induced by Ultraviolet Radiation
7.1 Integument
7.2 Eye
7.3 Immune System
7.4 Ultraviolet Radiation Carcinogenesis
Epidemiologic Evidence
Animal Models
Mechanisms
Skin Neoplasms
Ocular Neoplasia
Part III Hyperthermia
8 Clinical Use of Hyperthermia
9 Mechanisms of Hyperthermia-Induced Injury
10 Response to Injury Induced by Hyperthermia
10.1 Reaction of Specific Organs and Tissue to Hyperthermia
Alimentary System
Musculoskeletal System
Nervous System
Eye
Cardiovascular System
Urinary System
Male Reproductive System
Integumentary System
Disclaimer
References
Chapter-11---Agrochemi_2023_Haschek-and-Rousseaux--s-Handbook-of-Toxicologic
Chapter-12---New-Frontiers-in-Endocrine-Disruptor-Research-_2023_Haschek-and
Chapter-13---Nanoparticu_2023_Haschek-and-Rousseaux--s-Handbook-of-Toxicolog
Chapter-14---Radiation-and-Other_2023_Haschek-and-Rousseaux--s-Handbook-of-T
Index
Backcover

Citation preview

HASCHEK AND ROUSSEAUX’S HANDBOOK OF TOXICOLOGIC PATHOLOGY FOURTH EDITION Volume III: Environmental Toxicologic Pathology and Selected Toxicant Classes Edited by

WANDA M. HASCHEK COLIN G. ROUSSEAUX MATTHEW A. WALLIG BRAD BOLON Associated Editors

KATHLEEN M. HEINZ-TAHENY DANIEL G. RUDMANN Illustrations Editor

BETH W. MAHLER

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/ permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/ or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-443-16153-7 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Kattie Washington Editorial Project Manager: Billie Jean Fernandez Production Project Manager: Sreejith Viswanathan Cover Designer: Matthew Limbert Typeset by TNQ Technologies

Dedication To teach is to learn .

(Japanese proverb) To our families, teachers, colleagues, and friends who have supported us in our journeys through life, encouraged us when needed, mentored us in our learning, challenged us in our teaching, joined us in our passion, followed us in our trailblazing, and inspired us in our scholarly pursuits . We are grateful for the opportunities we have enjoyed to advance pathology and toxicology as distinct and blended disciplines, both for our own betterment and in service to our local and global communities.

Contents 2. Food and Toxicologic Pathology

Contributors xvii About the Editors xix Preface xxi

OLGA M. PULIDO, COLIN G. ROUSSEAUX AND PHAEDRA I. COLE

1. 2.

PART 1 Toxicologic Pathology in Environmental and Food Protection 1. Environmental Toxicologic Pathology and Human Health SUSAN A. ELMORE AND GARY A. BOORMAN

1. 2.

Introduction 3 History of Carcinogenic Testing in Animal Species 4 3. Principles of Evaluations for Carcinogenic Potential 6 4. Examples of Environmental Pollutants 8 4.1. Workplace Exposure 9 4.2. General Environmental Contaminants 10 4.3. Air Pollutants 13 4.4. Water Pollutants 15 4.5. Ground and Soil Contamination 16 4.6. Radiofrequency Radiation 17 4.7. Microplastics and Nanoplastics 20 5. The Role of Lifestyle and the Environment on Human Health 22 6. Methods of Toxicity and Carcinogenicity Testing 23 6.1. Fish Models 23 6.2. Transgenic Mouse Models 24 7. Current Considerations for Environmental Toxicity and Carcinogenicity Testing 25 7.1. Mechanism of Action versus Mode of Action 25 7.2. Human Relevancy 25 7.3. Alternative Testing Strategies 26 8. New Directions for Environmental Toxicity and Carcinogenicity Testing 27 8.1. Safe and Sustainable Alternatives 28 References 29

3.

4.

5.

vii

Introduction 34 1.2. Overview 35 Chemicals Intentionally Added to Food 37 2.1. Preservatives 37 2.2. Food Coloring 37 2.3. Flavor Enhancers 37 2.4. Emulsifiers, Stabilizers, and Thickeners 38 2.5. Functional Foods 38 2.6. Medicated Feed 38 2.7. Dietary Supplements 39 Contamination of Food 39 3.1. Environmental Contaminants 40 3.2. Food Packaging and Food Processing Contaminants/Food Contact Substances 41 3.3. Natural Toxins as Food Contaminants 43 Compounds with Toxic Properties Naturally Present in Certain Foods 46 4.1. Cyanogenic Glycosides 46 4.2. Glucosinolates Brassica sp. 47 Novel Foods 47 5.1. Genetically Modified Food 48 5.2. Novel Food ColorsdAnthocyanins 49 5.3. Novel Preservativesd Amygdalin 49 5.4. Novel EmulsifiersdYeast 49 5.5. Novel SweetenersdStevia 49 5.6. Novel ProteinsdCell-Based Meats 49 5.7. Novel OilsdOlestra 50 5.8. Novel Carbohydratesd PrecticX 50 5.9. Recombinant Bovine Somatotropin 50 5.10. CannabisdCannabidiol 50 5.11. Nanomaterials 50

viii

CONTENTS

5.12.

Probiotics and PrebioticsdIntelligent Labs Probiotics with Prebiotics 51 6. Adverse Reactions to Food Constituent 51 6.1. Food Allergies 51 6.2. Allergy-like Food Poisoning 53 6.3. Adverse Reactions to Gluten and Gluten-Related Disorders 53 6.4. Exposure of a Susceptible Population 57 6.5. Direct Chemical Toxicity 57 6.6. Nonallergic Food Hypersensitivity and Intolerance 58 6.7. Food Color and Food Allergy 58 7. Mechanism of Action of Clinical Disorders Related to Food 59 7.1. Gut Microbiota and Adverse Reactions to Food 59 7.2. Neurotransmission 60 7.3. Channel Blockers 69 7.4. Endocrine Modifiers 69 8. Safety Assessment of Food 70 8.1. Risk/Safety Assessment in Food 70 8.2. Food Additives 71 8.3. Food Contaminants 72 9. Regulation of Food 76 9.1. History of Food-Related Disease 76 9.2. History of Food Regulation 76 9.3. Food Regulations Around the World 81 10. Challenges and Future Developments in Food Safety 90 11. Conclusions 92 Glossary 93 References 94

3. Nutritional Toxicologic Pathology MATTHEW A. WALLIG, AMY USBORNE AND KEVIN P. KEENAN

1. 2. 3. 4.

5.

Introduction 105 Caloric Excess and Obesity 106 Caloric Restriction 110 Macronutrients and Micronutrients 4.1. Introduction 111 4.2. Macronutrients 113 4.3. Micronutrients 119 Dietary Contaminants 166 5.1. Analyses for Contaminants 5.2. Pesticides 166

5.3.

Mycotoxins, Heavy Metals, Phytoestrogens, and Other Contaminants 167 References 168

PART 2 Selected Toxicant Classes in the Environment 4. Herbal Remedies COLIN G. ROUSSEAUX

1. 2. 3.

4.

5. 6.

7.

111 8.

166

Introduction 184 1.1. Nomenclature 184 Apothecary to Pharmacy 189 Evidence for Herbal Remedy Efficacy 190 3.1. Empirical Evidence e Traditional Knowledge (Botanical) 190 3.2. Experimental Evidence e Controlled (Single Active Compound) 192 The Active Pharmaceutical Ingredient(s) 192 4.1. Influencing Factors on the Concentration of the API(s) in the Plant 193 4.2. Dose and Response 193 4.3. Contaminants 194 4.4. Adulterants 194 4.5. A Comparison Between Properties of Herbal Remedies and Conventional Drugs 196 4.6. Acceptability of Herbal Remedies 196 Quality, Efficacy and Safety 197 Quality 199 6.1. The Active Pharmaceutical Ingredient 200 6.2. Quality Control 200 6.3. Manufacturing Processes and Controls 200 Efficacy and Effectiveness 200 7.1. Traditional Knowledge of Efficacy 201 7.2. Experimental Evidence 201 7.3. Randomized Clinical Trials Using Herbal Remedies 201 Safety 203 8.1. Safety, Side Effects and Toxicity 203 8.2. Adverse Reactions 203 8.3. Interactions 204 8.4. Herb-Herb Interaction 204

ix

CONTENTS

8.5. 8.6. 8.7.

9.

10.

Direct Toxicity 209 Indirect Toxicity 209 Hypersensitivity e Idiopathic Allergic Reactions 211 Toxicology of Herbal Remedies 211 9.1. Lethality 211 9.2. Genotoxicity and Carcinogenesis 211 9.3. Herbal Toxicokinetics 212 9.4. Microbiome 216 9.5. Herbal Pharmacodynamics 218 9.6. Organ Toxicity 218 Toxicologic Pathology of Select Herbal Remedies 223 10.1. Aloe vera e Aloe barbadensis 223 10.2. Cannabis e Cannabis sativa and Cannabis indica 237 10.3. Chamomile e Chamomilla recutita 238 10.4. Coffee e Coffea arabica and C. Canephora 239 10.5. Cocoa e Theobroma cacao 245 10.6. Echinacea e Echinacea purpurea 248 10.7. Ephedra e Ephedra sinica 248 10.8. Garlic e Allium sativum 250 10.9. Ginkgo Biloba e Ginkgo biloba 252 10.10. Ginger e Zingiberis rhizome 256 10.11. Ginseng e Panax ginseng 257 10.12. Goldenseal e Hydrastis canadensis 258 10.13. Green Tea e Camellia sinensis 259 10.14. Indole-3-Carbinol e Brassica sp. Glucosinolates 260 10.15. Kava kava e Piper methysticum 262 10.16. Milk Thistle e Silybum marianum 266 10.17. Mint Mentha sp. [Contains Pulegone] 267 10.18. Rattlepods, Yellow Burrweed, and GroundseldCrotalaria, Amsinckia, and Senecio Containing [Contains Riddelliine a Pyrrolizidine Alkaloid] 268 10.19. Saw Palmetto e Serenoa repens 270 10.20. Senna e Senna alexandrina 271 10.21. St. John’s Wort e Hypericum perforatum 271

10.22.

Tobacco e Nicotiana tabacum 272 10.23. Turmeric Oleoresin e Curcuma longa 274 11. International Regulatory Overview 275 11.1. Select List of Countries and Their Regulatory Requirements 276 12. Discussion 279 13. Summary 285 References 286

5. Phycotoxins VAL BEASLEY, WAYNE CARMICHAEL, WANDA M. HASCHEK, KATHLEEN M. COLEGROVE AND PHILIP SOLTER

1.

2.

3.

4.

Introduction 306 1.1. Harmful Algal Blooms 306 1.2. Aquatic Hypoxia 308 1.3. Some Important Marine and Freshwater Toxins 309 1.4. The Need for Greater Access to and Reliance on Diagnostic Expertise and Instrumentation 310 1.5. A Future with Fewer Harmful Algal Blooms and Phycotoxin Poisonings 311 1.6. Rationale for the Subsequent Discussions of Phycotoxins 311 Saxitoxins 314 2.1. Source/Occurrence 314 2.2. Toxicology 315 2.3. Clinical Signs and Pathology 315 2.4. Human Exposure and Disease 315 2.5. Diagnosis, Treatment, and Control 316 Cyclic Imines 317 3.1. Source/Occurrence 317 3.2. Toxicology 318 3.3. Clinical Signs and Pathology 318 3.4. Human Exposure and Disease 319 3.5. Diagnosis, Treatment, and Control 319 Domoic Acid 319 4.1. Occurrence and Species Susceptibility 319 4.2. Toxicology 321 4.3. Clinical Signs and Pathology 321 4.4. Human Exposure and Disease 324 4.5. Diagnosis, Treatment, and Control 325

x 5.

6.

7.

8.

9.

10.

CONTENTS

Brevetoxins 325 5.1. Source/Occurrence 325 5.2. Toxicology 326 5.3. Clinical Signs 326 5.4. Gross and Histologic Findings 327 5.5. Human Exposure and Disease 328 5.6. Diagnosis, Treatment, and Control 329 Ciguatoxins 330 6.1. Source/Occurrence 330 6.2. Toxicology 331 6.3. Maitotoxins 331 6.4. Clinical Signs and Pathology 331 6.5. Human Exposure and Disease 331 6.6. Diagnosis, Treatment, and Control 332 Okadaic Acid and Dinophysistoxins 7.1. Source/Occurrence 333 7.2. Toxicology 333 7.3. Clinical Signs and Pathology 334 7.4. Human Exposure and Disease 334 7.5. Diagnosis, Treatment, and Control 335 Azaspiracid Toxins 335 8.1. Source/Occurrence 335 8.2. Toxicology 336 8.3. Clinical Signs and Pathology 336 8.4. Human Exposure and Disease 338 8.5. Diagnosis, Treatment, and Control 338 Cylindrospermopsins 339 9.1. Source/Occurrence 339 9.2. Toxicology 340 9.3. Clinical Signs and Pathology 341 9.4. Human Exposure and Disease 344 9.5. Diagnosis, Treatment, and Control 344 Microcystins and Nodularins 345 10.1. Source/Occurrence 345 10.2. Toxicology 347 10.3. Clinical Signs and Pathology 348

10.4.

333

Human Exposure and Disease 351 10.5. Diagnosis, Treatment, and Control 354 11. Anatoxins 355 11.1. Source/Occurrence 355 11.2. Toxicology 356 11.3. Clinical Signs and Pathology 356 11.4. Human Exposure and Disease 357 11.5. Diagnosis, Treatment, and Control 358 12. Guanitoxin [Formerly Anatoxin-A(S)] 358 12.1. Source/Occurrence 358 12.2. Toxicology 359 12.3. Clinical Signs and Pathology 359 12.4. Human Exposure and Disease 360 12.5. Diagnosis and Treatment 361 13. Lyngbyatoxins and Aplysiatoxins 361 13.1. Source/Occurrence 361 13.2. Toxicology 362 13.3. Clinical Signs and Pathology 363 13.4. Human Exposure and Disease 363 13.5. Diagnosis, Treatment, and Control 364 14. b-Methylaminoalanine 365 14.1. Introduction 365 14.2. Sources/Occurrences/ Exposures 365 14.3. Toxicology 365 14.4. Animal Studies 366 14.5. Mechanism of Action 366 14.6. Human Exposure and Disease 367 14.7. Analytical Methods for Detection and Quantification 367 14.8. Conclusion 368 15. Emerging Phycotoxins 368 15.1. Vacuolar Myelinopathy and Aetokthonotoxin 368 15.2. Palytoxins 370 15.3. Yessotoxins 372 16. Conclusions and Future Needs 373 References 374

xi

CONTENTS

6. Mycotoxins GENEVIEVE S. BONDY, KENNETH A. VOSS AND WANDA M. HASCHEK

1. 2.

3.

4.

5.

6.

7.

Introduction 394 Aflatoxins 401 2.1. Source/Occurrence 401 2.2. Toxicology 401 2.3. Manifestations of Toxicity in Animals 406 2.4. Human Risk and Disease 409 2.5. Diagnosis, Treatment, and Control 411 Ochratoxins 411 3.1. Source/Occurrence 411 3.2. Toxicology 412 3.3. Manifestations of Toxicity in Animals 414 3.4. Human Risk and Disease 416 3.5. Diagnosis, Treatment, and Prevention 417 Patulin 417 4.1. Source/Occurrence 417 4.2. Toxicology 417 4.3. Manifestations of Toxicity in Animals 418 4.4. Human Exposure and Disease 419 4.5. Diagnosis, Treatment, and Control 419 Trichothecene Mycotoxins 419 5.1. Sources/Occurrence 419 5.2. Toxicology 420 5.3. Manifestations of Toxicity in Animals 425 5.4. Human Risk and Disease 430 5.5. Diagnosis, Treatment, and Prevention 433 Zearalenone 433 6.1. Source/Occurrence 433 6.2. Toxicology 434 6.3. Manifestations of Toxicity in Animals 436 6.4. Human Risk and Disease 439 6.5. Diagnosis, Treatment, and Prevention 440 Fumonisins 440 7.1. Source/Occurrence/ Exposure 440 7.2. Toxicology and Mode of Action (MOA) 441 7.3. Manifestations of Toxicity in Animals 446 7.4. Human Risk and Disease 452 7.5. Diagnosis, Treatment, and Prevention 454

Regulations and Guidances 454 Alkaloids 455 Introduction 455 Source/Occurrence 455 Toxicology 457 Manifestations of Toxicity in Animals 460 8.5. Human Risk and Disease 463 8.6. Pharmaceutical Use 464 8.7. Diagnosis, Treatment and Prevention 465 9. Emerging Mycotoxins 465 9.1. Introduction 465 9.2. Alternaria Toxins 466 9.3. Aspergillus and Penicillium Toxins 468 9.4. Tremorgenic Mycotoxins 471 9.5. Fusarium Toxins 472 9.6. Diagnosis, Treatment, and Control 476 10. Summary/Conclusion 477 Acknowledgments 477 References 477 8.

7.6. Ergot 8.1. 8.2. 8.3. 8.4.

7. Poisonous Plants BRYAN L. STEGELMEIER AND T. ZANE DAVIS

1. 2.

3.

4.

Introduction 490 Selected Hepatotoxic Plants 490 2.1. Dehydropyrrolizidine Alkaloid eContaining Plants 490 2.2. Saponin-Containing Plants 494 2.3. Plants Containing Fungal Hepatotoxins 495 2.4. Alsike Clover 498 2.5. Lantana 498 2.6. Cocklebur (Xanthium strumarium) and Other Potent Hepatotoxic Plants 499 Selected Neurotoxic Plants 500 3.1. Plant-Induced Storage Diseases (Swainsonine/Calestegine/ Castanospermine) 500 3.2. Ryegrass Toxicity 506 3.3. Larkspur 509 3.4. Centaurea spp. 510 3.5. Nitro-Toxins 511 3.6. Hemlocks 512 3.7. Lupines 513 3.8. Death Camas 513 Selected Myotoxic Plants 514 4.1. Cardioactive Glycoside-Containing Plants 514 4.2. Rayless Goldenrod and White Snakeroot 515

xii

CONTENTS

4.3. Other Myotoxic Plants 517 Selected Teratogenic Plants 521 5.1. Lupine 521 5.2. Veratrum californicum 523 5.3. Poison Hemlock 524 6. Selected Nephrotoxic Plants 524 6.1. Oak 525 6.2. Lily and Grapes 526 6.3. Oxalate-Containing Plants 527 6.4. Amaranthus spp. 529 6.5. Calcinogenic Glycoside-Containing Plants 530 7. Other Toxic Plants 532 7.1. Pine Needles 532 7.2. Cyanogenic Plants 533 7.3. Nitrate-Accumulating Plants 535 7.4. Photosensitizing Plants 536 7.5. Bracken Fern 539 7.6. Ricinus spp. 541 8. Additional Resources 542 References 542

Systemic (Multi-Organ) Failure 604 5. Diagnosis and Treatment of Zootoxin-Mediated Diseases 604 5.1. Diagnosis 604 5.2. Treatment 607 6. Regulatory Guidance Regarding Zootoxins 609 6.1. Sources and Major Indications of Medicinal Zootoxins 610 6.2. Practices for Developing ZootoxinBased Medical Products 611 6.3. Practices for Developing Antivenom Products 614 7. Summary 615 Glossary 616 Acknowledgments 617 References 617

8. Animal Toxins

1. 2.

5.

BRAD BOLON, KATHLEEN HEINZ-TAHENY, KARA A. YEUNG, JUSTIN OGUNI, TIMOTHY B. ERICKSON, PETER R. CHAI AND CHARLOTTE E. GOLDFINE

1. 2.

3.

4.

Introduction 547 Sources of Exposure 550 2.1. Poisoning 551 2.2. Envenomation 554 2.3. Deliberate Administration 557 Zootoxin Classification 559 3.1. Zootoxin Classification by Source 562 3.2. Zootoxin Classification by Molecular Structure 568 3.3. Zootoxin Classification by Function 569 3.4. Zootoxin Classification by Mechanism of Action 573 Clinical Presentations and Pathologic Manifestations of Zootoxin-Mediated Diseases 595 4.1. Blood Vessels and Blood Components 595 4.2. Epithelium (Cutaneous and Mucosal Surfaces) 598 4.3. Kidney 600 4.4. Liver 601 4.5. Lung 601 4.6. Muscle (Cardiac and Skeletal) 602 4.7. Neuromuscular Junction and Other Peripheral Synapses 603

4.8.

9. Bacterial Toxins BRAD BOLON, FRANCISCO A. UZAL AND MELISSA SCHUTTEN

3. 4.

5.

6.

Introduction 629 Exotoxins 633 2.1. Sources of Exposure 633 2.2. Toxicology 640 Endotoxins 651 3.1. Sources of Exposure 651 3.2. Toxicology 653 Clinical Presentations and Pathologic Manifestations of Bacterial Toxin-Mediated Diseases 657 4.1. Enteric Effects (Intestine) 658 4.2. Fascial Effects (Connective Tissue) 658 4.3. Hepatic Effects (Hepatocytes and Hepatic Immune Cells) 660 4.4. Microvascular Effects (Blood Vessels) 660 4.5. Myotoxic Effects (Cardiac and Skeletal Muscle) 661 4.6. Neurotoxic Effects (Brain and Terminal Nerve Synapses) 662 4.7. Pneumotoxic Effects (Lung) 663 4.8. Systemic Effects (Sepsis and Toxic Shock Syndrome) 664 4.9. Miscellaneous Effects Attributed to Bacterial Toxins 664 Diagnosis and Treatment of Bacterial Toxine Mediated Diseases 665 5.1. Diagnosis 665 5.2. Treatment 667 Regulatory Guidance Regarding Bacterial Toxins 668

xiii

CONTENTS

6.1.

Food and Beverage Production and Water Treatment 668 6.2. Manufacturing Biomedical Products 668 6.3. Safety Assessment of Immunotoxins 670 7. Summary 671 Glossary 672 Acknowledgments 673 References 673

10. Metals SHARON M. GWALTNEY-BRANT

1. 2.

3.

4.

5.

6.

7.

8.

Introduction 680 Antimony 680 2.1. Sources and Exposure 680 2.2. Toxicology 680 2.3. Manifestations of Toxicosis 682 2.4. Diagnosis and Treatment 683 Arsenic 683 3.1. Inorganic Arsenic 683 3.2. Organic Arsenic 686 3.3. Arsine 687 3.4. Diagnosis and Treatment 687 3.5. Chemical Warfare Considerations 688 Beryllium 688 4.1. Sources and Exposure 688 4.2. Toxicology 688 4.3. Manifestations of Toxicosis 688 4.4. Diagnosis and Treatment 690 Bismuth 691 5.1. Sources and Exposure 691 5.2. Toxicology 691 5.3. Manifestations of Toxicosis 691 5.4. Diagnosis and Treatment 692 Cadmium 692 6.1. Sources and Exposure 692 6.2. Toxicology 693 6.3. Manifestations of Toxicosis 695 6.4. Diagnosis and Treatment 699 Chromium 700 7.1. Sources and Exposure 700 7.2. Toxicology 701 7.3. Manifestations of Toxicosis 701 7.4. Diagnosis and Treatment 702 Lead 702 8.1. Sources and Exposure 702 8.2. Toxicology 703 8.3. Manifestations of Toxicosis in Animals 705 8.4. Human Exposure and Disease 706 8.5. Diagnosis and Treatment 708

9.

Mercury 709 9.1. Sources and Exposure 709 9.2. Toxicology 709 9.3. Elemental Mercury 711 9.4. Inorganic Mercury 711 9.5. Organic Mercury 712 9.6. Diagnosis and Treatment 714 10. Plutonium 715 10.1. Sources and Exposure 715 10.2. Toxicology 715 10.3. Manifestations of Toxicosis 716 10.4. Diagnosis and Treatment 716 11. Thallium 716 11.1. Sources and Exposure 716 11.2. Toxicology 717 11.3. Manifestations of Toxicosis 717 11.4. Diagnosis and Treatment 719 12. Uranium 719 12.1. Sources and Exposure 719 12.2. Toxicology 719 12.3. Manifestation of Toxicosis 720 12.4. Diagnosis and Treatment 721 13. Summary and Conclusions 721 References 722

11. Agrochemicals ELIZABETH F. MCINNES, SABITHA PAPINENI, MATTHIAS RINKE, FREDERIC SCHORSCH AND HEIKE A. MARXFELD

1. 2.

3.

4.

Introduction 727 Herbicides 728 2.1. Introduction 728 2.2. Inhibition of Cell Division and Growth 729 2.3. Activation of Reactive Oxygen Species 730 2.4. Inhibition of Cellular Metabolism 735 Fungicides 736 3.1. Introduction 736 3.2. Triazole-Containing Azole Fungicides (Conazoles)/DMI-Fungicides (Demethylation Inhibitors)/C14Demethylase Inhibitors 737 3.3. Succinate Dehydrogenase Inhibitor Fungicides 740 3.4. Strobilurins or Quinol Oxidation Site of Complex III Inhibitor Fungicides 741 Insecticides 741 4.1. Introduction 741 4.2. Organophosphates and Carbamates 742 4.3. Organochlorines 744 4.4. Pyrethrins and Pyrethroids 745

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CONTENTS

4.5. New Insecticides 746 Rodenticides 748 5.1. Introduction 748 5.2. Anticoagulant Rodenticides 5.3. Cholecalciferol 753 5.4. Inorganic Compounds: Metal Phosphides 754 5.5. Alphachloralose 756 5.6. Bromethalin 757 5.7. Corn Cob 757 5.8. Strychnine 758 6. Conclusions 758 References 758

8.2.

5.

749

12. New Frontiers in Endocrine Disruptor Research PAUL S. COOKE, CHERYL S. ROSENFELD, NANCY D. DENSLOW, CHRISTOPHER J. MARTYNIUK, ANA M. MESA, JOHN A. BOWDEN, TRUPTI JOSHI, JUEXIN WANG, JUAN J. ARISTIZABAL-HENAO AND ANATOLY E. MARTYNYUK

1. 2.

3. 4.

5. 6. 7. 8.

Introduction 766 Environmental Chemicals Can Disrupt Endocrine Signaling 766 2.1. History of Endocrine Disruptor Research 766 2.2. Types of Chemicals With EndocrineDisrupting Activity 768 2.3. Routes of Exposure to Endocrine Disruptors 769 2.4. Regulatory Approaches to Endocrine Disruption 771 Mechanisms of Endocrine Disruption 773 Examples of Disruption of Endocrine Pathways by Some Environmental Contaminants and Emerging Endocrine Disruptors 773 4.1. Phthalates Disrupt Several Endocrine Pathways 774 4.2. Emerging Endocrine Disruptors: Glyphosate 774 4.3. Emerging Endocrine Disruptors: General Anesthetics as Endocrine Disruptors 775 Epigenetic Effects of EDCs 776 From Reactive to Proactive Endocrine Disruptor Analysis 778 Emerging Models in EDC Research 778 7.1. Zebrafish Model 778 7.2. CRISPR Screening 779 Omics Technologies to Evaluate Endocrine Disruption 780 8.1. Transcriptomics and Proteomics 780

Lipidomics and Metabolomics 781 8.3. Microbiome 783 8.4. Exposomics 786 9. New Frontiers in Bioinformatics and Integrative and Functional Enrichment Omics Approaches 788 9.1. Integrative Correlation Analyses 788 9.2. Integrative MultiOmics Pathway Resolution 789 10. Machine Learning and EDCs 789 10.1. How Machine Learning Works 789 10.2. Examples of Current Deep Learning Programs for Toxicology 793 11. Conclusions 793 Abbreviations 794 References 794

13. Nanoparticulates ANN F. HUBBS, DALE W. PORTER, ROBERT R. MERCER, VINCENT CASTRANOVA, LINDA M. SARGENT AND KRISHNAN SRIRAM

1.

Background 797 1.1. Definitions 797 1.2. Historical Perspective 798 1.3. Development of Nanotechnology 800 1.4. Current and Future Nanotechnology Applications 801 1.5. Human Exposures 803 2. Experimental Toxicologic Pathology of NPs 804 2.1. Enhanced Toxicity of Nanoscale Particulates 804 2.2. Visualizing NPs in Tissue 805 2.3. Cytopathology 809 2.4. Target Organ and Tissue Toxicity 818 2.5. Human Relevance of Experimental Studies in Animals 827 3. Future Trends in Nanopathology and Nanotoxicology 828 4. Conclusions 829 Acknowledgments 830 References 830

14. Radiation and Other Physical Agents ERIC D. LOMBARDINI AND MICHELLE E. PACHECO-THOMPSON

1.

Introduction 840 1.1. Sources and Occurrence 841 1.2. Impact of Household Technologies and Nonionizing Radiation 845

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CONTENTS

Part I Ionizing Radiation 846 2. Nature and Action of Ionizing Radiation 846 3. Mechanisms of Ionizing Radiation Injury 853 3.1. Interaction of Ionizing Radiation with Biological Materials 853 3.2. Subcellular and Cellular Effects of Ionizing Radiation 854 3.3. Cell and Tissue Radiosensitivity to Ionizing Radiation 856 3.4. General Tissue and Organ Effects of Ionizing Radiation 857 3.5. Molecular Mediators 858 3.6. Acute Radiation Syndromes and Combined Injury 859 3.7. Ionizing Radiation Carcinogenesis 860 4. Response to Injury Induced by Ionizing Radiation 862 4.1. Vascular and Connective Tissue Effects of Ionizing Radiation 862 4.2. Hematopoietic and Lymphoid Systems 865 4.3. Alimentary System 872 4.4. Nervous System 877 4.5. Endocrine System 880 4.6. Special Senses 882 4.7. Respiratory System 885

4.8. Musculoskeletal System 892 4.9. Cardiovascular System 896 4.10. Urinary System 899 4.11. Fetal Effects 903 4.12. Reproductive Tract 903 4.13. Integumentary System 905 Part II Ultraviolet Radiation 908 5. Nature and Action of Ultraviolet Radiation 908 6. Mechanisms of Ultraviolet Radiation Injury 909 7. Response to Injury Induced by Ultraviolet Radiation 910 7.1. Integument 910 7.2. Eye 913 7.3. Immune System 914 7.4. Ultraviolet Radiation Carcinogenesis 916 Part III Hyperthermia 919 8. Clinical Use of Hyperthermia 919 9. Mechanisms of Hyperthermia-Induced Injury 919 10. Response to Injury Induced by Hyperthermia 920 10.1. Reaction of Specific Organs and Tissue to Hyperthermia 920 Acknowledgments 924 References 924

Index

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Contributors Juan J. Aristizabal-Henao Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States Val Beasley University of Illinois at Urbana-Champaign, Urbana, IL, United States Brad Bolon

Ann F. Hubbs NIOSH, Centers for Disease Control and Prevention, Morgantown, WV, United States Trupti Joshi Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States Kevin P. Keenan Formerly Charles River Laboratories, Frederick, MD, United States

GEMpath, Inc., Longmont, CO, United States

Genevieve S. Bondy Formerly Food Directorate, Health Canada, Ottawa, ON, Canada

Eric D. Lombardini Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand

Gary A. Boorman Covance Laboratories Inc., Chantilly, VA, United States

Christopher J. Martyniuk Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States

John A. Bowden Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States Wayne Carmichael United States

Anatoly E. Martynyuk McKnight Brain Institute, University of Florida, Gainesville, FL, United States Heike A. Marxfeld BASF SE, Ludwigshafen, Germany

Wright State University, Dayton, OH,

Elizabeth F. McInnes

Syngenta, Berkshire, United Kingdom

Vincent Castranova NIOSH, Centers for Disease Control and Prevention, Morgantown, WV, United States

Robert R. Mercer NIOSH, Centers for Disease Control and Prevention, Morgantown, WV, United States

Peter R. Chai Brigham and Women’s Hospital, Boston, MA, United States

Ana M. Mesa Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States

Phaedra I. Cole Global Pharmacokinetics, Dynamics, Metabolism and Safety, Zoetis, Kalamazoo, MI, United States

Justin Oguni The Veterinary Clinic West, Marietta, GA, United States

Kathleen M. Colegrove University of Illinois at UrbanaChampaign, Urbana, IL, United States

Michelle E. Pacheco-Thompson Department of Defense Food Analysis and Diagnostic Laboratory, San Antonio, TX, United States

Paul S. Cooke Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States

Sabitha Papineni Labcorp Early Development Laboratories Inc., Greenfield, IN, United States

T. Zane Davis USDA/ARS Poisonous Plant Research Laboratory, Logan, UT, United States Nancy D. Denslow Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States Susan A. Elmore United States

ElmorePathology, LLC, Chapel Hill, NC,

Timothy B. Erickson Brigham and Women’s Hospital, Boston, MA, United States Charlotte E. Goldfine Brigham and Women’s Hospital, Boston, MA, United States Sharon M. Gwaltney-Brant Veterinary Information Network, Mahomet, IL, United States Wanda M. Haschek University of Illinois at UrbanaChampaign, Urbana, IL, United States Kathleen Heinz-Taheny Eli Lilly and Company, Indianapolis, IN, United States

Dale W. Porter NIOSH, Centers for Disease Control and Prevention, Morgantown, WV, United States Olga M. Pulido University of Ottawa, Ottawa, ON, Canada Matthias Rinke

Formerly of Bayer AG, Wuelfrath, Germany

Cheryl S. Rosenfeld Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States; Thompson Center for Autism and Neurobehavioral Disorders, Columbia, MO, United States Colin G. Rousseaux

University of Ottawa, Ottawa, ON, Canada

Linda M. Sargent NIOSH, Centers for Disease Control and Prevention, Morgantown, WV, United States Frederic Schorsch Bayer S.A.S, R&D Crop Science, Sophia Antipolis, France Melissa Schutten United States

Genentech, Inc., South San Francisco, CA,

Philip Solter University of Illinois at Urbana-Champaign, Urbana, IL, United States

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CONTRIBUTORS

Krishnan Sriram NIOSH, Centers for Disease Control and Prevention, Morgantown, WV, United States Bryan L. Stegelmeier USDA/ARS Poisonous Plant Research Laboratory, Logan, UT, United States Amy Usborne Formerly Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, United States Francisco A. Uzal United States

University of California, Davis, CA,

Kenneth A. Voss Formerly United States Department of Agriculture, Agricultural Research Service, Athens, GA, United States Matthew A. Wallig University of Illinois at UrbanaChampaign, Urbana, IL, United States Juexin Wang Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States Kara A. Yeung Brigham and Women’s Hospital, Massachusetts General Hospital, Boston, MA, United States

About the Editors

EDITORS Wanda M. Haschek-Hock, BVSc, Ph.D., is a Diplomate of the American College of Veterinary Pathologists (DACVP), Fellow of the International Academy of Toxicologic Pathology (FIATP), and Honorary Member of the Latin American Society of Toxicologic Pathology. She is Professor Emerita at the University of Illinois College of Veterinary Medicine, Department of Pathobiology. Wanda has over 40 years of experience in comparative, respiratory, and toxicologic pathology with a research focus on natural toxins and food safety. She is a former President of the Society of Toxicologic Pathology (STP) and of the Society of Toxicology’s (SOT) Comparative and Veterinary Specialty Section and has served as an Associate Editor for Toxicological Sciences and Toxicologic Pathology, as Councilor of the American College of Veterinary Pathologists (ACVP), and as Member of the American Board of Toxicology (ABT). She served as an Editor for the three editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology. She is a recipient of the STP’s Lifetime Achievement Award, the SOT Midwest Regional Chapter’s Kenneth DuBois Award, and the University of Sydney Faculty of Veterinary Science Alumni Award for International Achievement in 2016. Colin G. Rousseaux, BVSc, Ph.D., DABT, FIATP, is also a Fellow of the Royal College of Pathology (FRCPath) and Fellow of the Academy of Toxicological Sciences (FATS). He is a Professor (Adjunct) in the Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of Ottawa, Canada. He has over 35 years of experience in comparative and toxicologic pathology with a research focus on herbal remedies, fetal development and teratology, and environmental pollutants. He has described, investigated, and evaluated numerous toxicologic pathology issues associated with pharmaceuticals, pesticides, and agrochemicals. He has served on the editorial board of Toxicologic Pathology. He is a former President of the STP. Colin served as an Editor for the three editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology.

Matthew A. Wallig, DVM, Ph.D., DACVP, is Professor Emeritus in the Department of Pathobiology, College of Veterinary Medicine, the Department of Food Science and Human Nutrition, as well as the Division of Nutritional Sciences at the University of Illinois. His research has focused on the chemoprotective properties and mechanisms of phytochemicals in the diet, in particular those in cruciferous vegetables, soy, and tomatoes. His current interests have expanded to include defining morphologic parameters for diagnostic quantitative ultrasound in pancreatitis, pancreatic and hepatic neoplasia, metastatic disease, and chronic hepatic diseases such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Matt has served as an Editor for the last two editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology. Brad Bolon, DVM, MS, Ph.D., DAVCP, DABT, FATS, FIATP, FRCPath, has worked [sic] as an experimental and toxicologic pathologist in several settings: academia, a contract research organization, pharmaceutical companies (in both biomolecule and traditional small molecule settings), and private consulting. His main professional interests are the pathology of genetically engineered mice (especially embryos, fetuses, and placentas) and toxicologic neuropathology to assess the efficacy and safety of many therapeutic entities (biomolecules, cell and gene therapies, medical devices, and small molecules). He is a former President of the STP and a Member of the American College of Toxicology (ACT), British Society of Toxicological Pathology (BSTP), and European Society of Toxicologic Pathology (ESTP). Brad served as an Editor for the third edition of the Fundamentals of Toxicologic Pathology and an Associate Editor for the third edition of Haschek and Rousseaux’s Handbook of Toxicologic Pathology.

ASSOCIATE EDITORS Kathleen M. Heinz-Taheny, DVM, Ph.D., DACVP, DABT, currently serves as a Associate Vice President, Toxicology, at Lilly Research Laboratories. She has 16 years of experience as a pathologist and toxicologist supporting pharmaceutical safety assessment and drug

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ABOUT THE EDITORS

discovery research. Katie’s interests include renal pathology and rodent renal models, immunohistochemistry including tissue cross-reactivity studies, and digital pathology along with image analysis. She has served multiple offices including President of the Association of Reptile and Amphibian Veterinarians and served as Cochair of the 2018 STP conference “Keeping it Renal,” as well as chair of the STP Annual Symposium Committee. In 2019, Katie codirected the Industrial Toxicology and Pathology Short Course with Wanda Haschek-Hock. Daniel G Rudmann, DVM, Ph.D., DACVP, FIATP, is currently a Scientific Director at Charles River Laboratories, Inc. He has 26 years of experience as a pathologist and toxicologist in pharmaceutical discovery and development across therapeutic modalities and has expertise in digital pathology and machine learning, and reproductive toxicologic pathology. He has served as an Associate Editor for the journal Toxicologic Pathology and as Chair or Cochair for various STP committees.

ILLUSTRATIONS EDITOR Beth W. Mahler is employed by Experimental Pathology Laboratories, Inc., located in Research Triangle Park, NC, and works as a contractor at the U.S. National Institute of Environmental Health Sciences (NIEHS) in the Cellular and Molecular Pathology Branch under the Division of the National Toxicology Program (NTP). She has over 42 years of experience as a certified histologist (HT) in the areas of histology, animal necropsy, embryo collection and sectioning, and digital photomicroscopy. Since 2006, she has served as the Illustrations Editor for the journal Toxicologic Pathology. Past illustrative editorship roles include Associate Editor of Pathology of the Mouse, edited by Dr. Robert R. Maronpot, and Illustrations Editor for previous editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology.

Preface

Since its inception three decades ago, Haschek and Rousseaux’s Handbook of Toxicologic Pathology has been a comprehensive resource covering fundamental knowledge and skills as well as key technical procedures essential for the proficient practice of toxicologic pathology. The reference has found a home in the libraries of numerous academic, government, and industrial institutions engaged in basic and applied biomedical research around the globe, and in doing so has become an indispensable reference for many toxicologic pathologists, toxicologists, regulators, physicians, biomedical researchers, and students. Indeed, the Handbook has been recognized by many as the most authoritative single source of information in the field due to the breadth and depth of coverage for this field . Prior to publication of the inaugural Handbook edition in 1991, information regarding toxicologic pathology had to be gleaned in a piecemeal manner by reading articles in various toxicology and the few toxicologic pathology journals. The success of the one-volume 1st edition and the expanded roles of toxicologic pathologists over time led in due course to the release of updated Handbook versions in subsequent years: a two-volume 2nd edition in 2002 and a three-volume 3rd edition in 2013. The many scientific advances, ongoing and extensive collaboration among global societies of toxicologic pathology, and new regulatory guidance that has occurred since release of the 3rd edition amply prove that another rendition is necessary to maintain, and indeed enhance, the value of this resource to practitioners of the toxicologic pathology craft. For this reason, the Editors and Associate Editors are pleased to offer this new and expanded Handbook to aid your explorations of the toxicologic pathology field. This 4th edition of Haschek and Rousseaux’s Handbook of Toxicologic Pathology has been extensively updated to continue its comprehensive coverage. This update required extensive expansion, which was initially conceived as a four-volume text. However, the unrelenting explosion of information in this field instead has necessitated that this new edition instead be rendered as a five-volume set where the contents of Volume 2 of the 3rd edition now are divided between Volumes 2 (“Toxicologic Pathology in Safety Assessment”) and 3 (“Environmental Toxicologic Pathology and Selected Toxicant Classes”). Volume 3 will be the first volume or book

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completely dedicated to environmental pathology since the 1990s. For the 4th edition, the new five-volume design distributes important concepts in the following way. • Volume 1 (released in 2022) covers “Principles and the Practice of Toxicologic Pathology,” including such key topics as basic concepts in biology, pathology, pharmacology, and toxicology as they intersect in the field of toxicologic pathology (Chapters 2e8); primary methods in toxicologic pathology (Chapters 9e16); overviews of major models employed in toxicologic research (Chapters 17e24); and essential practices in generating and interpreting toxicologic pathology data (Chapters 25e29). New chapters in Volume 1 address absorption, distribution, metabolism, and excretion (ADME) principles for biomolecules; toxicologic pathology considerations in developmental and reproductive toxicity (DART) testing; digital pathology; and various animal models including rodents, rabbits, dogs, nonhuman primates, and alternative models (e.g., in silico, ex vivo). Because of the recent decision to divide Volume 2, leading to a five-volume 4th edition, chapter cross-referencing in the initially released Volume 1 (by volume and chapter number) may be incorrect when referring to contents of other volumes. • Volume 2 (released in 2023) addresses “Safety Assessment and Toxicologic Pathology.” Safety assessment covers the application of toxicologic pathology in developing specific product classes (Chapters 1e12) and principles of data interpretation (Chapters 13e17). New chapters in this section consider fundamental attributes of novel therapeutic classes (nucleic acid- and protein-based pharmaceutical agents, gene therapy and gene editing, stem cell and other cell therapies, medical devices, vaccines); agricultural and bulk chemicals; and differentiation of adverse from nonadverse effects. These chapters address both pathology and regulatory issues. Previous chapters on such topics as drug discovery and development, toxicity and carcinogenicity testing, report preparation, and risk assessment and communication have undergone extensive revision that includes in-depth discussion of new developments in the field.

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PREFACE

• Volume 3 is dedicated to “Environmental Toxicologic Pathology and Selected Toxicant Classes.” This volume covers toxicologic pathology as it relates to food (Chapter 2), nutrition (Chapter 3), and selected toxicant classes in the environment that affect human and animal health (Chapters 4e14). New chapters include herbal products (Chapter 4) and animal- and bacteria-derived toxins (Chapters 8 and 9); for these chapters, particular attention has been paid to the role of such substances in formulating new therapies (either as the active agent or as important contaminants of the manufacturing process). All previous chapters (e.g., endocrine disruptors, heavy metals, nanoparticulates, poisonous plants, radiation) have been extensively revised and updated. Organspecific environmental diseases will be addressed in volumes 4 and 5. • Volumes 4 and 5 provide deep and broad treatment of “Target Organ Toxicity” for major systems, emphasizing the comparative and correlative aspects of normal biology and toxicant-induced dysfunction, principal methods for toxicologic pathology evaluation, and major mechanisms of toxicity. New topics addressed in Volume 4 include the toxicologic pathology of adipose tissue, tendons, and teeth. New information regarding product development and regulatory issues has been incorporated in the organ system chapters for both volumes. The expanded coverage of nonclinical testing principles and practices, the new material on various models of toxicity, and the toxicologic pathology of novel toxicant

classes (especially new therapeutic modalities) will be a particular strength of this new Handbook edition and should and will continue to justify its long-standing reputation for excellence. The Editors do accept that information relevant to toxicity research, hazard identification, and risk assessment and management is ever growing and they acknowledge that readers may benefit by extending their search for up-to-the-minute information in this area to include other textbooks and journals of toxicology and toxicologic pathology. We would like to thank the dedicated efforts of the Associate EditorsdStacey Fossey and John Vahle (Volume 1), Katie Heinz-Taheny and Dan Rudmann (Volumes 2 and 3), Molly Boyle and Mark Hoenerhoff (Volume 4), and Jeff Everitt and Karen Regan (Volume 5); our incomparable Illustrations Editor, Beth Mahler; and the many chapter authors for their outstanding contributions in bringing this book to fruition. In addition, we wish to specifically acknowledge the significant involvement of many leading toxicologic pathologists as essential subject matter experts in contributing to this and previous editions; many are now retired, and a growing number are deceased including our friends and mentors Charles C. Capen, Victor J. Ferrans, Gordon C. Hard, Adalbert Koestner, Robert W. Leader, Daniel Morton, John F. Van Vleet, and Hanspeter R. Witschi. Wanda M. Haschek Colin G. Rousseaux Matthew A. Wallig Brad Bolon

P A R T 1

TOXICOLOGIC PATHOLOGY IN ENVIRONMENTAL AND FOOD PROTECTION

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C H A P T E R

1 Environmental Toxicologic Pathology and Human Health Susan A. Elmore1, Gary A. Boorman2 1

ElmorePathology, LLC, Chapel Hill, NC, United States; 2Covance Laboratories Inc., Chantilly, VA, United States

O U T L I N E 1. Introduction

3

2. History of Carcinogenic Testing in Animal Species

4

3. Principles of Evaluations for Carcinogenic Potential

6

4. Examples of Environmental Pollutants 4.1. Workplace Exposure 4.2. General Environmental Contaminants 4.3. Air Pollutants 4.4. Water Pollutants 4.5. Ground and Soil Contamination 4.6. Radiofrequency Radiation 4.7. Microplastics and Nanoplastics

8 9 10 13 15 16 17 20

5. The Role of Lifestyle and the Environment on Human Health

22

23 23 24

7. Current Considerations for Environmental Toxicity and Carcinogenicity Testing 7.1. Mechanism of Action versus Mode of Action 7.2. Human Relevancy 7.3. Alternative Testing Strategies

25 25 25 26

8. New Directions for Environmental Toxicity and Carcinogenicity Testing 8.1. Safe and Sustainable Alternatives

27 28

References

29

and Drug Administration (FDA). The first large-scale testing program was initiated by the National Cancer Institute (NCI), a subdivision of the NIH, in the 1970s, as part of a congressionally mandated war on cancer. In 1978, the bioassay program, as it was known, was transferred to the National Toxicology Program (NTP), located within the National Institute of Environmental Health Sciences (NIEHS), both of which are subdivisions of the NIH. To date, over 500 2-year toxicity and carcinogenicity

1. INTRODUCTION A major effort during the past 50 years has been to identify carcinogens and toxicants that occur in the environment and workplace, using animal models, toxicology, and molecular biology. Federal agencies that participated in this effort included the National Institutes of Health (NIH), the National Institute for Occupational Safety and Health (NIOSH), the Environmental Protection Agency (EPA), and the Food Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00001-0

6. Methods of Toxicity and Carcinogenicity Testing 6.1. Fish Models 6.2. Transgenic Mouse Models

3

Copyright Ó 2023 Elsevier Inc. All rights reserved.

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1. ENVIRONMENTAL TOXICOLOGIC PATHOLOGY AND HUMAN HEALTH

studies have been conducted by the NCI/NTP, and numerous environmental and workplace hazards have been identified, resulting in decreased human exposure and, thus, improved human health. A similar number of studies have been conducted by other countries, especially Europe and Japan. For example, the Fraunhofer Institute for Toxicology and Experimental Medicine in Hannover, Germany, focused on inhalation research, and the Netherlands Organisation for Applied Scientific Research (TNO) has evaluated nanoparticles and food stuffs. The British Industrial Biological Research Association (BIBRA) has provided reviews and evaluations on many long-term cancer studies conducted by chemical and pharmaceutical firms. The Japanese Ministry of Health, Labor and Welfare has also conducted many short-term and long-term studies in rodents and in cell cultures and these can be accessed from their online Japan Existing Chemical Database (JECDB). Improvements in study design, animal models, study evaluations and reporting, quality assurance procedures, and risk assessment methodologies have occurred as part of these efforts. Toxicity and carcinogenicity studies conducted worldwide are used for regulatory decisions in numerous countries. A joint global initiative called “International Harmonization of Nomenclature and Diagnostic Criteria” (INHAND) has harmonized diagnostic criteria and terminology for rodent, dog, rabbit, non-human primate and minipig studies for the global community. An additional INHAND publication for fish species is currently underway. Other international efforts are focused on finding ways to refine studies and reduce the number of animals used to identify potential human hazards (see Nomenclature and Diagnostic Resources in Anatomic Toxicologic Pathology, Vol 1, Chap 25). Websites for Government and Other Agencies Involved in the Identification of Environmental Toxicants and Carcinogens 1. BIBRA (British Industrial Biological Research Association). https://www.bibra-information.co.uk/. Accessed August 25, 2022. 2. EPA (Environmental Protection Agency). https:// www.epa.gov/. Accessed August 25, 2022. 3. FDA (U.S. Food and Drug Administration). https:// www.fda.gov/. Accessed August 25, 2022.

4. INHAND (International Harmonization of Nomenclature and Diagnostic Criteria). https:// www.toxpath.org/inhand.asp. Accessed August 25, 2022. 5. JECDB (Japan Existing Chemical Database). https:// dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp. Accessed August 25, 2022. 6. NCI (National Cancer Institute). https://www.cancer. gov/. Accessed August 25, 2022. 7. NIEHS (National Institutes of Environmental Health Sciences). https://www.niehs.nih.gov/. Accessed August 25, 2022. 8. NIH (National Institutes of Health). https://www.nih. gov/. Accessed August 25, 2022. 9. NIOSH (National Institute for Occupational Safety and Health). https://www.cdc.gov/NIOSH/. Accessed August 25, 2022. 10. NTP, National Toxicology Program. https://ntp.niehs. nih.gov/. Accessed August 25, 2022. 11. TNO (The Netherlands Organisation for Applied Scientific Research). https://en.wikipedia.org/wiki/ Netherlands_Organisation_for_Applied_Scientific_ Research. Accessed August 25, 2022.

2. HISTORY OF CARCINOGENIC TESTING IN ANIMAL SPECIES It is generally recognized that the English surgeon, Percivall Pott, was the first to suggest that an environmental agent could cause cancer in humans. Dr. Pott reported in 1775 that scrotal cancer among chimney sweeps was likely related to exposure to soot (Poirier, 2016). The initial discovery that cancer could be induced in animals by chemical compounds came in 1915 when cancer developed after the painting of coal tar on the ears of rabbits by Yamagiwa and Ichikawa, two Japanese investigators (Yamagiwa and Ichikawa, 1918). Over the next 50 years, more examples of chemically induced cancers in animals and humans were reported. In 1974, vinyl chloride was demonstrated to induce hemangiosarcomas of the liver in rats (Block, 1974; Kielhorn et al., 2002). Shortly thereafter, workers clearing out vats containing vinyl chloride were reported to develop hemangiosarcomas in the liverdan extremely rare human cancer. Demonstration of hemangiosarcomas in rodents prior to their recognition in vinyl chloride workers provided convincing evidence that rodent models could prove useful in

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2. HISTORY OF CARCINOGENIC TESTING IN ANIMAL SPECIES

identification of carcinogens that might occur in the environment and workplace. The concept in the 1950 and 1960s was that the elimination of cancer-causing chemicals from the environment would reduce the cancer burden in humans, with the World Health Organization suggesting that more than 75% of human cancers were environmentally related and thus could be prevented. Since thousands of chemicals were known to be in the environment and workplace, the goal of scientists at the National Cancer Institute was to evaluate hundreds of chemicals in screening studies (Boorman et al., 1994). The chemicals were to be screened in rodents, giving the maximum dose compatible with long-term survival. These animals were compared to controls that were often shared among several studies. Initially the rodents were examined only macroscopically, and chemicals that did not cause grossly visible masses (assumed to be tumors) in the animals at maximum lifetime exposure were considered relatively safe. Chemicals that caused tumors noted grossly were selected for further mechanistic studies. What was not anticipated was the immediate call for regulations to limit environmental or workplace exposure for chemicals found to cause masses in rodents, rather than waiting for the more definitive 2-year and mechanistic studies. Attempts to make regulatory decisions on these screening studies revealed that these initial studies were completely inadequate for complex risk assessment evaluations, including setting acceptable exposure limits. Setting standards from a single positive mouse result without metabolism and distribution studies to shed light on possible reasons for sex and species differences was difficult and not appropriate. Further, in many screening studies the animals were dosed by gavage or intraperitoneal injection, providing very little data on acceptable levels for air or water pollutants. The United States led an effort to evaluate chemicals for potential carcinogenic activity as part of the Carcinogenesis Program of the National Cancer Institute (NCI) beginning in 1962. The NCI is part of the National Institutes of Health (NIH), and was established under the National Cancer Institute Act of 1937 as the US government’s principal agency for cancer research and training.

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The National Cancer Act of 1971 broadened the scope and responsibilities of the NCI and created the National Cancer Program. Part of the effort included converting Fort Detrick, previously a US Army biological warfare research center, into a cancer research center. The NCI budget was markedly increased, and as part of the increased budget a program to test chemicals was established. The bioassay program included toxicologists, chemists, laboratory animal veterinarians, health and safety personnel, and veterinary pathologists working as a team in the selection of chemicals for evaluation, study design, selection of animal models, study conduct, and study evaluation (see Carcinogenicity Assessment, Vol 2, Chap 5). Various government agencies, including the EPA, NIOSH, and FDA, provided advice on chemical selection, animal models, and study design. Within a few years, approximately 200 chemicals were started on 2-year studies in rats and mice. The rapid increase in testing needs and lack of government facilities led the NCI staff to conduct these studies under contract by various laboratories and, occasionally, universities. As positive results were found for some important commercial chemicals, firms with a vested interest challenged study results, often employing pathologists who found that some findings were incorrect, poorly documented, or subject to another interpretation. The early history of the bioassay program has been nicely documented by Norbert Page, who was director of the program in the early 1970s (Page, 1977). This is an excellent resource for readers who want to learn more about the early history of chemical testing. Many countries became involved in this effort to identify chemicals that had carcinogenic activity either through individual studies at universities or institutes more focused on animal studies including the Fraunhofer Institute of Toxicology and Aerosol Research (Hannover, Germany), National Institute of Hygiene (Tokyo, Japan), Ramazzini Institute (Bologna, Italy), Huntingdon Research Centre (Huntingdon, UK), National Center for Toxicological Research, NCTR (Jefferson, AK, USA), and many others. Mainly in response to the thalidomide tragedy, in 1962, an amendment was added to the Food, Drug and Cosmetic Act requiring safety studies

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for all new drugs in the United States. This greatly increased the number of rodent carcinogenicity studies. Long-term animal studies are very complex, and the quality of the study and the interpretation of results varied widely. In 1965 the World Health Organization established The International Agency for Research on Cancer (IARC) which has helped provide standards for the conduct and evaluation of rodent studies (Parce et al., 2015). To address the quality of the NCI studies, NCI contracted with Experimental Pathology Laboratories Inc., a contract research organization (CRO), to conduct a quality assurance (QA) pathology peer review of the NCI studies (see Pathology and GLPs, Quality Control and Quality Assurance, Vol 1, Chap 27). Discrepancies between study and peer-review pathologists were usually resolved by a group of pathologists referred to as a Pathology Working Group (PWG). The use of pathology QA and PWGs have resulted in a marked improvement in the quality of the pathology data from toxicology studies (see Pathology Peer Review, Vol 1, Chap 26). Jerry Hardisty, of Experimental Pathology Laboratories, was one of the leaders in developing and refining the pathology review process and introducing the regulatory community to the benefits of a procedure that improves the quality of the data used in the protection of human health. Currently, nearly all studies submitted to regulatory agencies have been submitted to QA and peer review and/or PWG procedures. The NCI bioassay program was transferred in 1978 to the National Toxicology Program (NTP), located at the National Institute of Environmental Health Sciences (NIEHS), to expand the studies beyond just cancer endpoints. Over the years, chemical evaluations have become broader, including assays other than those for genetic alterations and general toxicity. Examples include assays for reproductive toxicity (see Male Reproductive System, Vol 5, Chap 9, and Female Reproductive System, Vol 5, Chap 10), immunotoxicity (Immune System, Vol 5, Chap 6), neurotoxicity (Nervous System, Vol 4, Chap 8), endocrine disruption (New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12 and Endocrine System, Vol 4, Chap 7), and developmental toxicity (Embryo, Fetus and Placenta, Vol 5, Chap 11). One of the strengths of the current

NCI/NTP program is that the toxicity and carcinogenicity studies are subject to a rigorous three-tier pathology peer review followed by a public peer review of the technical reports. This public peer review stimulates efforts to continue to improve the quality of the studies. While some initial studies were published in peer-review journals, it was decided to make all the data available through the US Government Printing Office as well as online (NTP Technical Reports Index). The complete raw data as well as the final technical reports are available to the public, regulatory agencies, industry, and other interested groups as soon as the data are finalized. The data are an excellent source for historical control data, for tumor incidence by class of chemicals, and for generating hypotheses for further testing.

3. PRINCIPLES OF EVALUATIONS FOR CARCINOGENIC POTENTIAL A variety of assays for genetic alterations revealed that all chemicals could be broadly separated into two classes: namely genotoxic and nongenotoxic (see Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8). The term “genotoxic carcinogen” indicates a chemical capable of producing cancer by directly altering the genetic material of target cells, while “nongenotoxic carcinogen” represents a chemical capable of producing cancer by a secondary mechanism, not related to direct DNA damage. Genotoxic carcinogens include polycyclic aromatic hydrocarbons (PAHs) such as benzopyrene, a component of coal-tar creosote used as a wood preservative, and dibenzanthracene, which is currently used as the main indicator of carcinogenic PAHs in air pollution; alkylating agents, such as nitrosamines used in the manufacture of some cosmetics, pesticides, and rubber products, and ethylene oxides used as a raw material in the large-scale production of chemicals such as ethylene glycol; and aromatic amines and amides, such as aniline dyes and benzidines, used in the production of dyes. Genotoxic carcinogens were generally accepted as posing a potential human health hazard, and exposure limits were set for environmental and workplace exposure. Limiting exposure to

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genotoxic carcinogens was generally accepted by the scientific community as reasonable for the protection of human health. Although nongenotoxic chemicals were shown to be negative for mutagenicity in a series of test systems, they could nevertheless result in cancer. Nongenotoxic chemicals have been shown to act as tumor promoters (e.g., 1,4-dichlorobenzene), endocrine modifiers (e.g.,17b-estradiol), receptor mediators (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin), immunosuppressants (e.g., cyclosporine), or inducers of tissue-specific toxicity and inflammatory responses (e.g., metals such as arsenic and beryllium). Nongenotoxic compounds that showed limited to moderate evidence of carcinogenic activity elicited considerable scientific debate. In many cases increased tumors occurred only at high exposures, were sometimes accompanied by target organ toxicity, were limited to a single species or, often, one sex, and involved a single tissue. In time it became clear that the mouse liver was a common site for an increase in benign and/or malignant tumors following exposure to nongenotoxic compounds. In some cases, regulatory agencies were willing to consider ancillary mechanistic data on these compounds that might show the compound posed little risk for humans. The liver (see Liver and Gallbladder, Vol 4, Chap 2) is the most common site for positive genotoxic and nongenotoxic tumor responses in rodents and has resulted in considerable effort to standardize liver diagnostic and terminology criteria. In the 1950s, a variety of terms, such as hepatoma Type I, hepatoma Type II, hepatoma Type A, and hepatoma Type B, as well as adenoma and carcinoma, were used in rodent studies. In both rats and mice, carcinomas were recognized by their pleomorphism and often trabecular patterns. However, criteria for separating benign tumors from hyperplasia were not well established. A meeting to resolve this issue for the rat resulted in the term “neoplastic nodule” for proliferative lesions of the rat liver that were not malignant. This term, although controversial, remained in use for years. The NTP held a series of meetings and published the results to establish the standard criteria of hyperplasia, adenoma, carcinoma, and hepatoblastoma. The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) has further established criteria for

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proliferative and nonproliferative lesions of the rat and mouse liver (Thoolen et al., 2010). The new international criteria recognizes both regenerative (in response to injury) and nonregenerative hyperplasia, hepatocellular adenoma, and hepatocellular carcinoma as proliferative lesions of hepatocytes. While the criteria have evolved and become generally accepted, the results, when restricted to the liver, can still be controversial. Positive results in the mouse liver may be accompanied by negative results in the rat, sometimes at higher exposures. Over the years this has resulted in less concern about chemicals that cause only mouse liver tumors, especially when the response is limited to one sex. Early in the testing program, when chemicals were often genotoxic and quite clearly carcinogenic, the convention was to separate all chemicals evaluated into two classes: carcinogens or noncarcinogens. As less toxic compounds were evaluated, often with only a positive response in the mouse liver, it became apparent that simply separating all compounds into carcinogenic or noncarcinogenic categories was inadequate. The NTP recognized this dilemma and now refers to “carcinogenic activity” by the sex and strain affected. Thus, a compound may show no carcinogenic activity in rats and male mice with some evidence of carcinogenic activity for the female mouse liver. This more nuanced approach recognizes that carcinogenic activity might be restricted to a single rodent strain or sex. During the course of analyzing and reporting over 500 2-year studies, it appeared that some chemicals had a sex- and/or species-specific effect. Thus, many research groups used the NTP results as a starting point for mechanistic research to help put the results into perspective. The Chemical Institute of Industrial Toxicology (CIIT) provided many mechanistic studies to complement NTP studies. A classic example involving a non-NTP study was the association of sodium saccharin with bladder tumors in male rats (see Lower Urinary Tract, Vol 5, Chap 3). In a chronic study and at high doses, precipitates were formed in the bladder (Cohen et al., 1995). But rodents, unlike humans, have a unique combination of high pH, high calcium phosphate, and high protein levels in their urine. Mechanistic studies have shown that one or more of the proteins that are prevalent in male

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rats combine with calcium phosphate and saccharin to produce microcrystals that damage the lining of the bladder (Whysner and Williams, 1996). This leads to increased cell proliferation and thus fewer opportunities for repair enzymes to repair damaged DNA during replication, leading to increased opportunity for genetic mutations and tumor formation. Therefore, with no other mechanistic pathways or mode of action identified, sodium saccharin– induced tumors were not considered relevant to human carcinogenesis. However, other potential mechanisms for urinary bladder carcinogenesis were shown to exist in rodents and humans. Chronic human exposure to persistent low-dose ionizing radiation from the Chernobyl accident resulted in the development of chronic proliferative atypical cystitis characterized by dysplasia and carcinoma in situ (Romanenko et al., 2009). The proposed mechanism is oxidative stress with upregulation of at least two signaling pathways (p38 mitogen-activated protein kinase and nuclear factor-kB cascades) and activation of growth factor receptors that result in microenvironment changes such as angiogenesis and remodeling of the extracellular matrix. Other examples of compound-related urinary bladder carcinogenesis include the chronic activation of PPAR-a and PPAR-g agonists within the urothelium, and the acid hydrolysis of N-hydroxy-arylamine-N-glucuronides to electrophilic N-hydroxy-arylamines and arylnitrenium ions in the urinary bladder lumen and subsequent absorption through the urothelium (Kadlubar et al., 1977; Oleksiewicz et al., 1977). Another example of a sex- and species-specific effect involves hydrocarbons and renal tumor formation. Unleaded gasoline and other nongenotoxic chemicals were shown to cause renal tumors in male but not female rats and not in either sex of mouse. A series of studies was undertaken to understand this sex- and speciesspecific hydrocarbon nephropathy and carcinogenesis (Doi et al., 2007; Swenberg, 1993). The formation and accumulation of the male rat– specific a2m-globulin protein was identified in the P2 segment of the proximal tubule with subsequent single cell necrosis, granular casts, regenerative tubules, linear mineralization in the papillae (hyaline droplet nephropathy), and, with chronic exposure, exacerbation of chronic progressive nephropathy. Chronic

exposure also led to increases in renal tumors and the proposed mode of action involved sustained tubular cell proliferation. Therefore, a subsequent NTP study investigated various chemicals that were associated with a2m-globulin accumulation. Two chemicals (propylene glycol mono-t-butyl ether and Stoddard solvent IIC) showed a dose-related increase in a2m-globulin protein without a corresponding treatmentrelated increase in renal tumors, although the magnitude of the a2m-globulin accumulation was equivalent to other studies that did have a clear tumor response. For those chemicals that did have hyaline droplet nephropathy in the prechronic study and renal tumors at 2 years, the results showed that, while hyaline droplet nephropathy may contribute to the renal tumor response, the critical component of the nephropathy most closely associated with the development of renal tumors could not be identified. Therefore, none of the nonneoplastic endpoints considered part of hyaline droplet nephropathy in the prechronic studies was consistently predictive of the ultimate tumor outcome in the 2-year studies (see Kidney, Vol 5, Chap 2). Websites for the History of Carcinogenic Testing in Animal Species 1. NTP Technical Reports Index. https://ntp.niehs.nih. gov/data/tr/index.html. Accessed August 25, 2022. 2. Brigs and Briggs, 1943. https://cancerres.aacrjournals. org/content/jcanres/3/1/1.full.pdf. Last Accessed August 25, 2022.

4. EXAMPLES OF ENVIRONMENTAL POLLUTANTS There are many examples of man-made environmental pollutants, including those from commercial or industrial waste, those released from consumer use (e.g., motor vehicles), and those that are used for protection of human health, such as water disinfection by-products. There can be exposure to people within a workplace, exposure to residents in the general vicinity of industrial waste products, or exposure to the general population, locally or globally. Due to the vast array of environmental pollutants, only a select few will be highlighted for discussion.

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4.1. Workplace Exposure One example of workplace exposure to a hazardous chemical is diacetyl poisoning (NIOSH, 2003). Diacetyl gives butter and certain food flavorings a distinctive buttery flavor and aroma. A number of employees at a microwave popcorn packaging plant in Missouri that used diacetyl for butter-like flavoring developed bronchiolitis obliterans, commonly referred to as “popcorn worker’s lung” (Kreiss et al., 2002). This is a rare, nonreversible, and life-threatening obstructive lung disease where the bronchioles are compressed and narrowed by fibrosis, usually with inflammation. In 2000, the Missouri Department of Health and Senior Services requested technical assistance from NIOSH in this investigation. They conducted a cross-sectional survey of 117 current factory workers that included interviews, pulmonary-function testing, and air sampling for volatile organic compounds and dusts. It was determined that the employees that mixed the butter flavoring had the highest exposure to diacetyl and the highest incidence of bronchiolitis obliterans. Based on this investigation, NIOSH published an alert in 2003 and an update in 2004 that described health effects that may occur because of workplace exposure, gave examples of workplace settings in which illness did occur, and recommended steps that companies and workers should take to prevent hazardous exposure (NIOSH, 2003, 2004). In

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2008, Dan Morgan, Gordon Flake, and colleagues at NIEHS/NTP investigated the respiratory toxicity of diacetyl in C57BL/6 mice and demonstrated that oropharyngeal aspiration of clinically relevant doses reaches the distal airways and results in lesions of endobronchiolar fibrohistiocytosis, suggestive of early stages of bronchiolitis obliterans (Morgan et al., 2008). In 2011, they demonstrated severe airway epithelial injury, aberrant repair, and bronchiolitis obliterans after diacetyl instillation in rats (Palmer et al., 2011) (Figure 1.1), which replicated the features of the human disease (Morgan et al., 2012). Diacetyl is currently approved by the FDA as a safe flavor ingredient, and to date there are no specific standards for occupational exposure to diacetyl. However, as a result of information regarding the adverse health effects of diacetyl inhalation, the Flavor and Extract Manufacturers Association recommended a reduction of diacetyl in butterlike flavorings, and the Occupational Safety and Health Association (OSHA) published hazard communication guidance for diacetyl and food flavorings containing diacetyl. Several manufacturers have since removed this chemical from popcorn products, and awareness has been raised for both employers and employees. Unfortunately diacetyl, as well as many other harmful chemicals, are constituents identified in e-cigarette liquids and aerosols (Eshraghian & Al-Delaimy, 2021; Farsalinos et al., 2015).

FIGURE 1.1 Lung: male Sprague–Dawley rats 7 days following a single intratracheal instillation of 125 mg/kg of diacetyl resulting in bronchiolitis obliterans. H&E stain. (A) Illustration of a bronchiole with polypoid intraluminal fibrosis and peribronchiolar inflammation. (B) Concentric intramural fibrosis within a bronchiole, typical of constrictive bronchiolitis obliterans. Images courtesy of Dr. Gordon Flake, NIEHS/NTP. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3rd ed, Academic Press, 2013, Figure 18.1, p. 1034, with permission.

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Asbestos is another classic example of workplace exposure to a hazardous chemical. “Asbestos” is a term for a set of six naturally occurring silicate minerals (chrysotile, amosite, crocidolite, tremolite, actinolite, and anthophyllite). The silicate tetrahedron (SiO4) is the basic chemical unit of all silicate minerals. Each silica mineral is classified based on the number of tetrahedra in the crystal structure and how they are arranged. Each also varies in toxicity related to form and size of fibers, surface chemistry, and biopersistence. Asbestos became increasingly popular among manufacturers and builders in the late 19th century due to its resistance to fire, heat, electrical, or chemical damage; sound absorption; tensile strength; and affordability. In 2009 approximately 2 million tons of asbestos were mined worldwide, with Russia as the largest producer (50%), followed by China (14%), Brazil (12.5%), Kazakhstan (10.5%), and Canada (9%). It has been used in various applications, such as building insulation and electrical insulation for hotplate wiring and fireproofing roofing and flooring, and the fibers were often mixed with cement or woven into fabric or mats. Prolonged inhalation of asbestos fibers can result in a variety of serious illnesses, including asbestosis warts, pleural plaques, diffuse pleural thickening, pneumothorax, asbestosis (a type of pneumoconiosis), lung cancer, and mesothelioma (Bianchi and Bianchi, 2007). Historically, miners were affected the most due to long exposures to high concentrations. During World War II thousands of tons of asbestos were used to wrap pipes, line boilers, and cover engine and turbine parts in ships. Approximately 100,000 people in the United States are expected to die from ship-building related asbestos exposure. Contamination of products such as vermiculite and talc has also increased public exposure. Exposure of construction workers (remodeling and demolition) and occupants in buildings containing asbestoscontaminated building materials remains a problem today. Although the European Union has banned all use of asbestos, including the extraction, manufacture, and processing of asbestos products, and Australia has entirely banned the use of asbestos, many countries have no regulation or allow the limited use of/exposure to asbestos. For example, the United States EPA issued the Asbestos Ban and Phase Out Rule in 1989, but this was subsequently overturned to allow many consumer products to

legally contain trace amounts of asbestos. Washington State banned asbestos in automotive brakes starting in 2014, and OSHA has set workplace exposure limits of 100,000 fibers with lengths greater than or equal to 5 microns per cubic meter of workplace air for 8-h shifts and 40-h work weeks. Despite the known carcinogenic effects of asbestos and regulatory efforts, 90,000 people die from asbestos-related diseases globally each year. Epidemiological studies of asbestos-exposed workers and supporting animal studies indicate that inhalation of asbestos is the principal route of exposure of public health concern (Addison and McConnell, 2008; Niklinski et al., 2004). Direct and indirect mechanisms have been proposed on the basis of in vitro cellular assays and acute and subchronic animal bioassays. Established mechanistic events leading to toxicity and cancer are: impaired fiber clearance leading to macrophage activation, inflammation, generation of reactive oxygen and nitrogen species, tissue injury, genotoxicity, aneuploidy and polyploidy, epigenetic alteration, activation of signaling pathways, and resistance to apoptosis (Straif et al., 2009). Respiratory responses to inhalation of asbestos fibers are substantially different between species, and the biological mechanisms responsible for these differences are unknown. The combination of cigarette smoking and asbestos exposure increases the risk of lung cancer in humans due to a synergistic effect, indicating that assessments of industrial health risks should take smoking and other airborne health risks into consideration when setting occupational asbestos exposure limits (Ngamwong et al., 2015).

4.2. General Environmental Contaminants Chemicals can become general environmental contaminants through either unintentional or intentional release into the water, soil, or air. Dioxin and dioxin-like compounds are but one example of well-known and persistent environmental pollutants. “Dioxin” is a common name for the polychlorinated dibenzo-p-dioxins (PCDDs), including 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD), and a group of structurally and chemically related compounds called “dioxin-like compounds” with toxic responses similar to TCDDs, such as polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and others (Dioxins, 2017; Table 1.1). Dioxins and dioxin-like compounds produce

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TABLE 1.1

Dioxins and Dioxin-like Compounds

POLYCHLORINATED BIPHENYLS

TCB

Tetrachlorinated biphenyl

PeCB

Pentachlorinated biphenyl

HxCB

Hexachlorinated biphenyl

HpCB

Heptachlorinated biphenyl

OCB

Octachlorinated biphenyl

PCB

Polychlorinated biphenyl

POLYCHLORINATED DIBENZO-P-DIOXINS

TCDD

Tetrachlorinated dibenzo-p-dioxin

PeCDD

Pentachlorinated dibenzo-p-dioxin

HxCDD

Hexachlorinated dibenzo-p-dioxin

HpCDD

Heptachlorinated dibenzo-p-dioxin

OCDD

Octachlorinated dibenzop-dioxin

PCDD

Polychlorinated dibenzop-dioxin

POLYCHLORINATED DIBENZOFURANS

TCDF

Tetrachlorinated dibenzofuran

PeCDF

Pentachlorinated dibenzofuran

HxCDF

Hexachlorinated dibenzofuran

HpCDF

Heptachlorinated dibenzofuran

OCDF

Octachlorinated dibenzofuran

PCDF

Polychlorinated dibenzofuran

Table reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3rd ed, Academic Press, 2013, Table 18.1, p. 1036, with permission.

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a similar spectrum of toxic effects, and the mechanism of toxicity is the high-affinity binding of the compound to the aryl hydrocarbon (Ah) receptor. An NTP 2-year gavage study of TCDD in female Harlan Sprague–Dawley rats revealed a variety of treatment-related neoplastic and nonneoplastic lesions in several organs (https://ntp.niehs.nih.gov/ntp/htdocs/ lt_rpts/tr521.pdf). Neoplasms included increased incidences of cholangiocarcinoma, hepatocellular adenoma, cystic keratinizing epithelioma of the lung, and gingival squamous cell carcinoma of the oral mucosa. The toxic equivalence factor (TEF) was developed to facilitate risk management and regulatory control of this class of compounds and is based on the ability to activate the Ah receptor. It expresses the toxicity of dioxins and dioxin-like compounds in terms of TCDD, the most potent form of dioxin. Therefore, a mixture of dioxins and dioxin-like compounds can be expressed as a single number called the “toxic equivalent” or TEQ. In addition to being carcinogenic, some dioxins, like TCDD, are potent dermatological (chloracne), reproductive, endocrine, developmental, cardiovascular, and immune system toxicants (Figure 1.2). This class of compound is found in the soil, with very low levels present in plants, water, and air. But dioxins accumulate in the adipose tissue of animals, with a half-life in the body estimated to be 7–11 years. More than 90% of human exposure occurs through food consumption, mainly dairy products, fish, and shellfish. Human exposure may also occur from mother to child via breast milk. PCDDs and PCDFs are unwanted by-products of industrial processes such as smelting, production of herbicides and pesticides, and chlorine bleaching of paper pulp. Sources of significant dioxin contamination are mainly incomplete incinerator burning, as well as release from long-term storage and improper disposal of waste industrial oils with high levels of PCDFs. There has been historical widespread use of PCBs in transformers, capacitors, heat transfer and hydraulic fluids, plasticizers, and numerous other applications. Once released into the environment, dioxins and dioxin-like compounds may end up in animal feed material. Some examples of

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FIGURE 1.2 Thymus: female Sprague–Dawley rats from an NTP TCDD study. H&E stain. (A) and (B) Normal thymus from a rat treated with vehicle control (corn oil: acetone, 99:1) for 31 weeks. (C) and (D) Atrophic thymus from a rat exposed to 100 ng/kg body weight of TCDD by gavage for 31 weeks. Compared to vehicle control (A and B), the thymus from the TCDD-exposed rat is smaller in size, weighs less, and is characterized by thinning and decreased cellularity of the cortex with a relative increase in medullary regions (C and D). Within the medulla, there is an increased cellularity, prominent epithelium, and occasional pigment-laden macrophages (D). The mechanism of action includes both direct AhR-mediated changes in developing cortical thymocytes and indirect effects on thymocytes via the AhR-expressing thymic epithelial and dendritic cells. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3rd ed, Academic Press, 2013, Figure 18.2, p. 1037, with permission.

isolated but serious incidents of environmental and human contamination include rice bran oil contaminated with PCBs and PCDFs in Japan in 1968 and in Taiwan in 1979; the 1976 release of large amounts of dioxins from a chemical factory in Seveso, Italy; the use of contaminated bentonite clay in the manufacture of animal feed in the United States in 1976; contaminated citrus pulp pellets used as animal feed exported from Brazil that resulted in contaminated milk in Germany in 1998; illegally disposed of PCBbased industrial oil waste that contaminated animal feed and, subsequently, poultry and

eggs in Belgium in 1999; the recall of tons of contaminated pork meat in Ireland in 2008 due to contaminated animal feed; and contaminated batches of Agent Orange, a defoliant used during the Vietnam war from 1962 to 1970. There is also increased exposure of residents in northwestern Wisconsin, especially the Hmong descendants, who engage in subsistence fishing in the Lower Fox River (Hutchison and Kraft, 1994; Schantz et al., 2010). During the 1950 and 1960s this river had the highest concentration of pulp and paper mills in the world, and PCBcontaminated waste was routinely discharged

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into the river. The EPA Superfund cleanup for this project began in 1999 and ended in 2020 (EPA Fox River Superfund Site). Perfluorinated substances (PFASs) are a group of more than 4600 man-made chemicals, many with little or no current information on biological effects (Pelch et al., 2019). They have been widely used in multiple industrial and commercial applications since the 1950s and most do not breakdown, resulting in environmental persistence. Examples of commercial uses include nonstick cookware, water-repellent clothing, stain-resistant fabrics and carpets, some cosmetics, some firefighting foams, and products that resist grease, water, and oil. PFASs are found in human and animal blood worldwide and are present in a variety of food products. Consumption of contaminated drinking water, eating fish caught from water contaminated by PFASs, accidentally swallowing contaminated soil or dust, eating food that was packaged in material containing PFAS, and the use of consumer products made with PFAS are considered the major sources of exposure for humans. Multiple worldwide epidemiological studies have reported on the negative effects that PFASs have on human health, including infertility, steroid hormone perturbation, thyroid, liver, and kidney disorders, and metabolic disfunctions (Steenland and Winquist, 2021; Sunderland et al., 2019). Animal toxicology studies have suggested potential suppression of humoral immunity, neuroendocrine effects, and exposure-related gestational and developmental effects. Cumulative evidence from mammalian animal studies has suggested that the liver is an important target organ with hepatotoxic effects that include liver enlargement, hepatocellular adenomas, and peroxisome proliferation [specifically peroxisome proliferator–activated receptor (PPAR)-a], suggesting a possible nongenotoxic carcinogenic mechanism. Unfortunately, new PFASs are constantly being developed, considered as “safe and sustainable alternatives” with few scientific studies to confirm safety (Sheng et al., 2018). Importantly, contaminants such as these can be used or released on a local level but have worldwide distribution. Current control measures to reduce human exposure include control of industrial processes to reduce formation of dioxins, reduction in the release of dioxins via controlled waste incineration with low

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emissions, and monitoring of food supplies for the presence of high levels of dioxins.

4.3. Air Pollutants (also see Respiratory Tract, Vol 5, Chap 4) Air pollution, via the release of chemicals and particulates into the atmosphere, is another serious threat to human health. Dr. Dan Costa, in a very nice brief history of air pollution in Casarett and Doull’s Toxicology, noted that the Roman philosopher Seneca, in 61 AD, felt an “alteration to his disposition” whenever he got away from Rome with the “stink of chimneys and whatever pestilential vapors and soot enclosed in them” (Costa, 2008). This suggests that nearly 2000 years ago Seneca understood that air pollution was detrimental to good health. An inversion fog next to a zinc plant in Donora, Pennsylvania in 1948 killed 20 people and sickened hundreds. A smog that occurred in London in December 1952, mainly from coal heating in homes, was so thick that children were hired to go ahead of cars to help them find their way. Initial estimates were that 100,000 people were made ill and another 4000 premature deaths occurred (Bell et al., 2004). More recent research suggests that the number of fatalities may have been as high as 12,000. This event led to greater public awareness, environmental research, and government regulation of air pollution, and led to Great Britain’s Clean Air Act in 1956. An air pollution control act was established by the US Congress in 1955 and by the UK parliament in 1956. The Clean Air Act of 1970 allowed the EPA to set standards for what kinds of toxic air pollutants could be released into the “ambient air”, either from factories or cars and trucks. The accidental release of methyl isocyanate vapors in 1984 by the Union Carbide India Limited (UCIL) pesticide plant in Bhopal, India, resulted in 3928 certified deaths but with an estimate from independent organizations ranging from 10,000 to 30,000 deaths and perhaps another 100,000–200,000 with permanent injuries. The UCIL plant produced the pesticide Sevin (brand name for carbaryl) using methyl isocyanate as an intermediate. This more recent event, considered one of the world’s worst industrial catastrophes, also raised global awareness of potentially hazardous air pollutants.

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Some of the most common gaseous pollutants today include carbon monoxide, sulfur dioxide, chlorofluorocarbons (CFCs), and nitrogen oxides produced by industry and motor vehicles. Photochemical ozone and smog are created as nitrogen oxides and hydrocarbons react to sunlight. Although ozone in the upper atmosphere is beneficial, preventing potentially damaging electromagnetic radiation from reaching the Earth’s surface, ozone in the lower atmosphere is considered a pollutant with potentially harmful effects on the respiratory systems of animals and humans (see Respiratory System, Vol 5, Chap 4). Long-term exposure to ozone has been shown to increase risk of death from respiratory illness (Jerrett et al., 2009). A study of 450,000 people living in cities with high ozone levels, such as Houston and Los Angeles, showed a significant correlation between ozone levels and respiratory illness over the 18-year follow-up period. This study revealed that chronic exposure to ozone resulted in a 30% increased risk of dying from lung disease. Two-year and lifetime inhalation studies of ozone conducted by the NTP revealed increased incidences of alveolar/bronchiolar adenoma or carcinoma in female B6C3F1 mice as well as a variety of nonneoplastic lesions in mice and/or rats, including goblet cell hyperplasia and squamous metaplasia in the nose, squamous metaplasia in the larynx, and inflammation (histiocytic infiltration), metaplasia (extension of bronchial epithelium into the centriacinar alveolar ducts), and interstitial fibrosis in the lung. In 2015, the EPA published revisions to the National Ambient Air Quality Standard (NAAQS) to set a lower 8-h primary standard level for ozone to provide increased protection for children and other “at risk” populations against an array of ozone-related adverse health effects, including decreased lung function, increased respiratory symptoms, possible cardiovascular-related morbidity, as well as total nonaccidental and cardiopulmonary mortality (EPA Federal Register 65292). Formaldehyde is another example of an air pollutant with known toxic effects. Exposure to the general population is by breathing contaminated indoor or outdoor air, and from tobacco smoke. Some of the major sources of formaldehyde include automobile and other combustion sources, and fumes released from new construction or home-finishing products. It can be a component of smog when sunlight and oxygen react with atmospheric methane and other

hydrocarbons. Formaldehyde has many applications, and is a building block in the synthesis of many other compounds of specialized and industrial significance. It is commonly used in the textile and automobile industries. Regardless of the method of exposure (inhalation, dermal, ingestion), formaldehyde is highly toxic to humans. In 2011, the NTP designated formaldehyde as a known human carcinogen based on sufficient evidence of carcinogenicity from studies in humans and supporting data on mechanisms of carcinogenesis (Formaldehyde, Report on Carcinogens). Epidemiological studies of workers exposed to high levels found that formaldehyde caused myeloid leukemia and rare cancers, including sinonasal and nasopharyngeal cancer. In laboratory animal studies, formaldehyde caused cancer primarily in the nasal cavity, and mechanistic studies determined that genotoxicity was the mechanism of action. Formaldehyde has been classified as a known human carcinogen by the International Agency for Research on Cancer and the National Toxicology Program, and is determined to be a probable human carcinogen by the US EPA. There have been marked improvements in the assessment of various air pollutants and their potential risk for humans. Early in the testing program, most chemicals, including air pollutants, were given by oral gavage because it was a simple route of administration. However, this did not result in primary exposures to the upper respiratory tract or lung. This made extrapolating results to humans and determining acceptable levels in the air difficult. The complexity of inhalation studies, especially particulates, resulted in only a few laboratories specializing in what was then considered the art and science of inhalation studies. Many laboratories equipped for inhalation studies now use this as a common mode of drug delivery. These laboratories have markedly enhanced our understanding of the complexity of the inhalation studies, differences in anatomy and airflow between species, metabolic capacity within the nasal cavities, and the many factors that influence particulate deposition in the airways (see Respiratory System, Vol 5, Chap 4). One simple example of the complexity of inhalation studies was the NTP ozone study, where ozone was uniformly distributed in an empty inhalation chamber but when rats were added, ozone which entered the chamber at 1 ppm was undetectable in the lower sampling ports. Only after

4. EXAMPLES OF ENVIRONMENTAL POLLUTANTS

adding a recirculating fan to increase airflow to 75 air changes per hour was it possible to keep ozone exposure levels uniform throughout the chamber.

4.4. Water Pollutants Public concerns spread beyond just potential carcinogens to any pollutants that might adversely affect humans or the environment. Rachael Carson’s Silent Spring, which appeared in serial form in The New Yorker and later as a bestselling book in 1970, attracted immediate attention and changed public opinion. Carson reported on how humans were misusing powerful, persistent chemical pesticides, such as the aerial spraying of dichlorodiphenyltrichloroethane (DDT) to kill mosquitos, before knowing the full extent of their potential harm to plant and animal life. This publication spurred the reversal in the overall United States’ national pesticide policy and led to a nationwide ban on DDT for agricultural uses. Environmental awareness may also have been due to astronauts on the moon showing images of the earth as a delicate blue/green planet in space. In April 1970 more than 20 million Americans celebrated the first Earth Day, the brainchild of Gaylord Nelson, then a US senator from Wisconsin, who witnessed the destructive effects of a 1969 massive oil spill in Santa Barbara, California. In December 1970, in response to the growing public demand for cleaner water, air, and land, President Richard Nixon and Congress established the US Environmental Protection Agency (EPA). In addition to conducting research, the EPA had the power to establish and enforce environmental standards. Within a few months of his appointment, William Ruckelshaus, the EPA’s first administrator, in a surprise announcement warned the cities of Atlanta, Detroit, and Cleveland of alleged violations of water pollution standards. During the early years of the EPA, based on their findings, many laws were passed that impacted on both air and water toxicants. The Clean Water Act of 1972 allowed the EPA to set standards for what kinds of pollutants could be released into lakes, streams, and rivers, and it forces polluters to obtain permits. Although the U.S. congress delegated power to the EPA to formulate rules that carry the force of law, they can still be overturned by a congressional law, because congress remains the higher power.

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Although the first Earth Day was celebrated in the United States, it became recognized worldwide by 1990 and is now coordinated globally by the Earth Day Network in more than 193 countries (https://www.earthday.org/about-us/; accessed August 25, 2022). There are many types and sources of man-made water pollution that still exist today. For example, water pollution may occur via the direct discharge of wastewater from commercial and industrial waste (intentionally or through spills) into surface waters without prior adequate treatment to remove harmful compounds; through the release of waste and contaminants (e.g., chemical fertilizers and pesticides) into surface runoff flowing to surface waters; through inappropriate or illegal waste disposal and leaching into groundwater; and via chemical contaminants, such as chlorine, from treated sewage. As one example, disinfection by-products (DBPs) such as halogenated acetic acids result from the treatment of water with chlorination and ozonation to destroy pathogens and oxidize odor-forming compounds. Several toxic effects, including testicular damage, have been attributed to the chloroacetic acids. Chloramine has become a popular disinfectant in the United States and has been found to produce Nnitrosodimethylamine, which is a possible human carcinogen, as well as highly genotoxic iodinated DBPs, such as the alkylating agent iodoacetic acid, when iodide is present in source waters. A more recent example of water pollution is the lead contamination of residential water in Flint Michigan that started in 2014 and lasted until 2019 (Ruckart et al., 2019). In 2013 the city ended its five-decade practice of piping treated water for residents from Detroit in favor of the cheaper alternative of pumping water from the Flint River. This change was considered temporary until a new water pipeline from Lake Huron was installed. Flint city officials failed to treat the highly corrosive river water, resulting in lead leaching out from the aging pipes to thousands of homes. The residential water looked, smelled and tasted foul and the incidence of elevated blood lead levels in children doubled in 1 year and tripled in some affected neighborhoods (for more information on lead toxicity see Metals, Vol 3, Chap 10). Access to safe drinking water is considered one of the major challenges to the world today, mostly due to the effects of an increasing

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population, the growing use of fertilizer with eutrophication of surface waters, and global climate change. The World Health Organization considers cyanobacteria (a phylum of bacteria also known as blue-green algae), which rapidly grow in warm nutrient-rich water and produce toxins such as microcystins (a bacterial phycotoxin), a major challenge for this century (see Phycotoxins, Vol 3, Chap 5). Cyanobacteria are a genetically diverse species of bacteria and occupy a broad range of habitats, including freshwater, saltwater, and terrestrial ecosystems. Aquatic cyanobacteria can produce extensive and highly visible blooms in both fresh water and saltwater. Although cyanobacteria are also called blue-green algae, there is no relationship between the cyanobacteria and other organisms called algae. The toxins produced by some cyanobacteria include neurotoxins, hepatotoxins (e.g., microcystins), cytotoxins, and endotoxins that can be toxic to humans as well as other animals and marine life in general. People swimming, water skiing, or boating in lakes with cyanobacteria may be exposed to microcystins or other toxins. There have been worldwide reports of livestock deaths after drinking microcystincontaminated water and human poisoning has been reported as welldsuch as the 1996 incident in Brazil where the water used for renal dialysis was contaminated, resulting in liver failure and death. As mentioned previously, there is a concern about drinking water DBPs which are found in low concentrations following treatment of water with chlorine or chloramines. An interesting dilemma is that efforts to reduce DBPs may allow more microcystins to remain in the drinking water, illustrating the need for a balance in efforts to provide safe drinking water through chemical treatments. Evaluation of compounds found in the water often utilizes rodents with drinking water exposures. The use of fish species has been suggested as an inexpensive animal model that also provides data that are directly relevant to species that inhabit water and the wetlands (see Animal Models in Toxicologic Research: Non-mammalian, Vol 1, Chap 22). An effort by the NTP utilizing two fish species (guppy and medaka) compared the results with traditional 2-year rodent studies following similar standards for chemistry, pathology, and quality assurance. One nongenotoxic and two genotoxic chemicals were evaluated. All three chemicals produced tumors at multiple sites in rats and mice. The fish in

flow-through systems required large amounts of chemical, making chemical disposal more costly and more problematic. One problem not anticipated was that nitromethane, which is rich in nitrogen, resulted in bacterial growth in the exposure aquariums. The solution was to increase water flow and increase tank cleaning, which increased cost and loss of fish. Guppy and medaka that die early are usually lost for examination, further limiting the data from these studies. An additional complication of fish studies is that fish size is determined in part by density, so early deaths may result in increased size in the remaining fish. Only 1,2,3trichloropropane increased liver tumors in both fish species (NTP TR 528). Nitromethane was negative in 14-month studies and 2,2-bis (bromomethyl)-1,3-propanediol was equivocal for liver tumors. This limited study using two fish models and three positive rodent carcinogens suggested that fish models have several technical issues to be addressed before fish could be considered a reliable replacement for rodents.

4.5. Ground and Soil Contamination Soil contamination can occur when chemicals are released by spill or underground leakage, or when chemicals are intentionally released, as with agricultural (pesticide) or industrial use. In 1980, birth defects in children of Love Canal residents in the Niagara Falls area of New York and the need for subsequent relocation focused attention on ground and soil contaminants (Kielb et al., 2010). This also raised awareness of the abandoned hazardous waste sites and was an impetus for the establishment in 1980 of the “Superfund” legislation, which is a common name for the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) (O’Neil, 2007). This law authorized the EPA to address hazardous waste sites across the country by identifying parties responsible for site contamination, and to either compel the parties to clean up such sites or do so itself (when responsible parties cannot be identified) using a special trust fund. The initial clean-up efforts were funded by a tax on petroleum and chemical industries that expired in 1995. To date, approximately 70% of Superfund cleanup activities have been paid for by the parties responsible for the contamination. There have been some notable successes, but the number, size, and complexity of the identified hazardous

4. EXAMPLES OF ENVIRONMENTAL POLLUTANTS

waste sites makes remediation difficult. Evaluation of the chemical mixtures for potential adverse health effects is a difficult and sometimes daunting task. Among the most significant soil contaminants today are hydrocarbons, heavy metals, herbicides, pesticides, and chlorinated hydrocarbons. The health effects of exposure to contaminated soil can vary greatly depending on pollutant type, dose, mode of exposure, and vulnerability of exposed population. Importantly, man-made contaminants released into the water, soil, or air have the potential to result in direct harm to humans, or may cause indirect harm via any number of harmful effects on the ecosystem. Air and water toxicants have often received more attention than soil contaminants. Funds for testing Superfund dumpsite chemicals have resulted in numerous chemicals found in the soil being tested for toxic effects. The separation into soil contaminants is often arbitrary, as volatile chemicals find their way into the air, and most soil contaminants, unless tightly bound, find their way into ground and surface waters. In an effort to reduce air pollution from coal-burning plants, there has been increasing use of nuclear plants to generate a cleaner source of energy. This has not been without hazards. An uncontrolled nuclear reaction in a nuclear reactor could result in widespread contamination of air, soil and water. The accident at the Chernobyl nuclear power plant in 1986 and, more recently, that at the Fukushima Daiichi nuclear power plant in Japan in 2011 are compelling examples (see Radiation and Other Physical Agents, Vol 3, Chap 14). Both accidents resulted in significant soil contamination. Radioactive wastes such as uranium mill tailings, spent (unused) reactor fuel, and other radioactive wastes can remain radioactive and dangerous to human health for thousands of years. A major concern is the anthropogenic radioisotope cesium-137, which binds to the soil and has a half-life of over 30 years, causing concerns for plants, animals, and humans for decades (Taira et al., 2011; Yasunari et al., 2011).

4.6. Radiofrequency Radiation (also see Radiation and Other Physical Agents, Vol 3, Chap 14) Cell phones and other commonly used wireless communication devices transmit

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information via nonionizing radiofrequency radiation (RFR). Because of the common use of wireless communication devices there is wide agreement within the international scientific community that RFR from wireless communications has the potential to pose a health risk to users. In 2011 the WHO International Agency for Research on Cancer (IARC) classified radiofrequency electromagnetic fields (RF-EMFs) as a “possible” (Group B) human carcinogen (Humans, 2013). At the time, there was limited evidence in experimental animals for the carcinogenicity of RFR. In the United States, cellular phones and other wireless communication devices are required to meet the RFR exposure guidelines of the Federal Communications Commission (FCC). The existing exposure guidelines recommend a maximum permissible exposure level of approximately 580 mW per square centimeter and are based on protection from acute injury from thermal effects of RFR exposure rather than on the nonthermal effects of chronic exposures (Federal Communications, 2013). 2G, 3G, and 4G cellular networks have been in use commercially since 1991, 2001, and 2009, respectively, and 5G is the latest generation of cellular technology, purported to provide higher data transfer rates, lower network lag, and increased network capacity. 2G and 3G cellular networks, which are predominately used for voice calls and texting, operate at frequencies between 800 and 1900 MHz (MHz). 4G and 4G-LTE networks, which include additional frequencies between 700 and 2500 MHz, were developed to support increased data needs like streaming video, internet access, and file downloads. 5G networks use millimeter waves (MMWs) and do not travel as far and do not penetrate the body as deeply as the RFR at lower frequencies used in the 2G, 3G, and 4G networks. However, preliminary observations suggest that MMW increase skin temperature, alter gene expression, promote cellular proliferation and synthesis of proteins linked with oxidative stress, inflammatory and metabolic processes, could generate ocular damages, and affect neuromuscular dynamics (Di Ciaula, 2018). There are two types of modulations: global system for mobiles (GSMs) and code division multiple access (CDMA). One of the biggest differences between these two modulations is

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that GSM phones use SIM cards to link a particular phone with its network, while CDMA phones do not require a SIM card because the phone itself is linked to the network. Toxic effects have been reported in RFRexposed laboratory animals and in vitro systems. A comprehensive review of the toxicity of RFR from in vitro models, laboratory animals, and humans was conducted and published in the International Agency for Research on Cancer (IARC) Monograph series (Humans, 2013). Literature reviews have concluded that there is inconsistent and weak evidence for cell phone RFR-associated genotoxicity from various frequencies and modulations of RFR from a variety of experimental systems, ranging from cell-free DNA preparations to cells of exposed animals and humans (Brusick et al., 1998; Repacholi et al., 2012; Verschaeve et al., 2010; Vijayalaxmi and Prihoda, 2012). One complicating factor in the study of the genotoxic effects of cell phone RFR is that, under certain conditions, RFR is sufficiently energetic to heat cells and tissues, and not all studies have considered this factor in their design (Asanami and Shimono, 1997; Komae et al., 1999; Speit et al., 2013). A variety of published health effects of radiofrequency radiation in humans and in animal studies have been reported (FDA, 2020). Epidemiological evidence of adverse health effects in humans is not consistent or convincing and it has been suggested that the lack of cancer in humans indicates an inability to study an exposure for the length of time necessary with an adequate sample size and unexposed comparators (Ahlbom et al., 2004; Miller et al., 2019). Almost no data are available for childhood exposure. An increasing number of people have developed a constellation of symptoms reportedly attributed to exposure to RFR (e.g., headaches, fatigue, appetite loss, insomnia), a syndrome termed Microwave Sickness or Electro-Hyper-Sensitivity (EHS) (Heuser and Heuser, 2017; Miller et al., 2019). However, it has been difficult to show under blind conditions that exposure to EMF can trigger these symptoms (Rubin et al., 2005). Many animal studies have indicated a direct link between exposure to RFR and adverse biological effects; however, some have reported no adverse effects. These contrasting results have

been considered possibly due, in part, to differences in study eligibility criteria, the number of studies included, when the review was conducted, and how studies were evaluated (Ioannidis, 2018). There is a concern that RFR exposure may result in reproductive toxicity. Human studies have shown an effect on sperm motility, viability, DNA fragmentation, and fertilizing ability (Adams et al., 2014; Houston et al., 2016; Kesari et al., 2018; Singh et al., 2018). Many in vivo animal studies have focused on the effects of RFR exposure to adverse female and male reproductive system outcomes. The main targets of the female reproductive system are endometrial tissue, ovarian follicle counts, granulosa cells, and quality of oocytes and embryos during pregnancy. However, inconsistency in study parameters and outcomes make it difficult to draw a firm conclusion (see review by Vornoli et al. (2019)). The main effects seen in rodent male reproductive toxicity include increased testicular proteins, free radical formation, reduced sperm quality, oxidative stress, reduced motility, hypospermatogenesis, and spermatozoa maturation arrest (Guo et al., 2019; Kesari and Behari, 2018; Kesari et al., 2010, 2011; Mailankot et al., 2009; Meo et al., 2011; Sepehrimanesh et al., 2017; Tas et al., 2014). There is a concern that low level chronic exposures may increase the risk of cancer by unknown mechanisms, but epidemiological studies may not provide meaningful data for many years because of the long latency period between exposure and tumor diagnoses, highlighting the need for animal studies. Unfortunately, the majority of animal exposure research is conflicting, and most has not been conducted with actual cellular phone radiation. A significant research effort, involving large well-planned animal experiments, was needed to provide the basis to assess the risk to human health of wireless communications devices. Therefore, in 1999 the U.S. Food and Drug Administration (FDA) nominated RFR used by cell phones for a National Toxicology Program (NTP) study. NTP conducted near life time, 2year studies in Hsd:Sprague Dawley SD rats and B6C3F1 mice using RFR at frequencies (900–1900 MHz) and modulations (GSMs [global system for mobiles] and CDMA [code division multiple access]) used in the US

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telecommunications industry at that time (https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/ tr595_508.pdf; https://ntp.niehs.nih.gov/ntp/ htdocs/lt_rpts/tr596_508.pdf?utm_source¼dire ct&utm_medium¼prod&utm_campaign¼ntpgo links&utm_term¼tr596). Increases in nonneoplastic lesions of the heart, brain, and prostate gland in male rats, and of the brain in female rats, occurred with exposures to CDMAmodulated RFR at 0.9 gigahertz (GHz). Exposure to GSM- or CDMA-modulated cell phone RFR at 1.9 GHz did not increase the incidence of any nonneoplastic lesions in male or female B6C3F1/N mice. Increases in nonneoplastic TABLE 1.2

lesions of the heart, brain, and prostate gland in male rats, and of the heart, thyroid gland, and adrenal gland in female rats occurred with exposures to GSM modulated RFR at 0.9 GHz. The carcinogenic results for rats and mice are listed in Tables 1.2 and 1.3, respectively. The Ramazzini Institute (RI) performed a lifespan carcinogenic study on Sprague–Dawley rats to evaluate the carcinogenic effects of RFR (Falcioni et al., 2018). Animals were exposed prenatally until natural death to mobile phone radiofrequency representative of 1.8 GHz GSM base station radiation emissions. Exposure was to 0, 5, 25 and 50 V/m with a whole-body

NTP Carcinogenesis Results From Male and Female HSD: Sprague–Dawley SD Rats Exposed to Whole-Body Radiofrequency Radiation (RFR) at 900 Megahertz and Modulations (GSM and CDMA) Used by Cell Phones

Diagnosis

Sex

Modulation

Finding

Heart: Malignant schwannoma

Male

GSM and CDMA

Clear evidence of carcinogenic activity

Heart: Malignant schwannoma

Female

GSM and CDMA

Equivocal evidence of carcinogenic activity

Brain: Malignant glioma

Male

GSM and CDMA

Related to RFR

Brain: Malignant glioma

Female

CDMA

Equivocal evidence of carcinogenic activity

Adrenal medulla: Benign, malignant, or complex pheochromocytoma (combined)

Male

GSM

Related to RFR

Adrenal medulla: Benign, malignant, or complex pheochromocytoma (combined)

Female

CDMA

Equivocal evidence of carcinogenic activity

Brain: Benign or malignant granular cell tumors

Male

GSM

May have been related to RFR

Prostate gland: Adenoma or carcinoma (combined)

Male

GSM

May have been related to RFR

Pituitary gland: Adenoma of the pars distalis

Male

GSM and CDMA

May have been related to RFR

Pancreas: Islet cell adenoma or carcinoma (combined)

Male

GSM

May have been related to RFR

Liver: Adenoma or carcinoma (combined)

Male

CDMA

May have been related to RFR

CDMA, code division multiple access; GSM, global system for mobiles. TR-595: https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr595_508.pdf. Explanation for evidence of carcinogenicity: https://ntp.niehs.nih.gov/whatwestudy/testpgm/cartox/criteria/index.html? utm_source¼direct&utm_medium¼prod&utm_campaign¼ntpgolinks&utm_term¼baresults.

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TABLE 1.3 NTP Carcinogenesis Results From Male and Female B6C3F1 Mice Exposed to Whole-Body Radiofrequency Radiation at 1900 Megahertz and Modulations (GSM and CDMA) Used by Cell Phones Diagnosis

Sex

Modulation

Finding

Skin: Fibrosarcoma, sarcoma, or malignant fibrous histiocytoma (combined)

Male

GSM

Equivocal evidence of carcinogenic activity

Lung: Alveolar/bronchiolar adenoma or carcinoma (combined)

Male

GSM

Equivocal evidence of carcinogenic activity

All organs: Malignant lymphoma

Female

GSM

Equivocal evidence of carcinogenic activity

Liver: Hepatoblastoma

Male

CDMA

Equivocal evidence of carcinogenic activity

All organs: Malignant lymphoma

Female

CDMA

Equivocal evidence of carcinogenic activity

TR-596: https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr596_508.pdf. Explanation for evidence of carcinogenicity: https://ntp.niehs.nih.gov/whatwestudy/testpgm/cartox/criteria/index.html? utm_source¼direct&utm_medium¼prod&utm_campaign¼ntpgolinks&utm_term¼baresults.

exposure for 19 h/day. There was a statistically significant increase in the incidence of Schwannomas in the hearts of treated male rats at the highest dose (50 V/m). Although not statistically significant, there was an increase in the incidence of Schwann cell hyperplasia in the hearts of treated male and female rats and malignant glial tumors in the brains of female rats at the highest dose (50 V/m). Long term animal studies published prior to the 2011 IARC review failed to conclusively demonstrate the carcinogenic potential of RFR exposure in experimental animals. Since that time, more in vivo bioassays, such as the NTP and RI studies, have been conducted with improved exposure systems and more accurate measures of RFR dosimetry. When the current NTP studies were being designed, 2G technology was the industry standard, and 3G technologies were under development. While newer technologies (4G and 5G) have continued to evolve, it is important to note that these technologies have not completely replaced the older technologies and have not been evaluated in animal studies (Hardell and Carlberg, 2020). Animal experiments are crucial because meaningful data will not be available from epidemiological studies for many years due to the long latency period between exposure to a carcinogen and the diagnosis of a tumor. The effects of whole-body RFR exposure are not predictive of the results for a local RFR exposure in the same animal, cannot

be directly related to the results of the local RFR exposure a human receives while using a cell phone, and as such is a limitation of rodent studies. Therefore cell phone exposure is not considered hazardous by the FDA (2020). However, animal studies do provide valuable information. Additional studies using animals that are genetically predisposed to cancer and endpoints other than cancer, such as reproductive and neurological effects would be helpful. Replication of prior studies that indicate adverse effects is also needed. As technology progresses, a significant research effort, involving large wellplanned animal experiments, is needed to provide the basis to assess the risk to human health of wireless communications devices.

4.7. Microplastics and Nanoplastics The collective term “microplastics” is used to describe a wide range of plastic particles with potential deleterious effects on human health. The sizes can range from a few microns to several millimeters in diameter and can vary from irregular fragments to spheres and fibers (Thompson, 2015). Microplastics are often released into the environment during the plastic production process. Environmental microplastics may also be produced by the breakdown of discarded plastics or from the release of microbeads used in cosmetics and toothpaste into wastewater. Microplastics are found in

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oceans, glaciers, arctic sea ice, soil, food, indoor air and air samples from national parks (Wetherbee et al., 2019). Degradation of microplastics leads to the formation of nanoplastics and this process may be augmented by organisms such as the Antarctic krill (Dawson et al., 2018). Particles less than 5 mm diameter are considered microplastics while particles less than 100 nm (1 mm) are considered nanoplastics. Micro- and nanoplastics are abundant and ubiquitous in our environment. For example, they are found in our food chain from drinking bottled water and eating ocean fish, in our air from 3D printers and release from synthetic clothing, in the air and soil along highways from wear and tear of tires, and in wastewater from washing clothes made of synthetic fibers (Napper and Thompson, 2016). Sludge from sewage treatment plants is often used as a fertilizer and, when spread on fields, end up contaminating the soil. Millions of tons of plastics find their way into the environment each year and the accumulating mass of plastics is creating an increasing health concern. In one study, plastic and plant fibers were found in more than 80% of human lung tissue and in more than 90% of lung cancers prompting the authors to speculate that these fibers may have played a role in the cancer (Pauly et al., 1998). Microplastics/nanoplastics have also been found in human stool samples (Yong et al., 2020). Microplastics have been shown to accumulate in the liver, kidney and gut of mice (Deng et al., 2017). Disposable plastic added to the environment has greatly increased in response to the emerging COVID-19 viral pandemic with millions of latex gloves, plastic face shields and masks discarded daily. The long life span of plastic and the continued breakdown into microplastics suggests that this accumulation and contamination will continue to be an issue of concern. Evaluation of the class of microplastics and nanoplastics for potential health effects poses an enormous challenge for the toxicologist. The first challenge is the variety of chemicals and chemical additives in microplastics. Numerous raw materials are used in the production of plastic and the additives for color, transparency, UV stabilization, antioxidants, flame retardants, stability, lubricants and fillers add complexity to the mixture. A second challenge is the shape and size of the plastic particles. Most human exposure is by inhalation and/or ingestion of

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multiple microplastics of different sizes, shapes and composition (Amato-Lourenco et al., 2020; Campanale et al., 2020). The World Health Organization expressed concern about the potential of nano- and microplastics on human health (Campanale et al., 2020). All biota, including ecosystems, are susceptible to the toxicological effect of microplastics (Enyoh et al., 2020). Other toxicity literature to date involves marine organisms where various toxicities have been shown (Deng et al., 2017; Napper and Thompson, 2016). Some of the additives used in plastics, such as phthalates, have been studied in traditional rodent studies (Amereh et al., 2020). In the past few years there have been several microplastic/nanoplastic studies involving mice (Yong et al., 2020). In general, mice are less sensitive to microplastics/nanoplastics than fish and the toxicity depends on the size and concentration of the microplastics. Inflammation of the intestine and liver are the most common responses while other studies are negative (Yong et al., 2020; Zheng et al., 2021). Li and colleagues demonstrated that polystyrene microplastics can cause cardiac fibrosis by promoting cardiomyocyte apoptosis in the rat (Li et al., 2020). Polystyrene nanoparticles induced thyroid dysfunction with lower levels of free T3 and T4 and renal disease in the rat (Amereh et al., 2019). Since microplastics are often colorless in histological sections, fluorescent labeling has been used to localize the particles but, unless the dye is stable, it may appear in lipid droplets confounding localizations of the particles (Schur et al., 2019). While human exposure is small, perhaps hundreds of particles per day by inhalation and less by ingestion, this ubiquitous exposure involves nearly the entire human population and exposure is increasing. The coming decades will be a challenge for toxicologists to understand the potential toxicity of nano and microplastics for plants, marine organisms, domestic species and humans as there are significant knowledge gaps in this field. Little is known about the quantity, size, shape and composition of microplastics in the environment, where it can be found and the amount and source of human exposure. Also, little is known about its origins, how the particles are transported, how they are transformed in the environment, and their ultimate fate. It appears that smaller microplastics are more toxic and with continued degradation the number of small

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particles will increase (Hwang et al., 2020; Kogel et al., 2020). This is clearly an area that needs continued attention from the toxicology community. A comprehensive recent review of toxicity of microplastics and nanoplastics in mammalian systems can be found in the Int. J. Environ. Res. Public Health (Yong et al., 2020).

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Websites for Examples of Environmental Pollutants 1. NIOSH, 2006. NIOSH Health Hazard Evaluation Report on Butter Flavoring, 2006. http://www.cdc. gov/niosh/hhe/reports/pdfs/2000-0401-2991.pdf. Accessed August 25, 2022. 2. NIOSH, 2003. NIOSH Alert on Flavorings, 2003. https://www.cdc.gov/niosh/docs/2004-110/pdfs/ 2004-110.pdf?id¼10.26616/NIOSHPUB2004110. Accessed August 25, 2022. 3. NIOSH, 2004. NIOSH Update on Flavorings, 2004. https://www.cdc.gov/niosh/updates/upd-01-15-04. html. Accessed August 25, 2022. 4. OSHAdFlavorings-Related Lung Disease. https:// www.osha.gov/flavorings-related-lung-disease. Accessed August 25, 2022. 5. Dioxins, 2017. http://www.niehs.nih.gov/health/topics /agents/dioxins/index.cfm, Accessed August 25, 2022. 6. PCBs. Hutchison and Kraft, 1994. https://www. sciencedirect.com/science/article/pii/ S0380133094711631. Accessed August 25, 2022. 7. EPA Fox River Superfund Site. https://cumulis.epa. gov/supercpad/cursites/csitinfo.cfm?id=0507723. Accessed August 25, 2022. 8. Ozone. EPA Federal Register 65292. https://www. govinfo.gov/app/details/FR-2015-10-26/2015-26594. Accessed August 25, 2022. 9. Formaldehyde, Report on Carcinogens. https://ntp. niehs.nih.gov/ntp/roc/content/profiles/ formaldehyde.pdf. Accessed August 25, 2022. 10. Fish studies. NTP TR 528. https://ntp.niehs.nih.gov/ ntp/htdocs/lt_rpts/tr528.pdf?utm_source¼direct&utm _medium¼prod&utm_campaign¼ntpgolinks&utm_ term¼tr528. Accessed August 25, 2022. 11. Thompson R.C. (2015) Microplastics in the Marine Environment: Sources, Consequences and Solutions. In: Bergmann M., Gutow L., Klages M. (eds.) Marine Anthropogenic Litter. Springer, Cham. https://doi. org/10.1007/978-3-319-16510-3_7. https://link. springer.com/chapter/10.1007/978-3-319-16510-3_7. Accessed August 25, 2022. 12. Plastics. Wetherbee, G., Baldwin, A., Ranville, J., 2019, It is raining plastic.: U.S. Geological Survey Open-File Report 2019e1048, one sheet, available at https://doi. org/10.3133/ofr20191048. https://pubs.er.usgs.gov/ publication/ofr20191048. Accessed August 25, 2022. 13. Napper IE and Thompson RC. Release of synthetic microplastic plastic fibers from domestic washing

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16.

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machines: Effects of fabric type and washing conditions. Marine Pollution Bulletin 112 (2016) 39e45. https://www.sciencedirect.com/science/article/pii/ S0025326X16307639. Accessed August 25, 2022. Luı´s Fernando Amato-Lourenc¸o, Luciana dos Santos Galvao, Letty A. de Weger, Pieter S. Hiemstra, Martina G. Vijver, Thais Mauad. An emerging class of air pollutants: Potential effects of microplastics to respiratory human health? Science of The Total Environment, Volume 749,2020,141676, ISSN 0048e9697. https://www.sciencedirect.com/science/article/pii/ S0048969720352050. Accessed August 25, 2022. Fatemeh Amereh, Akbar Eslami, Simin Fazelipour, Mohammad Rafiee, Mohammad Ismail Zibaii, Mohammad Babaei, Thyroid endocrine status and biochemical stress responses in adult male Wistar rats chronically exposed to pristine polystyrene nanoplastics, Toxicology Research, Volume 8, Issue 6, November 2019, Pages 953–963, https://doi.org/10. 1039/c9tx00147f. Accessed August 25, 2022. Fatemeh Amereh, Mohammad Babaei, Akbar Eslami, Simin Fazelipour, Mohammad Rafiee, The emerging risk of exposure to nano(micro)plastics on endocrine disturbance and reproductive toxicity: From a hypothetical scenario to a global public health challenge. Environmental Pollution, Volume 261, 2020,114158, ISSN 0269–7491. https://www. researchgate.net/publication/339157075_The_ emerging_risk_of_exposure_to_nanomicroplastics_ on_endocrine_disturbance_and_reproductive_ toxicity_From_a_hypothetical_scenario_to_a_global_ public_health_challenge. Accessed August 25, 2022. Haibin Zheng, Jun Wang, Xuanyi Wei, Le Chang, Su Liu, Proinflammatory properties and lipid disturbance of polystyrene microplastics in the livers of mice with acute colitis, Science of The Total Environment, Volume 750, 2021, 143085, ISSN 00489697. August 25, 2022. EFSA Panel on Contaminants in the Food Chain. https://efsa.onlinelibrary.wiley.com/doi/full/10. 2903/j.efsa.2016.4501. Accessed August 25, 2022. FDA, 2020. Review of published literature between 2008 and 2018 of Relevance to Radiofrequency Radiation and Cancer. https://www.fda.gov/media/ 135043/download. Accessed August 25, 2022.

5. THE ROLE OF LIFESTYLE AND THE ENVIRONMENT ON HUMAN HEALTH An area of increasing interest in environmental hazards is the role that lifestyle can play in susceptibility to cancer and other diseases. “Lifestyle” is a broad term that is used to describe the way a person lives. Outcomes of lifestyle choices that may contribute to disease include stress, diet, lack of exercise, sun exposure, and exposure

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to a variety of chemicals because of drug abuse, alcohol consumption, and smoking (Key et al., 2002; Muscat and Wynder, 1991; Narayanan et al., 2010; Muscat and Wynder, 1991) (see also Food Toxicologic Pathology, Vol 3, Chap 2 and Nutritional Toxicologic Pathology, Vol 3, Chap 3). It was recognized more than 50 years ago that smoking was associated with an increase in lung cancer; the huge impact of smoking on overall public health and the financial costs to society have been documented. The 2010 report of the Surgeon General, “How Tobacco Smoke Causes Disease,” discusses the biology and behavior basis for smoking-attributable diseases (Surgeon General’s Report, 2010). According to this report, cigarettes are responsible for one in five deaths each year in the United States. In addition to lung cancer, cigarette smoke and second-hand smoke is now known to be a cofactor in multiple diseases, including breast cancer, cardiovascular diseases and pulmonary diseases, and in reproductive and developmental effects. The risk for some of these diseases, such as breast cancer and cardiovascular disease, is also influenced by diet and exercise. It took several decades before it was recognized that lifestyle choices such as sun exposure, excessive alcohol consumption, or diet could increase the incidence of cancers. Ultraviolet radiation, mostly UVB but also UVA and UVC, from chronic or severe sun exposure or ultraviolet radiation from sunlamps and tanning beds is known to increase the risk of skin cancer and ocular diseases (see Radiation and Other Physical Agents, Vol 3, Chap 14). Alcohol causes cancers of the liver, oral cavity, pharynx, larynx, and esophagus, and causes a small increase in the risk of breast cancer. Poor diet may result in obesity, which increases the risk of diabetes as well as cancers of the esophagus, colorectum, breast, endometrium, and kidney. More recently, increased fat consumption, reduced fiber consumption, high meat diets and sugarsweetened carbonated soft drink consumption are reported to be associated with increased risk of colorectal cancer (Hodge et al., 2018; Pacheco et al., 2019). Genetic predisposition is another consideration for certain cancers. Therefore, it can be difficult to discern the causative agent (i.e., genetics, lifestyle, or environmental exposure) of certain types of cancer, and quite often the cause may be multifactorial. The National Cancer Institute defines risk

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factors as anything that may increase the chance of developing cancer, and these can be grouped into four types: behavioral risk factors (e.g., smoking, diet, exercise, alcohol consumption), environmental risk factors (e.g., asbestos, radon, second-hand smoke, UV radiation, pollution, and pesticides), biological risk factors (i.e., gender, race, age, skin complexion) and genetic risk factors (i.e., hereditary factors that increase risk for cancers such as breast, colorectal, and prostate and are more likely to be found in families). However, there is increasing recognition of the complexity of cancer development and the potential for a combination of these factors to interact with one another to determine cancer risk. Websites 1. Surgeon General’s Report. Centers for Disease Control and Prevention (US); National Center for Chronic Disease Prevention and Health Promotion (US); Office on Smoking and Health (US). How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. Atlanta (GA): Centers for Disease Control and Prevention (US); 2010. Preface. Available from: https:// www.ncbi.nlm.nih.gov/books/NBK53011/. Accessed August 25, 2022.

6. METHODS OF TOXICITY AND CARCINOGENICITY TESTING 6.1. Fish Models Fish models such as rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes), zebrafish (Danio rerio), and various other aquarium fish have been useful in mechanistic studies (see Animal Models in Toxicologic Research: Nonmammalian, Vol 1, Chap 22). Extensive studies of the genetics of the zebrafish have made this fish an attractive model for evaluation of chemicals. Rainbow trout have been used for the study of carcinogenicity of several food and environmental contaminants. Advantages of this and other fish models have been noted, including significant savings in cost and time over rodent studies, sensitive early life-stage bioassay, sensitivity to many classes of carcinogens, well-described tumor pathology, and responsiveness to tumor promoters and inhibitors. However, three long-term medaka and guppy (Poecilia reticulata) studies that were

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chosen by the NTP as an exploration of alternate or additional models for examining chemical toxicity and carcinogenicity proved insensitive to the known rodent carcinogens 2,2bis(bromomethyl)-1, 3-propanediol and 1,2,3trichloropropane (National Toxicology, 2005). As with most fish, there are some aspects of the trout model that limit its application as a surrogate for human cancer research, such as lack of complete organ homology and physiologic differences. Despite this drawback, fish models are gaining acceptability in toxicological research of environmental chemicals due to extensive homology between fish and human genomes, and the low dose and short duration of exposure required for tumor induction. Emphasis on the development of fish models as useful adjuncts to conventional rodent models in the study of environmental carcinogenesis is expected to continue, especially for contaminants that have an impact on aquatic systems.

6.2. Transgenic Mouse Models Transgenic mouse models have been suggested as a more rapid method to replace or supplement the traditional rodent models. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) has become a popular method of mouse genome editing. The advantages of this system include lower costs, shorter timelines, and the capacity to alter multiple genes simultaneously. This system consists of an RNA-guided DNA endonuclease (Cas9) and corresponding guide RNAs (CRISPRs) that allow for one-step generation of mutant mice. (see Carcinogenicity Assessment, Vol 2, Chap 5; and Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). The transgenic models including knockout mice are very informative for mechanistic studies and have some utility as a model for screening unknown chemicals and compounds (Tennant et al., 1995). The Tg RasH2 transgenic mouse has been developed as an alternative and/or supplemental assay for lifetime mouse bioassays because it is sensitive to both genotoxic and nongenotoxic carcinogens and develops spontaneous and chemically induced neoplasms earlier in life and in greater numbers than wild-type mice (Morton et al., 2002). Pharmaceutical firms in

many cases are using a standard 2-year rat model coupled with a transgenic mouse assay. An important issue for pharmaceutical firms is the occurrence of human idiosyncratic toxicity, often involving the liver, that occurs in clinical trials or, more unfortunately, after the compound has been released for general use. In hindsight, it probably was unrealistic to expect one animal model to predict human risks from exposures as disparate as asbestos fibers, sunlight, and dioxins for a genetically diverse human population. Humans also vary in eating habits and exercise, and subject themselves to other exposures such as alcohol and smoking over a lifetime. Humanized mice carrying functioning human genes as well as liver and kidney microphysiological systems, also known as organ-on-a-chip, have enabled targeted testing and measurement of functional endpoints. Recent approaches to better represent human heterogeneity have used multiple mouse strains to help predict the diversity in human responses. In one example, 36 different strains of mice of varying sensitivity to acetaminophen were analyzed for genetic polymorphisms that correlated with acetaminophen sensitivity (Harrill et al., 2009). At the same time, volunteers were exposed to standard acceptable exposures of acetaminophen. A subgroup of the volunteers showed a spike in liver enzymes, in some cases at a level such that exposures were discontinued. It was shown that the genetic alterations in mice predictive for acetaminophen sensitivity were also found in the humans that showed increased acetaminophen sensitivity. This has encouraged some to speculate that using multiple strains of mice might provide some insight into idiosyncratic drug reactions, especially those involving the liver of patients. The Complex-Trait Consortium (CTC) is an international group of scientists (geneticists, molecular biologists, bioinformaticists, statisticians, etc.) working to identify networks of genes and allelic variants that modulate complex phenotypes in diverse environments (CTC, 2019). In 2002, the CTC members proposed to develop a new mouse genetics resource called the “Collaborative Cross” (Chesler et al., 2008; Churchill et al., 2004). This resource was intended to model the complexity of the human genome and supports analyses of common human diseases with complex etiologies. The overall goal was to understand the role of

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genetics in common diseases through the development of a large panel of recombinant inbred mouse strains, called the Collaborative Cross genetic reference panel, derived from a genetically diverse set of founder strains and designed specifically for complex trait analysis. The Collaborative Cross project was formally initiated in 2004 at the Jackson Laboratory through the generation of a full, reciprocal diallel cross among eight founder strains. A diallel cross investigates the genetic underpinnings of quantitative traits and involves the crossing of each of several individuals with two or more others in order to determine the relative genetic contribution of each parent to specific characteristics in the offspring. The CTC is now a partnership among several national and international universities and research institutions and includes three distinct breeding arms located in the United States, Israel, and Australia. All three populations are traceable to the F1 mice generated by the eight founder strains at the Jackson Laboratory and sent in 2004 to: (1) the Oak Ridge National Laboratory (since relocated to the University of North Carolina at Chapel Hill); (2) the International Livestock Research Institute in Kenya (since relocated to Tel Aviv University in Israel); and (3) the Western Australia Institute for Medical Research in Perth (Collaborative Cross). Because the genetics of the mouse are known and represent nearly 90% of the known variation in laboratory mice, this effort is intended to provide power to understand the complexity of disease and the role of biological networks. The overall goal is that this panel of mouse strains will reflect the diversity of human genetics and be used to understand, treat, and ultimately prevent human diseases. The mice are available for distribution free of any intellectual property constraints, to serve as a community resource for systems genetics studies.

Complex Trait Consortium and Collaborative Cross Websites 1. CTC, Complex Trait Consortium, 2019. http://www. complextrait.org. Accessed August 25, 2022. 2. Collaborative Cross. http://materiais.dbio.uevora.pt./ MA/Artigos/Collaborative_Cross.pdf. Accessed August 25, 2022.

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7. CURRENT CONSIDERATIONS FOR ENVIRONMENTAL TOXICITY AND CARCINOGENICITY TESTING 7.1. Mechanism of Action versus Mode of Action Within the past decade there has been increased attention on determining not just the carcinogenic potential of a chemical, but also its mechanism of action. Moreover, a broader review of the “mode of action” of a potential carcinogen has been the focus of federal agencies investigating environmental and workplace chemicals. Understanding the mode of action can aid in identifying processes that may cause chemical exposures to differentially affect a particular population segment or life stage. In 2005 the EPA provided a framework for the critical analysis of mode of action information to address the extent to which the available information supports the hypothesized mode of action, whether alternative modes of action are also plausible, and whether there is confidence that the same inferences can be extended to populations and life stages that are not represented among the experimental data (EPA Risk Assessment). This mode of action analysis is based on physical, chemical, and biological information, and includes a variety of data such as tumor types, whether tumors are responsive to endocrine influence, similarity of metabolic activation and detoxification between humans and tested species, influence of route of exposure, development of tumors that invade locally or systemically or lead to death, tumor latency, etc. A few examples of possible modes of carcinogenic action include mutagenicity, mitogenesis, inhibition of cell death, cytotoxicity with reparative cell proliferation, and immune suppression. In contrast, “mechanism of action” implies a more detailed understanding and description of events, often at the molecular level.

7.2. Human Relevancy According to the International Agency for Research on Cancer (IARC), all known human carcinogens that have been studied adequately for carcinogenicity in experimental animals have produced positive results in one or more

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animal species (IARC Monographs, 2006). For some of these chemicals (aflatoxins, vinyl chloride, diethylstilbestrol), carcinogenicity in experimental animals was known or highly suspected before epidemiology data confirmed their carcinogenicity in humans. However, not all agents that cause cancer in experimental animals will also cause cancer in humans, although it is considered biologically plausible that agents for which there is sufficient evidence of carcinogenicity in experimental animals may also present a carcinogenic hazard to humans. Accordingly, in the absence of additional scientific information, these agents are considered to pose a carcinogenic hazard to humans. Examples of additional scientific information are data that demonstrate that a given agent causes cancer in animals through a species-specific mechanism that does not operate in humans, or data that demonstrate that the mechanism in experimental animals also operates in humans. The inappropriate use of experimental animal responses can have adverse public health implications; therefore, modes of action in rodent cancer studies should be periodically reexamined for consistency and coherence with data from emerging studies.

7.3. Alternative Testing Strategies In the past few decades there has been increasing emphasis on alternative tests and testing strategies aimed at reducing the number of animals used in research, improving the ability to extrapolate from rodents to humans, decreasing the time and costs of studies, and, when possible, replacing animals with other models (Sistare et al., 2011). As an example, the U.S. EPA is prioritizing ongoing efforts to develop and use new approach methods to test chemicals for health effects. In 2019, the EPA announced that it will reduce animal testing and funding 30% by 2025 and eliminate it altogether by 2035. Additional technologies such as microphysiological systems, toxicogenomics, proteinomics, metabonomics, high-throughput screening, bioinformatics, systems biology and computer-based systems offer potential supplements and alternatives to animal use (Collins et al., 2008; Schmidt, 2009; Shukla et al., 2010) (see Toxicogenomics: A Primer for Toxicologic Pathologists, Vol 1, Chap 15, and Alternative Models in Biomedical Research: In Silico, In Vitro, Ex Vivo, and Non-Traditional In Vivo Approaches, Vol 1, Chap 24). The 2-year bioassay is

still considered the gold standard but is not infallible. It has become very expensive, takes years to complete, utilizes large number of animals, and in the end provides only very limited information for the resources expended. Compounds that show limited or no toxicity in preclinical studies may cause adverse effects in a subset of patients, resulting in their withdrawal from the market. It has been shown that multiple mouse strains or hybrid crosses containing most of the mouse genome have the ability to predict genetic polymorphisms in humans responsible for these unique sensitivities. Predicting chemical safety with multiple mouse strains is complex, and efforts are proceeding slowly to enhance the ability of animal studies to be more predictive for subpopulations that might be sensitive to a compound or environmental agent. Other areas that hold promise are the use of the rapidly developing genomic technologies that can provide information on thousands of gene products altered by exposure to a compound, and the use of biomarkers of target organ toxicity. The NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), representing 15 US federal regulatory and research agencies, are involved with the development and review of new and revised safety testing methods with regulatory applicability, including alternative test methods that may reduce, refine, or replace the use of animals in order to advance animal welfare while ensuring human health and safety (NICEATM: Alternative Methods, 2020). The statutory duties of this committee are: (1) advising on test method development and validation; (2) conducting technical reviews of new safety testing methods; (3) transmitting formal recommendations to federal agencies; (4) promoting regulatory acceptance of valid methods; and (5) fostering national and international harmonization. To date, NICEATM and ICCVAM have contributed to the national and/ or international regulatory acceptance of 48 alternative test methodsd27 in vitro methods that replace or reduce animal use, and 21 in vivo methods that significantly reduce the number of animals used or further reduce or avoid the potential for pain and distress. The International Cooperation on Alternative Test Methods (ICATM), with members that currently include the United States, the European Union, Japan,

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Canada, and the Republic of Korea, is a voluntary collaboration with a similar goal of international acceptance of validated alternative test methods. Recently, collaborations between the NIH, NTP, National Human Genome Research Institute (NHGRI), National Chemical Genomics Center (NCGC), EPA, and FDA resulted in a potential new paradigm shift in the assessment of chemical hazard and risk. This new approach to toxicity and carcinogenicity testing, called “Tox21,” includes the use of a high-speed robotic screening system, biochemical- and cell-based assays, assays involving three-dimensional models of different human tissues and organs, and assays using lower but complex organisms such as worms and fish (Schmidt, 2009; Tox21, 2011). At least 10,000 compounds will be tested for potential toxicity. The compounds tested by this system include environmental contaminants as well as those found in industrial and consumer products, food additives, and drugs. Comparison with historical animal data will be done to determine if hypothesized improvements will be realized. The overall goal is to develop more efficient and less time-consuming approaches to predict how chemicals may affect human health. This program will test whether high-throughput and computational toxicology approaches can yield predictive data comparable to that from animal studies. In the short term, this new approach to toxicity testing will help prioritize chemicals for animal testing when compared to the traditional method of relying on production volume, likelihood for human exposure, or structural similarity to other chemicals with known liabilities. It is anticipated that this approach will move toxicology from a predominately observational science using in vivo models to a predominately predictive science based on in vitro biological observations that are target-specific and mechanism-based. The animal bioassay may never be completely replaced due to the complexity of living organisms and disease processes, but efforts to reduce and refine its use will continue. These examples show the expanded efforts to improve current testing strategies, and it is anticipated that the art and science of evaluating potential toxicity and carcinogenicity of intentional or unintentional exposures will continue to improve and will hopefully provide better public health protection for humans.

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Websites for Current Considerations for Environmental Toxicity and Carcinogenicity Testing 1. EPA Risk Assessment. http://www.epa.gov/ cancerguidelines/. Accessed August 25, 2022. 2. IARC Monographs (2006). http://monographs.iarc.fr/ ENG/Preamble/CurrentPreamble.pdf. Accessed August 25, 2022. 3. NICEATM: Alternative Methods, 2020. http://iccvam. niehs.nih.gov/. Accessed August 25, 2022. 4. Tox21, 2011. http://www.niehs.nih.gov/news/news room/releases/2011/december07/index.cfm. Accessed August 25, 2022.

8. NEW DIRECTIONS FOR ENVIRONMENTAL TOXICITY AND CARCINOGENICITY TESTING The general approach to environmental toxicology testing by government agencies has always been to provide objective evaluations of substances of public health concern and to strengthen the science base for risk assessment. To date there are more than 80,000 chemicals registered for use in the United States, and each year an estimated 2000 more are manufactured and introduced for use in everyday items. Since it is impossible to evaluate all these chemicals, assessments are done to evaluate the available evidence that substances may cause adverse health effects and whether these substances may be of concern given what is known about current human exposure levels. Agencies such as the NTP are moving toxicology testing from a predominantly observational science at the level of disease-specific models to a predominantly predictive science focused upon a broad inclusion of target-specific, mechanism-based, biological observations (Andersen et al., 2010; Auerbach et al., 2010; Harrill et al., 2009; Powell et al., 2006). The types of studies are also expanding from the standard prechronic (2-week), subchronic (3-month), and chronic (2-year) studies to include organ systems’ toxicity (immunotoxicity, developmental toxicity, neurotoxicity, and reproductive toxicity) and an increased emphasis on nonneoplastic endpoints. The NTP has included the Modified OneGeneration Reproductive Study design to include (1) treatment exposure during pregnancy and early life; (2) evaluation of critical windows of

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exposure, which indicates the need for a larger focus on evaluating the potential for postnatal adverse effects; and (3) updates to standard study designs with more functional endpoints to assess how agents affect the reproductive and endocrine status of animals. The types of compounds studied for carcinogenicity and toxicity have traditionally been those from intentional or nonintentional release into the environment, or from worker exposure in manufacturing processes. But studies of potentially harmful environmental substances are expanding with the inclusion of nontraditional substances such as dietary supplements and herbal medicines (see Herbal Remedies, Vol 3, Chap 4), radiofrequency radiation emissions from cellular phones, phototoxicology and photocarcinogenicity studies of various substances, water disinfection by-products, DNA-based products used as therapies in a wide range of human diseases, Nucleic Acid Pharmaceutical Agents, Vol 2, Chap 7 and Gene Therapy/Gene Editing, Vol 2, Chap 8), and endocrinedisrupting agents (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). However, the overall goal remains the same: to evaluate substances of public health concern and provide reliable scientific information used by regulatory authorities to establish exposure guidelines for protection of public health.

8.1. Safe and Sustainable Alternatives Substances identified as hazardous are often replaced by new or existing substances, likely due to voluntary (e.g., public pressure, economic consequences) or mandatory reasons (e.g., regulatory ban), or both. The challenge for the toxicologic community is that, although nextgeneration chemicals and products are often purported to be “better” and “safer” than those they are replacing, information about the replacement’s potential to lead to effects that could pose similar or greater harm to human health (i.e., regrettable substitutions) is often limited or not accessible. The risk is that the substitute material might actually be worse than the material that it replaces. In the United States and worldwide, there have been many instances of “regrettable substitutions”. DDT, an agricultural pesticide banned by the Stockholm Convention due to its biological persistence, bioaccumulation, and toxicity, was

replaced by organophosphate pesticides, a group of chemicals that have shown both acute and chronic effects (Minton and Murray, 1988). The brake cleaner dichloromethane was phased out as a brake cleaner due to its environmental effects but the replacement, n-hexane, was subsequently determined to be neurotoxic (Hogue, 2017). Bisphenol-A, an endocrine-disrupting chemical used in consumer and industrial products, was replaced by the relatively data-poor compounds bisphenol-S (BPS) and BPS derivatives for some applications (Pal et al., 2017; Qiu et al., 2019). Companies manufacturing products that must meet flammability standards are switching to other flame-retardant chemicals that are technically and economically feasible, yet some of those substitutes may also be toxic (Brown, 2012). One last example is the replacement of acrylamide, which is a potent neurotoxin, with the safer Nvinyl formamide. However, the synthesis of the replacement required the use of the highly toxic hydrogen cyanide (Hogue, 2017). There has historically been limited emphasis placed on strategies and methods that proactively evaluate the potential for human health effects of alternatives (e.g., relative potencies, margins of exposure to biological and toxicological responses). This cyclic public health challenge needs better solutions that enable industry, regulators, and the public to find a better way forward. This provides an opportunity to consider technology change rather than just the substitution of one chemical for another. Importantly the full life cycle of the replacement product should be considered. Assessments should evaluate exposure to a substance during each step of a product’s life cycle, from manufacturing through use, recycling and disposal since exposure to chemicals can increase or decrease over time. Assessments that take into account process changes to reduce use of hazardous chemicals can create additional opportunities for “green chemistry” and green engineering solutions. Green chemistry is the design of products and processes that reduce or eliminate the use or generation of hazardous substances and was developed in the business and regulatory communities as a natural evolution of pollution prevention initiatives (Green Chemistry: Theory and Practice, Anastas and Warner, 2000). Anastas and Warner developed a list of what would make a chemical process or product “greener,” called the “12 Principles of Green Chemistry”

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REFERENCES

(12 Principles of Green Chemistry). Importantly, they list less hazardous chemical synthesis, designing safer chemicals, use of safer solvents and auxiliaries, design for degradation after use, and inherently safer chemistry for accident prevention. Chemical alternatives assessment (CAA) is a process for identifying, comparing, and selecting safer alternatives to chemicals of concern on the basis of their hazards, performance, and economic viability (CDESC, 2014). CAAs is a framework that can be used to inform government and industry decision-makers on how to develop and adopt safer alternatives. Below is a list of select CAA frameworks that currently exist just in the United States: • The Lowell Center for Sustainable Production’s AA Framework; https://www. cleanproduction.org/images/ee_images/ uploads/resources/lowell_center_aa_ framework_2009.pdf. Accessed August 25, 2022. • BizNGO’s CAA Protocol; https://www.bizngo. org/static/ee_images/uploads/resources/ BizNGOChemicalAltsAssessmentProtocol_V1. 1_04_12_12-1.pdf. Accessed August 25, 2022. • The Interstate Chemicals Clearinghouse Guidance for AA and Risk Reduction; http://theic2.org/article/download-pdf/file_ name/IC2_AA_Guide_Version_1.0.pdf. Accessed August 25, 2022. • U.S. EPA Design for the Environment’s AA Methodology; https://www.epa.gov/ saferchoice/design-environment-alternativesassessments. Accessed August 25, 2022. • California DTSC (Department of Toxic Substances Control). https://dtsc.ca.gov/ scp/. Accessed August 25, 2022. • Toxic Use Reduction Institute (TURI) Alternatives Assessment Process Guidance; https://www.turi.org/Our_Work/ Alternatives_Assessment/Alternatives_ Assessment. Accessed August 25, 2022. Some of the attributes among these frameworks include exposure at the use phase, cost and availability, other life cycle impacts, social impacts, and comparison of materials and/or processes. Some of the key issues of alternative analysis elements are technical performance, environmental impacts, human health impacts, financial assessments, life cycle considerations, sustainability and social impacts. Unfortunately, there is no concerted national or international effort to identify chemicals with “safer” human health

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and environmental profiles. Some of the key issues that will need to be addressed in the future include exposure versus hazard considerations, how to handle data gaps/uncertainty, decision rules for resolving trade-offs among different categories, incorporation of new data streams, and the need for research and innovation.

Websites for New Directions for Environmental Toxicity and Carcinogenicity Testing 1. Hogue, Cheryl (2017). “Assessing Alternatives To Toxic Chemicals”. Chemical and Engineering News. https:// cen.acs.org/articles/91/i50/Asessing-AlternativesToxic-Chemicals.html. Accessed August 25, 2022. 2. CDESCS, 2014 (Committee on the Design Evaluation of Safer Chemical Substitutions). A Framework to Guide Selection of Chemical Alternatives. https://www.nap. edu/catalog/18872/a-framework-to-guide-selection-ofchemical-alternatives. Accessed August 25, 2022. 3. 12 Principles of Green Chemistry. https://www.acs. org/content/acs/en/greenchemistry/principles/12-pri nciples-of-green-chemistry.html. Accessed August 25, 2022.

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2 Food and Toxicologic Pathology Olga M. Pulido1, Colin G. Rousseaux1, Phaedra I. Cole2 1

University of Ottawa, Ottawa, ON, Canada, 2Global Pharmacokinetics, Dynamics, Metabolism and Safety, Zoetis, Kalamazoo, MI, United States

O U T L I N E 1. Introduction 1.2. Overview

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2. Chemicals Intentionally Added to Food 2.1. Preservatives 2.2. Food Coloring 2.3. Flavor Enhancers 2.4. Emulsifiers, Stabilizers, and Thickeners 2.5. Functional Foods 2.6. Medicated Feed 2.7. Dietary Supplements

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3. Contamination of Food 3.1. Environmental Contaminants 3.2. Food Packaging and Food Processing Contaminants/Food Contact Substances 3.3. Natural Toxins as Food Contaminants

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4. Compounds with Toxic Properties Naturally Present in Certain Foods 4.1. Cyanogenic Glycosides 4.2. Glucosinolates Brassica sp. 5. Novel Foods 5.1. Genetically Modified Food 5.2. Novel Food ColorsdAnthocyanins 5.3. Novel PreservativesdAmygdalin 5.4. Novel EmulsifiersdYeast 5.5. Novel SweetenersdStevia 5.6. Novel ProteinsdCell-Based Meats 5.7. Novel OilsdOlestra 5.8. Novel CarbohydratesdPrecticX 5.9. Recombinant Bovine Somatotropin 5.10. CannabisdCannabidiol 5.11. Nanomaterials 5.12. Probiotics and PrebioticsdIntelligent Labs Probiotics with Prebiotics

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00002-2

6. Adverse Reactions to Food Constituent 6.1. Food Allergies 6.2. Allergy-like Food Poisoning 6.3. Adverse Reactions to Gluten and Gluten-Related Disorders 6.4. Exposure of a Susceptible Population 6.5. Direct Chemical Toxicity 6.6. Nonallergic Food Hypersensitivity and Intolerance 6.7. Food Color and Food Allergy 7. Mechanism of Action of Clinical Disorders Related to Food 7.1. Gut Microbiota and Adverse Reactions to Food 7.2. Neurotransmission 7.3. Channel Blockers 7.4. Endocrine Modifiers

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8. Safety Assessment of Food 8.1. Risk/Safety Assessment in Food 8.2. Food Additives 8.3. Food Contaminants

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9. Regulation of Food 9.1. History of Food-Related Disease 9.2. History of Food Regulation 9.3. Food Regulations Around the World

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10. Challenges and Future Developments in Food Safety

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11. Conclusions

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Glossary

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References

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1. INTRODUCTION Availability and consumption of safe and nutritious food is essential for human life and health, for without it, survival is impossible. Obtaining food is a primary drive for all biotadno food they die, a little they survive, and if there is more than enough they reproduce. The rise of agricultural knowledge and practices can be traced back to ancient times during which humankind progressed from nomadic, hunting, and gathering tribes to more settled societies, supported by herds of domesticated animals and cultivated crops. We have changed from our nomadic past and have a surfeit of food from an integrated food industry (Figure 2.1). The supply of food to individuals relies on a precarious supply chain that faced challenges during the SARS-CoV-2 pandemic, a coronavirus that caused COVID-19, which may mean shortages in the near future (Chiwona-Karltun et al., 2021). Nutritive and nonnutritive biomolecules, including proteins, carbohydrates, fats, vitamins, trace elements, fiber, and antioxidants, are integral components of foods. These will not be addressed in this chapter and the reader is directed to

Nutritional Toxicologic Pathology, Vol 3, Chap 3, for further information. This chapter introduces chemical food and feed safety, focusing on food toxicology, pathology, and regulatory approaches to minimize human health risks. Food can be harmful and pose health risks even though it is nutritionally adequate. For example, food may contain microbiological contaminants (bacteria, molds, and yeasts) (Bintsis, 2018; FDA, 2020a; WHO, 2021a); physical contaminants (dead insects, glass, wood, hair, plastic, and metal fragments); chemical and environmental contaminants, including manmade and natural toxicants; plant toxins; and food compounds toxic to specific susceptible sectors of the population (Table 2.1). Food toxicology is the study of the nature, properties, adverse effects, and detection of toxic substances in food and their disease manifestation. Food safety refers to the conditions and practices that preserve the quality of food and prevent contamination and foodborne illnesses. Feed safety refers to animal feed safety, the safety of which is a global food safety requirement.

FIGURE 2.1 The hominid development to the modern human. Reprinted with permission from Haridy R: Ancient antistarvation mechanism may be driving modern obesity epidemic, New Atlas, 2019. https://newatlas.com/ancientmechanism-fat-burning-protein-evolution-obesity-epidemic/60637/ (Accessed November 7, 2021).

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Although this chapter focuses on human food safety, the reader should note that animal feed safety is a necessity for animal health and welfare as well as human health. The effects of compounds in the feed may result in residues that need to be assessed, e.g., antibiotics. In fact, the overuse of antibiotics amplified the issue of antimicrobial resistance, which will be discussed later in the chapter. Similar to food production, feed production is subject to the quality assurance of integrated food safety systems (FAO, 2021b). Abbreviations used in this chapter can be found in the glossary section.

1.2. Overview Food toxicology focuses on compounds that are intentionally added to food, contaminants, and inherent toxicity of the food. These toxicants and toxins will be discussed in detail below. The effects of compounds intentionally added to food or that are contaminants are diverse. Some have direct acute toxicity, e.g., domoic acid in mussels (see Phytotoxins, Vol 3, Chap 5) and aflatoxin (see Mycotoxins, Vol 3, Chap 6); chronic toxicity, e.g., carcinogenesis caused by aflatoxins; heavy metals, e.g., lead (see Metals, Vol 3, Chap 10, Rai et al., 2019); fetal development, e.g., mercury (see Embryo, Fetus, and Placenta, Vol 5, Chap 11); endocrine effects, e.g., zearalenone (see Endocrine system, Vol 4, Chap 7); and endocrine disruption (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12), e.g., some persistent organic pollutants (see Environmental Toxicologic Pathology, Vol 3, Chap 1). Some poisonous compounds are part of the food itself, e.g., cyanogens (see Poisonous Plants, Vol 3, Chap 7), or from pathogens using the food as a substrate (see Bacterial toxins, Vol 3, Chap 9). Global food availability, food choices, nutritional status, and cultural eating habits may predispose some consumers to a higher risk of exposure to hazardous chemicals in food, e.g., northern communities (i.e., subarctic or subpolar populations) are especially exposed, as cultural dietary practices can increase exposure to toxicants (Chu et al., 2008). Specifically, fish and marine mammals tend to contain much higher

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concentrations of mercury and persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) due to bioaccumulation. The elderly is generally more susceptible to toxicants and toxins, e.g., the algal toxin domoic acid (DA) (Pulido, 2008, 2014). Children are even more vulnerable than older adults, partly due to a higher intake of air, water, and food in relation to their body weight, but more importantly, immature metabolism. The profile of toxic effects may differ from adults, as damage caused by toxins and toxicants in fetal and infant periods is dependent on exposure via the placenta or breast milk (see Embryo, Fetus and Placenta, Vol 5, Chap 11). In fact, the fetus may save the mother from toxicity by acting as a sink, e.g., mercury poisoning and Minamata Bay (Eto, 2000). Organ systems with a long developmental period, such as the nervous system (see Nervous System, Vol 4, Chap 8) (De Vellis and Chapter, 2005; Neal-Kluever et al., 2018), are particularly susceptible to toxic insult. Chronic exposure during critical organ development can lead to irreversible tissue damage and long-term impairment, such as the fetal alcohol syndrome (Brown et al., 2019; Mattson et al., 2019), chronic lead exposure in children (Rocha and Trujillo, 2019), and mercury (Eto, 2000)dthe “mad hatter” syndrome. Vulnerable populations include infants, children, pregnant women, workers, and the elderly (Koman et al., 2019) (see Environmental Toxicologic Pathology, Vol 3, Chap 1). Due to the bioaccumulation of many POPs, incremental increased exposure may become sufficient to cause adverse effects in adults (Ball et al., 2019; Di Monte et al., 2002), a phenomenon referred to as the “boomerang effect” (WHO, 2021b). This effect has been recognized in the 1976 Toxic Substances Control Act (TSCA) in the United States, which mandates protection of susceptible and highly exposed populations (EPA, 2016). Animal feeds may contain intentional additives, such as growth hormones and antibiotics, that leave residues in the fed animal at slaughter. For this reason, maximum residue limits for

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TABLE 2.1

Chemicals Foreign to Food

ContaminantsdUnintentional AdditivesdIntentional

Anthropogenic

Food Processing

Natural Sources

To improve appearance: e.g., colorants, glazing agents, waxes

To extend storage stability: e.g., preservatives, antioxidants

To improve and modify consistence: e.g., emulsifiers, stabilizers, thickener, etc.

Residues of agricultural chemicals: e.g., fungicides, herbicides, insecticides

Residues of disinfectants, chemical intermediates, metabolites

Algal and marine toxins

To improve and modify food: e.g., sweeteners, aromas, essences

To improve nutritional and biological value: e.g., vitamins

As adjuvant: Enzymes

Residues of animal production: e.g., estrogens, antibiotics, tranquilizers

Residues of adjuvants

Mycotoxins

Antibiotics

Contaminants following microbiological processes: e.g., solvents, glazing agents, coagulators, neutralizing agents, acids and alkalis, enzymes, catalysts, bacteria

Phytotoxinsdplants

Environmental pollutants: e.g., persistent organic pollutants (POPs)

Oxidation and food spoilage products

Opportunistic pathogens

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Chemicals Foreign to Food

2. CHEMICALS INTENTIONALLY ADDED TO FOOD

a specific additive have been developed for most countries. We will discuss residues and their regulations later in the chapter. Examples of agents causing food safety issues related to animal feed include melamine and cyanuric acid; bovine spongiform encephalopathy; footand-mouth disease; dioxin, mycotoxins, E. coli O157:H7 contamination; clenbuterol and rBST; as well as the development of antimicrobial resistance (FAO, 2021a).

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acid, sodium benzoate, and benzoates; sulphur dioxide and sulphites; nitrites and nitrates; lactic acid; propionic acid; and sodium propionate. Common antioxidants include ascorbic acid, sodium ascorbate, butylated hydroxytoluene, butylated hydroxyanisole, gallic acid, sodium gallate, sulphur dioxide, sulphites, and tocopherols. Nitrates and nitrites are used as preservatives in cured and processed meats, fish, and cheese. Although not carcinogenic, they may react with secondary amines or amides to form carcinogenic N-nitroso compounds (NOCs). For this reason, the International Agency for Research on Cancer (IARC) classified ingested nitrates and nitrites, in situations that would lead to endogenous nitrosation (production of NOCs), as “probable human carcinogens” (Group 2A) (IARC, 2010).

Substances that are added to food to maintain or improve the safety, freshness, taste, texture, or appearance of food are known as food additives. Some food additives have been in use for centuries for preservationdsuch as salt (in meats such as bacon or dried fish), sugar (in marmalade), or sulphites (in wine), others improve appearance (e.g., colorants, glazing agents, and waxes), improve and modify food palatability (e.g., sweeteners, aromas, and essences), improve and modify consistency (e.g., emulsifiers, stabilizers, and thickeners), and improve nutritional value (e.g., vitamins). Other additives are often used as adjuvants (e.g., enzymes) (Guan and YaDeng, 2016), and pharmaceuticals in animal products. The most common food additives are monosodium glutamate (MSG), artificial food coloring, sodium nitrite, guar gum, high-fructose corn syrup, artificial sweeteners, and trans fat (Health Canada, 2016).

Food colors are used to add, maintain, or restore the color uniformity of food products and thereby making them more appealing to the consumer. Coloring agents are considered food additives and are regulated as such. Source of food colors can be “natural” or “synthetic.” “Natural” color additives are generally pigments derived from plant or animal sources by extraction or other physical processing. The perception that natural food colors are innocuous is not always true, e.g., saffron and curcumin (Indian saffron) have pharmacological properties (de Mejias et al., 2020; Sharifi-Rad et al., 2020).

2.1. Preservatives

2.3. Flavor Enhancers

Preservatives and preservation techniques prevent foods from spoiling and oxidizing quickly, thus, preventing microbiological contamination and spoiling of the food. Spoiling of food usually results from inadequate preservation leading to growth of human pathogens, e.g., Salmonella typhimurium, Listeria monocytogenes, and Clostridium botulinum, or production of microbial toxins, e.g., Staphylococcus aureus enterotoxin, and oxidation. Common antimicrobial preservatives are sorbic acid, sodium sorbate, and sorbates; benzoic

Flavorings are used in small amounts and are not intended to be consumed alone. There are certain natural food flavors which are derived from herbs, spices, and substances having an exclusively sweet, sour, or salty taste. These natural food flavors are not included in the definition of flavorings for regulatory purposes. There are two categories of flavorings used in foods: natural and synthetic flavorings. In the United States, synthetic flavorings are further subdivided into artificial and nature-identical flavorings.

2.2. Food Coloring

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Natural Flavoring Substances Natural flavoring substances are obtained by physical, microbiological, or enzymatic processes. These natural flavorings can be either used in their natural or processed form for consumption by human beings. However, they cannot contain any nature-identical or artificial flavoring substances. Artificial Flavoring Substances Flavoring substances that are not identified in a natural product intended for consumptiondwhether or not the product is processeddare artificial flavoring substances. These food flavorings are typically produced from crude oil or coal tar by fractional distillation and additional chemical manipulation of these naturally sourced chemicals.

2.4. Emulsifiers, Stabilizers, and Thickeners Emulsifiers A food emulsifier, also called an emulgent, is a surface-active agent, surfactant, that acts as a border between two immiscible liquids such as oil and water, allowing them to be blended into stable emulsions. Emulsifiers also reduce stickiness, control crystallization, and prevent separation of the components. Emulsifiers made from plant, animal, and synthetic sources commonly are added to processed foods such as mayonnaise, ice cream, and baked goods to create a smooth texture, prevent separation, and extend shelf life. Commonly used emulsifiers in modern food production include mustard, soy and egg lecithin, mono- and diglycerides, polysorbates, carrageenan, guar gum, and canola oil. Emulsifiers create two types of emulsions: either droplets of oil dispersed in water or droplets of water dispersed in oil. Within the emulsion, there is a continuous and dispersed phase. In an oil-in-water emulsion, the continuous phase is the water, and the dispersed phase is the oil; conversely, in a water-in-oil emulsion, the oil is the continuous phase. In addition, application of mechanical force from a blender or homogenizer, which breaks down the dispersed phase into tiny droplets that become suspended in the continuous phase, can form emulsions.

Stabilizers Stabilizers are substances that maintain the emulsion and physical characteristics while increasing stability and thickness of the food. Ingredients that normally do not mix, such as oil and water, need stabilizers, e.g., low-fat foods are dependent on stabilizers due to the increased water content. Lecithin, agar-agar, carrageenan and pectin stabilizers are common in margarine, dairy products, salad dressings, and mayonnaise. Thickeners Thickeners range from flavorless powders to gums that thicken the food product. Variables affecting choice of thickener include pH, frozen state, clarity, and taste. Starches, pectin, and gums are the most common commercial thickeners used in soups, sauces, and puddings.

2.5. Functional Foods In addition to their nutritional benefit, functional foods possess added benefits when consumed regularly. These physiological effects are related to the presence of bioactive compounds that have been shown to have several specific properties, such as antioxidant, antimutagenic, antibacterial, or antiinflammatory activities. Probiotics and prebiotics are considered functional foods (Section 2.12). Probiotics contain live microbes, which possess the potential risk of infection or in situ toxin production. Since numerous types of microbes are used as probiotics, safety is also intricately tied to the nature of the specific microbe being used. Genetic stability of the probiotic over time, deleterious metabolic activities, and the potential for pathogenicity or toxicity must be assessed depending on the characteristics of the genus and species of the microbe being used (Sanders et al., 2010). Fortified foods are enriched with specific nutrients, usually vitamins and minerals, which are potentially deficient in the diet, e.g., milk fortified with vitamin D.

2.6. Medicated Feed Adding medication to animal feed is used to treat animal diseases in large groups of animals, in particular feedlot cattle, pigs, and poultry. Farmers can either incorporate the medicines

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into the feed or drinking water on the farm or use commercial medicated feed. Medicated feed is an effective way for a farmer to treat their herd without individual application of the medication. Most medications are distributed to many tissues, some of which will be consumed. For this reason, clearance of the medication before slaughter is essential. Maximum residue limits (MRLs) are regulatory limits imposed to ensure that those consuming the food are not exposed to the medication in any significant quantity. This will be discussed in more detail under maximum residue limits and antimicrobial resistance.

2.7. Dietary Supplements Dietary supplements (United States) or food supplements (other countries) are intended to add nutritional value to the diet. They are popular worldwide because they do not require a medical prescription and are sold in general stores and through Internet websites. Often advertised as being “natural,” consumers perceive them as healthier and safer than pharmaceuticals, aiming to treat common conditions such as obesity, erectile disfunction, diabetes, hypertension, and memory loss. They are defined as concentrated sources of nutrients, e.g., vitamins, minerals, proteins, or other herbal substances with nutritional or physiological effects, to supplement the diet. Unfortunately, adulteration of dietary supplements is an increasing problem worldwide (see Herbal Remedies, Vol 3, Chap 4). Up to six active pharmacological compounds (adulterants) have been found in a single food sample (Muschietti et al., 2020). The presence of synthetic pharmaceutical analogues in supplements is a serious health risk and consumers are unaware of their existence. They may induce side effects via their pharmacological activity or by interacting with prescription drugs. A major incident of food adulteration in China was made public in September 2008. Kidney and urinary tract toxicity, including kidney stones, affected about 300,000 Chinese infants and young children, with 6 reported deaths. Melamine and cyanuric acid had been deliberately added to milk to boost protein content measurements (Figure 2.2). Subsequently, melamine and

FIGURE 2.2 Chemical (Author’s diagram).

structure

of

melamine

cyanuric acid was detected in many milk and milk-containing products, as well as other foods and feed products, causing health issues in humans and animals (see Kidney, Vol 5, Chap 2) (Gossner et al., 2009). Melamine exerts a number of pathological changes, including nephrolithiasis (Figures 2.3 and 2.4), chronic kidney inflammation (Brown et al., 2007), and bladder carcinoma, all have been studied in animals (Hau et al., 2009). In addition, the microbiome plays a significant role in melamine toxicity (Zheng et al., 2013). Furthermore, in addition to melamine contamination of milk products, global contamination of feeds and pet food resulted from melamine adulteration (Figure 2.5).

3. CONTAMINATION OF FOOD There are numerous ways food can be contaminated (see Table 2.1) at every step of production from raw material on the farm to the consumer i.e., raw materials, food processing and packaging. Contaminants can come from anthropogenic activities, e.g., water contamination; residues of agrochemicals (fungicides, herbicides, and insecticides); residues of animal production, e.g., estrogens, antibiotics, and tranquilizers; residues of adjuvants; environmental pollutants, e.g., POPs; residues of disinfectants, chemical intermediates, and metabolites; contaminants created as a result of food processing: e.g., acrylamide. In addition, unintentional chemical contamination can arise from environmental,

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FIGURE 2.3 Kidney urolithiasis following melamine toxicitydhuman. Prominent hydronephrosis with calculi. Reproduced with permission from Anderson P: KidneydGross natural color portion of staghorn calculus remain with hydronephrosis, Reprinted with permission from Pathology Education Informational Resource (PEIR) Digital Library, 2013. https://peir.path.uab.edu/library/picture.php?/ 4415 (Accessed November 7, 2021) [4415].

food processing or food packaging sources (Buckley and Woteki, 2020). Health risks posed by chemicals that are not deliberately added to food are addressed by regulatory authorities utilizing a lexicon of terms, e.g., tolerable daily intakes (TDIs), tolerable weekly intakes (TWIs), or MRLs. If the contaminant in question is carcinogenic and genotoxic, margins of exposure (MOEs) are calculated to assess the cancer risk in exposed individuals. Any potentially carcinogenic compound would not be considered for use as a food additive by US FDA because the Delaney Clause states that no substance, demonstrated to cause cancer in humans or animals, may be added to food. Consequently, regulatory decisions are not based on scientific risk assessment but rather as a ‘matter of law,’ e.g., FDA (2018a). This zero-risk standard of the Delaney clause has been amended to apply to pesticide residues (EPA, 1996a) but not to food.

3.1. Environmental Contaminants Environmental contaminants are chemicals that accidentally or deliberately are released into the environment e.g., industrial pollution and agrichemicals (see Agrochemicals, Vol 3, Chap 11). Some of these contaminants are

FIGURE 2.4 Histologic diagnosis of melamineassociated renal failure based on renal crystal characteristicsdcat. (A) Dilated distal cat tubule contains clusters of round green melamine/cyanuric acid crystals with radiating spokes and concentric striations (arrow). Surrounding proximal tubules appear unaffected (hematoxylin and eosin; bard45 mm). (B) Dilated distal cat tubule contains fragmented or globular dense green melamine/cyanuric acid crystals (long arrows). Note attenuation of the lining epithelium with wide separation of nuclei (short arrow) and mitotic figure (arrowhead) indicative of tubular epithelial necrosis and regeneration (hematoxylin and eosin; bard45 mm). Reprinted with permission from Brown CA, Jeong KS, Poppenga RH et al.: Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007, J Vet Diagn Invest 19(5):525–531, 2007 [Page 529].

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FIGURE 2.5 Flow chart of the melamine-contamination chain from adulteration. Solid lines indicate contaminated products as observed during the 2008 incident. Dashed lines indicate possible contamination but not reported during the 2008 incident. Reprinted with permission from Gossner CM, Schlundt J, Embarek B et al.: The melamine incident: implications for international food and feed safety, Environ Health Perspect 117(12):1803–1808, 2009. [Page 1806].

chemically stable with a low degradation rate and may bioaccumulate. A wide variety of environmental contaminants have been detected in foods such as lead, arsenic, bromate, brominated flame retardants, chlorinated naphthalene, dioxins and furans, mercury, polychlorinated biphenyls (PCBs), perchlorate, per- and polyfluoroalkyl substances (Thompson and Darwish, 2019; Rather et al., 2017) (see also Environmental Toxicologic Pathology, Vol 3, Chap 1). A specific example of an environmental contaminant is per- and polyfluoroalkyl substances (PFASs), a family of human-made chemicals. Because of their durability, resistance to grease, oil, water, and heat, they are used in a wide range of products, e.g., stain- and waterresistant fabrics, carpeting, cleaning products, paints, and fire-fighting foams. Today, there are nearly 5000 different types of PFAS (FDA, 2021a), many of which are found in surface waters. PFASs bioaccumulate. Bioaccumulation of PFASs has been confirmed through blood analysis in humans and animals. Whether PFAS accumulation causes serious health conditions is still under investigation (NTP, 2019; Sinclair et al., 2020; FDA, 2021a). So far lack of consistent results has hindered determination of health risks following exposure (Bach et al., 2016). However,

EPA’s recent release of a Systematic Review Protocol for a planned Integrated Risk Information System (IRIS) Assessments of PFBA, PFHxA, PFHxS, PFNA, and PFDA does help (FDA, 2021a; EPA, 2019). A prolonged period of latency, sometimes decades, between toxic exposure and disease, e.g., Parkinson’s disease (Petersen et al., 2008) and cancer, may be seen with some contaminants, Unfortunately, many confounding variables make causality conclusions contentious. Compounds linked to Parkinson’s disease include, but are not limited to, manganese, lead, and solvents, e.g., trichloroethylene (Johns Hopkins Medicine, 2021; Di Monte et al., 2002). Biomonitoring, where an organism provides quantitative information on the quality of the environment around it, helps define exposure (EPA, 2021).

3.2. Food Packaging and Food Processing Contaminants/Food Contact Substances There are a number of contaminants that either form from food processing or leaching from food containers. The major contaminants include bisphenols, phthalates, perfluorocarbons, and

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perchlorate (Kahn et al., 2020). For definition of terms see FDA (2018a). Bisphenols: The use of bisphenols resulted in unintentional food contamination. This contamination increased beginning in the 1960s, when bisphenol A (BPA) was identified as a useful ingredient in the manufacture of polycarbonate plastics and polymeric metal can coatings. BPA can bind to the estrogen receptor and is classified as an endocrine disruptor (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). Nonhuman laboratory studies and human epidemiologic studies suggest links between BPA exposure and numerous endocrine-related end points, including reduced fertility, altered timing of puberty, changes in mammary gland development, and development of neoplasms (Rubin, 2011; Moon et al., 2021) as well as diabetes and obesity. Phthalates: Phthalate esters have a diverse array of uses in consumer products and can leach into food. Animal and human studies have shown that phthalates are antiandrogenic and adversely affect male fetal genital development. These chemicals exert direct testicular toxicity, thereby reducing circulating testosterone concentrations and increasing the risk of hypospadias and cryptorchidism at birth. Phthalates are also associated with changes in men’s hormone concentrations and changes in sperm motility and quantity (Meeker and Ferguson, 2014). In addition, the metabolites of phthalates are linked to oxidative stress, which can lead to changes to metabolic health outcomes (Attina and Trasande, 2015). Perfluorocarbons (PFCs): PFCs have wide utility in stain-resistant sprays for carpets and upholstery, fire-retarding foams, nonstick cooking surfaces, and grease proofing of paper and paperboard used in food packaging. Studies have associated PFOA and PFOS exposure with adverse health outcomes, such as reduced immune response to vaccines, metabolic changes, and decreased birth weight (Trasande et al., 2018). Perchlorate: Perchlorate most commonly enters the food supply through its presence as a contaminant in water or as a component of nitrate fertilizers. In addition, perchlorate is an indirect food additive causing contamination of food through its use as an antistatic agent for plastic packaging in contact with dry foods with surfaces that do

not contain free fat or oil (such as sugar, flour, and starches); or through degradation from hypochlorite bleach, which is used as a cleaning solution in food manufacturing. Perchlorate is known to disrupt thyroid hormone production through interference with the sodium iodide symporter (NIS), which allows essential iodide uptake in the thyroid gland (Steinmaus et al., 2016; Endocrine System, Vol 4, Chap 7). Food Packaging In general, packaging protects the packaged foodstuff from spoilage by external agents such as pests, odors, microorganisms, light, and oxygen, and is, therefore, beneficial. However, the quality and safety of food can be reduced by the leaching of chemicals from packaging to food. This release of chemicals from packaging to the food is known technically as migration (Barnes et al., 2007). Metals, glass, ceramics, plastics, rubber, and paper can all release minute amounts of their chemical constituents when they touch certain types of foods. Postmarket concerns of approved levels of a compound that was considered safe have arisen and resulted in regulatory changes. For example, Canada was the first country to declare BPA a toxic substance and the European Union and Canada banned BPA use in baby bottles, but the United States considers it is safe at the current levels in foods (FDA, 2018c). It is also noteworthy that chemicals used as BPA alternatives have also been reported to pose health risks, but the risk/ benefit of these chemicals has yet to be determined (FDA, 2018c; Jacobson et al., 2019). Food Processing When foods are heat processed, chemical reactions between components of the food result in desired flavor, appearance, and texture changes. Naturally occurring components in the food (such as glucose and amino acids) can generate potentially harmful compounds, collectively referred to as food processing–induced chemicals (FPICs). Many heat-processing induced chemicals have been shown to be genotoxic and carcinogenic (Abramsson-Zetterberg, 2018). While exposure is considered unavoidable, improving the understanding of the processes by which these products are formed can help limit the conditions under which they are formed. Examples of FPIC include acrylamide (FDA,

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2019a), benzene, chloropropanols, ethyl carbamate, furans, heterocyclic aromatic hydrocarbons, nitrosamines, polycyclic aromatic hydrocarbons (PAHs), and semicarbazide (Abramsson-Zetterberg, 2018). These compounds are prevalent in bread and baby food, so they are consumed in high quantities by the most vulnerable consumer groups (CDC, 2018). Variability of the FPICs and their combinations make risk assessment challenging, e.g., home cooking of potato products (FAO, 2007). Acceptable levels have been calculated for many contaminants, below which signs of toxicity should not be evident; however, less data are available for mixtures in which combined molecules may be less or more toxic than each individual molecule. In addition to food and drug regulatory authorities, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) lists contaminants and undertakes risk assessments (Thompson and Darwish, 2019) to set standards for specific additives and contaminants. Food Contact Substances Food contact substances (FCSs) are defined as any substance intended for use as a component of materials used in manufacturing, packing, packaging, transporting, or to hold food if such use is not intended to have any technical effect (FDA, 1997, 2018b). FCSs are typically included (Koszucka and Nowak, 2019) in coatings, plastics, paper, adhesives, colorants, antimicrobials, and antioxidants. FCSs are legally considered food additives (Hattan and Rulis, 1999). Currently in the US, FCSs are evaluated primarily through US FDA’s food contact notification (FCN) program (FDA, 2018b; Choudhuri et al., 2019; Bailey et al., 2005).

3.3. Natural Toxins as Food Contaminants Natural toxins are compounds produced by living organisms, which are typically not harmful to the organisms themselves but can enter the food chain and be poisonous to humans or animals when consumed. Common sources of such toxins include plants, fungi, algae, and bacteria (Fletcher and Netzel, 2020; WHO, 2018).

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Algal Compounds in Food PHYCOTOXINS IN FOOD

Some algal compounds are beneficial and are added to food, either during processing as a pigment or for their nutritional value. They are often marketed as a food supplement or functional food, e.g., spirulina has a high content of protein, chlorophylls, carotenoids, and phycobiliproteins (Lafarga et al., 2020). Not all compounds produced are beneficial and some are toxic (phycotoxins). Phycotoxins ([phyco ¼ seaweeds and algae] plus toxins) are potent natural toxins synthesized by certain marine and freshwater algae or cyanobacterial species (see Phycotoxins, Vol 3, Chap 5). Under the right environmental conditions these algae grow to produce “Harmful Algal Blooms” (HABs). These algal blooms often cause water discoloration (Figure 2.6); hence, the terms “Red Tides” or “Green Tides” in marine waters. Phycotoxins are grouped by chemical structure, mechanisms of action, target tissues, and biological and health effects. Cyanotoxins, produced by cyanobacteria, are preferential contaminants of soft water reservoirs, including lakes, rivers, etc., and drinking water, potentially with direct risk to human health. Marine biotoxins produced by dinoflagellates and diatoms enter the food chain and can accumulate in tissues of aquatic organisms such as bivalve mollusks and fish. These toxins are a constant threat to public health and the economy, requiring multidisciplinary action at the local, federal, and international levels to manage their potentially harmful effects. Rasmussen et al. (2016) reviewed in detail the chemical diversity, origin, and analysis of phycotoxins that are harmful to humans and may cause massive fish kills. Exposure to “phycotoxins and HABs,” can occur through oral, respiratory or dermal contact in aquatic or terrestrial environments (Pulido, 2016). The highest risks for human exposure and phycotoxin induced toxic effects are due to ingestion of seafood contaminated with marine algal toxins and respiratory exposure through aerosols. Exposure to water contaminated with cyanotoxins through drinking water, freshwater fish, and dermal exposure, e.g., recreational activities in contaminated lakes, may cause severe disease (see Phycotoxins, Vol 3, Chap 5).

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FIGURE 2.6 Blue-green algal bloom. Environmental pollution centers: the issue of toxic algal blooms (https:// www.environmentalpollutioncenters.org/news/the-issue -of-toxic-algal-blooms/2018) (Accessed November 7, 2021). CYANOTOXINS

Cyanotoxins are secondary metabolites from cyanobacteria that may be toxic to living organisms including humans. They are divided into chemical groups, i.e., cyclic peptides (microcystin and nodularin), alkaloids (saxitoxins, cylindrospermopsin, aplysiatoxin, lyngbyatoxin-a), lipopolysaccharides (LPSs) (Pulido, 2016) and guanitoxin (GNT, formerly known as anatoxin-a(S)), a cyanobacterial organophosphate toxin that is the most potent natural neurotoxin produced by fresh-water cyanobacteria (Fiori et al., 2020). Classification can also be based on biological effects in target tissues such as hepatotoxins, neurotoxins, cytotoxins, dermatoxins, and irritant toxins (see Phycotoxins, Vol 3, Chap 5). MARINE ALGAL TOXINS

Marine algal toxins are largely produced by dinoflagellates and diatoms. Bivalve mollusks, such as mussels, oysters, clams, cockles, and scallops are filter-feeding organisms that can easily accumulate these toxins resulting in seafood poisoning when ingested (Lawrence et al., 2011). The conditions that trigger the occurrence of toxic phytoplankton species or production of toxins are not fully understood. Climate change has been implicated in the apparent increase in HABs worldwide (FAO, 2020b). Algal toxins have contributed to the decline in fish and shellfish populations and have been implicated in increased mortality of other species

such as gastropods, crustaceans, and other animals, dependent on the marine food web. They accumulate through the food chain and may pose an acute threat to consumers, although less is known about health risks following chronic exposure (Lawrence et al., 2011). Clinically, these toxins can cause seafood poisoning syndromes including diarrheic shellfish poisoning (DSP) by okadaic acid (OA); paralytic shellfish poisoning (PSP) by saxitoxins (STX); amnesic shellfish poisoning (ASP) by domoic acid (DA); neurologic shellfish poisoning (NSP) by brevetoxin-b (BTX); azaspiracid shellfish poisoning (AZP) by azaspiracid (AZA); and ciguatera fish poisoning (CFP) by ciguatoxins (CTX). The toxic syndromes, palytoxicosis and clupeotoxism, are caused by palytoxin (PLTX) following exposure to PLTX via inhalation, cutaneous, and ocular exposure during handling of soft corals in aquariums (Pelin et al., 2016). DA and its associated syndrome ASP will be discussed as an introductory example of the health impacts of an algal toxin. DOMOIC ACID AND AMNESIC SHELLFISH POISONING

Domoic acid was first identified as a causative agent of a human shellfish poisoning outbreak in 1987, in Canada, after contaminated blue mussels (Mytilus edulis) were consumed. The poisoning was characterized by a constellation of clinical symptoms and signs involving multiple organ systems: the GI tract, central nervous system (CNS), and cardiovascular systems. Among the most prominent symptoms described was memory impairmentdamnesic shellfish poisoning. Several species of Pseudonitzschia (Bates et al., 2018) and other marine organisms, such as the red alga Chondria armata, produce DA. This toxin can enter the food web by contaminating shellfish, crustaceans, and other types of seafood. The most common vector is the blue mussel (Figure 2.7). Although bivalve mollusks can wash out the toxin with time, harvesting and consumption of the shellfish at the time of contamination can lead to severe human or animal disease by activation of GluRs and, inducing excitatory excitioxicity, discussed in detail elsewhere (Pulido, 2008, 2014; Rousseaux, 2008a,b).

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clinically significant memory problems have been seen in a dose response manner following the ingestion of razor clams with presumably safe levels of DA (Grattan et al., 2016, 2018; Stuchal et al., 2020). Vigilance will be necessary as HABs blooms appear to be increasing worldwide, including those of the Pseudonitzschia species which produce DA (FAO, 2020b).

FIGURE 2.7 Prince Edward Island blue mussels (Mytilus edulis) also known as the common mussel (www.peimussel.com). Accessed November 22, 2021, with permission.

Following the ASP incident in Canada, a maximum residual limit (MRL) of 20 mg DA/ g (20 mg/kg) shellfish meat (flesh), based on the consumption of 250 g of shellfish meat by a person weighing 60 kg was established. Subsequent toxicological studies in experimental animals supported this limit, which has been adopted by other countries, thus becoming the standard international regulatory MRL. In 2004, the Joint FAO/WHO/IOC ad hoc Expert Consultation on Biotoxins in Bivalves Mollusks conducted risk assessments for a number of biotoxins present in bivalve mollusks, including DA toxin (Lawrence et al., 2011). The experts agreed that the derived provisional acute reference dose (RfD) for DA toxin, based on an adult body weight of 60 kg, was 100 mg/kg. This risk assessment highlighted more susceptible populations including pregnant women, infants and children, people with premorbid pathology and adults >65 years of age. Since the establishment of an MRL, no other episodes of severe acute DA intoxication have been reported in humans. However, intoxication in wild animals, including sea mammals, has been extensively documented (see Phycotoxins, Vol 3, Chap 5). These animals present with acute DA toxicosis and chronic clinical manifestations. Neurological symptoms include epilepsy, supporting the possible link for the developmental neurotoxicity by DA, and other chronic effects (Ramsdell and Gulland, 2014). Less is known about human chronic exposure to DA, although

Mycotoxins Significant health risks exist when compounds synthetized by fungi (mycotoxins) contaminate crops. These mycotoxins are potentially toxic to animals and humans. Fungi such as Fusarium, Aspergillus, Penicillium, and Alternaria species are recognized producers of mycotoxins of concern for food safety and public health. Aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic toxins, and ergot alkaloids are the mycotoxins of greatest agro-economic importance (see Mycotoxins, Vol 3, Chap 6). Considerable attention has been given to aflatoxins found commonly on agricultural crops such as maize (corn), peanuts, cottonseed, and tree nuts, due to their carcinogenic potential. Legislation is in place in many countries to control levels of aflatoxin contamination. In the United States, pet food is periodically found to contain toxic concentrations of aflatoxin resulting in dogs showing clinical signs ranging from sluggishness, loss of appetite, vomiting, jaundice, and/or diarrhea as well as death (FDA, 2021b). Fumonisins are a group of mycotoxins frequently found in maize (corn). Fumonisins B1 and B2 are cancer-promoting compounds with a long-chain hydrocarbon unit similar to that of sphingosine and sphinganine. Fumonisin B1 (FB1) is the most toxic and has been shown to promote tumors in rats, and to cause equine leukoencephalomalacia and porcine pulmonary edema (see Mycotoxins, Vol 3, Chap 6). Bacterial Toxins More than 90% of the cases of food poisoning each year are caused by Staphylococcus aureus, Salmonella, Clostridium perfringens, Campylobacter, Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus, and entero-pathogenic Escherichia coli. These bacteria are commonly found on many raw foods. The two main classes of bacterial toxins are exotoxins (secreted by many gram-positive and a few gram-negative bacteria)

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and endotoxins (produced only by gramnegative bacteria). Bacterial toxins are encountered when invading bacteria release toxins into improperly preserved food. Primary mechanisms for bacterial toxin activity include enzymatic disruption of host cell chemistry, membrane damage to host cells, and immune activation to incite a tissue-disrupting immunological response (see Bacterial Toxins, Vol 3, Chap 8).

4. COMPOUNDS WITH TOXIC PROPERTIES NATURALLY PRESENT IN CERTAIN FOODS There are many plants that contain toxic components (see Poisonous Plants, Vol 3, Chap 7). Examples include cyanide in almonds, cherry and apple seeds; lectins in kidney beans; glycosides in green potatoes; oxalic acid in rhubarb; a-amanitin in mushrooms, particularly the death cap (Amanita phalloides), and the destroying angel (Amanita virosa); and urushiol in mangoes and cashews.

4.1. Cyanogenic Glycosides Several plants and associated plant-based foods naturally contain cyanogenic glycosides. Approximately 25 cyanogenic glycosides are known. Cyanogenic glycosides are plant constituents produced by over 2000 species of plants along with a corresponding hydrolytic enzyme (beta-glycosidase) (Cressey and Reeve, 2019). The combination of cyanogenic glycosides with a hydrolytic enzyme is the plant’s protective mechanism against predators. When the plant cell structure is disrupted by a predator, these compounds interact, and the hydrolysis of the cyanogenic glycoside leads to the formation of a cyanohydrin that spontaneously decomposes to hydrogen cyanide (HCN), an aldehyde, and ketone. The rate of cyanide production from cyanogenic glycosides due to bacterial b-glycosidase activity depends on the sugar moiety in the molecule and the stability of the intermediate cyanohydrin following hydrolysis by bacterial b-glucosidase. Cyanogenic glycosides with a gentiobiose sugar, amygdalin, linustatin, and neolinustatin, undergo a two-stage hydrolysis, with gentiobiose initially being hydrolyzed to

glucose to form prunasin, linamarin, and lotaustralin, respectively (Cressey and Reeve, 2019). The glycosides, cyanohydrins, and hydrogen cyanide are collectively known as cyanogen. The major cyanogenic glycosides known to be present in edible plants are amygdalin (almonds), dhurrin (sorghum), linamarin (cassava, lima beans), lotaustralin (cassava, lima beans), prunasin (stone fruit), and taxiphyllin (bamboo shoots). Among the cyanogenic plants of concern are cassava and bamboo shoots, common crops in Africa, Asia, and Latin America (Parmar et al., 2017; Pinto-Zevallos et al., 2016). Cassava (Figure 2.8) is also known by other common names, including manioc, manihot, and yucca. There are a number of varieties of cassava with a range of cyanide content, from low (referred to as “sweet cassava”) to higher (referred to as “bitter cassava”) (Taylor, 2021). Bitter cassava requires more extensive processing than sweet cassava to remove the cyanogenic potential. The toxicity of a cyanogenic plant depends primarily on the potential concentration of hydrogen cyanide that may be released upon consumption. If the cyanogenic plant is inadequately detoxified during processing or preparation of the food, the potential hydrogen cyanide concentration can be high. If cassava or bamboo shoots are eaten either raw or after inadequate processing, evidence of toxicity may ensue.

FIGURE 2.8 Cassava root. Reprinted with permission from Taylor G: Growing Cassava: planting guide, care, problems and harvest, Morning Chores https://morningchores.com/ growing-cassava/2021. (Accessed November 5, 2021).

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Available information on the kinetics of decomposition of plant cyanogenic glycosides to hydrogen cyanide in the human gut has been compiled to determine if human gut microflora is able to release cyanide sufficiently quickly from foods containing cyanogenic glycosides to potentially cause toxic effects in humans (JECFA, 2012). In humans, cyanide exposure can lead to acute intoxication, including death. Chronic exposure can lead to long-term toxicity with a variety of associated diseases, including “konzo,” an upper motor neuron disease characterized by irreversible, nonprogressive symmetric spastic paraparesis with an abrupt onset. Cyanide is associated with “Tropical Ataxic Neuropathy,” a neuropathy causing several neurological syndromes such as optical atrophy, angular stomatitis, sensory gait ataxia, and neurosensory deafness (Rivadeneyra-Domı´nguez and Rodrı´guez-Landa, 2020). Finally, chronic cyanide exposure may exacerbate goiter and cretinism caused by iodine deficiency (Espe´rance Kashala-Abotnes et al., 2019).

Isothiocyanates are irritating to mucous membranes but not readily consumed by humans in sufficient quantities to be toxic; however, ruminants can die of acute isothiocyanate poisoning following ingestion of glucosinolates. In addition, the metabolite isothiocyanate has antithyroid effects by interfering with the synthesis of thyroid hormones (see Endocrine System, Vol 4, Chap 7). Oxazolidine-2-thiones are closely related to isothiocyanates. They are produced by the conversion of the glucosinolate progoitrin in rapeseed meal to goitrin which in turn is hydrolyzed to these compounds. Oxazolidine-2-thiones depress growth and increase the incidence of goiters, by blocking the incorporation of iodine into thyroxine precursors and by suppressing thyroxine secretion from the thyroid. Nitriles depress growth and cause liver and kidney lesions. In severe cases they cause liver necrosis, bile duct hyperplasia, and megalocytosis of tubular epithelium in the kidney. Thiocyanates inhibit iodine uptake by the thyroid leading to reduced iodination of tyrosine then resulting in decreased production of the important thyroid hormone thyroxine (Endocrine System, Vol 4, Chap 7).

4.2. Glucosinolates Brassica sp. Glucosinolates are found in several oil meals that have been used traditionally in the northern states, Canada, and Europe as protein supplements for livestock. Some examples include mustard and most importantly, rapeseed meal. Glucosinolates are also found in brassicas such as cabbage, broccoli and Brussels sprouts. As such, they are frequently consumed as a normal part of human diet and may be beneficial as glucosinolates found in cruciferous vegetables have an antibiotic-like effect and may help ward off bacterial, viral, and fungal infection in the intestines and other parts of the body. A number of recent studies have also suggested that a diet rich in cruciferous vegetables may lower your risk of certain cancers (RivadeneyraDomı´nguez and Rodrı´guez-Landa, 2020). Glucosinolates are hydrolyzed in the rumen by either the enzyme glucosinolase or thioglucosidase, into glucose, hydrogen sulfate, and one of the following aglycone derivatives: isothiocynates, thiocyanates, nitriles, or related compounds such as oxazolidine-2-thiones. The enzymic reaction occurs when plant tissue is crushed, for example, by mastication, or ruminal activity.

5. NOVEL FOODS Novel foods include foods and ingredients that have not been used to any significant extent in a particular country prior to a specific date specified in a food regulation. Novel foods include pure chemicals, genetically modified foods, cloned animals, whole foods new to a particular world region, and foods processed by a new technology. A novel food can be a newly developed, innovative food or food produced using new technologies and production processes. Examples of Novel Food include new sources of vitamin K (menaquinone) or extracts from existing food (Antarctic Krill oil rich in phospholipids from Euphausia superba), agricultural products from third world countries (chia seeds, noni fruit juice), or food derived from new production processes (UV-treated food (milk, bread, mushrooms, and yeast)) (Table 2.2). In Canada, a food is considered novel if it contains a microorganism that does not yet have a history of safe use as a food; a food that

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TABLE 2.2 Select Novel Foods and Examples Novel Food

Examples

New or intentionally modified molecular structure

Tagatose and salatrim

Consist of or are isolated from microorganisms, fungi or algae

Algae oil from the microalgae Ulkenia sp.

Consist of or are isolated from materials of mineral origin

Clinoptilolite (zeolite)

Consist of or are isolated from plants and parts of plants

Noni juice (Morinda citrifolia), chia seeds (Salvia hispanica)

Consist of or have been isolated from animals or their parts

Insects, oil from Antarctic krill (Euphasia superba), peptides from the fish Sardinops sagax

Cell and tissue cultures from animals, plants, microorganisms, fungi or algae

Extract from cell cultures of Echinacea angustifolia, in vitro meat

Food resulting from a production process not used for food production resulting in a change in composition or structure

High pressure pasteurized fruit preparations, UVtreated mushrooms (Agaricus bisporus), UV-treated baker’s yeast (Saccharomyces cerevisiae), UV-treated milk

Consist of engineered nanomaterials Vitamins, minerals and other substances

Iron (II) ammonium phosphate, vitamin K2 (menaquinone), chromium picolinate

Used exclusively in food supplements (not permitted in food categories other than food supplements)

Maqui berry (Aristotelia chilensis), rose root (Rhodiola rosea)

has been manufactured, prepared, preserved or packaged by a process that has not been previously used for that food and causes the food to undergo a major change; or a food that comes from a plant, animal or microorganism that has been genetically modified so that the plant, animal or microorganism, shows characteristics that it didn’t before, doesn’t show characteristics that it did before or has one or more characteristic that no longer falls within the expected range. The following are examples of novel foods.

also “recombinant DNA technology” or “genetic engineering” (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). Currently available GM foods are mostly derived from plants, but in the future foods derived from GM microorganisms (GMOs) or GM animals are likely to be introduced on the market. Most existing genetically modified crops have been developed to improve yield through the introduction of resistance to plant diseases or of increased tolerance to herbicides. GM foods can improve yields and reliability. In the future, genetic modification could be aimed at altering the nutrient content of food, reducing its allergenic potential, or improving the efficiency of food production systems. However, creating GMOs has caused political opposition in some groups. Objections made by some that genetically modifying organisms equates to the manipulation of life while others argue that it is essentially an extension of traditional plant cultivation and animal breeding techniques. There has been much debate about

5.1. Genetically Modified Food Genetically modified (GM) foods are foods derived from organisms whose genetic material (DNA) has been modified to that which is not natural, e.g., through the introduction of a gene from a different organism. The technology for the creation of GM is often called “modern biotechnology” or “gene technology,” sometimes

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the risks to the environment and human health from GMs and GMOs, which has led to the development of regulatory frameworks for the evaluation of genetically modified crops (Karalis et al., 2020). Internationally, FAO/WHO Codex guidelines are available for risk analysis of GM food (FAO/WHO, 2011).

5.2. Novel Food ColorsdAnthocyanins Anthocyanins are the largest group of watersoluble natural pigments that have been used with commercial success. They are responsible for most of the red, purple, and blue colors exhibited by flowers, fruits, and other plant tissues but they suffer color instability, spontaneous degradation, and conversion to uncolored forms under heat, light, and moderate pH. Many efforts have been made to search for highly stable anthocyanins from fruit, vegetables, or ornamental plants, and to improve the stability of anthocyanins. Production of anthocyanins from cell cultures has been investigated. Also, there is the current medical interest in anthocyanins as they possess potent antioxidant properties, which help explain the health benefits of red grapes and wines.

5.3. Novel PreservativesdAmygdalin Amygdalin based G-6-P synthase inhibitors have been proposed as novel preservatives for food and pharmaceutical products. G-6-P synthase enzyme has been involved in the synthesis of the microbial cell wall, and its inhibition may lead to the antimicrobial effect. These synthesized compounds exhibit good antioxidant, antimicrobial, and better preservative efficacy in food preparation as compared to the standard compounds.

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5.5. Novel SweetenersdStevia Novel sweeteners comprise a loosely defined group of sweeteners that, while providing differences in sweetness and caloric content, are derived from natural sources. Trehalose, tagatose, stevia, and others are in this category. Novel sweeteners are hard to classify because of what they are made from and how they are made. Stevia is an example. The FDA has approved highly refined stevia preparations as novel sweeteners but has not approved wholeleaf stevia or crude stevia extracts for this use. Tagatose is also considered a novel sweetener because of its chemical structure. Tagatose is a low-carbohydrate sweetener similar to fructose that occurs naturally but is manufactured from the lactose in dairy products. The FDA categorizes tagatose as a generally recognized as safe (GRAS) substance.

5.6. Novel ProteinsdCell-Based Meats Novel proteins are any type of protein that does not come from an animal such as beef, lamb, chicken, pork, dairy products, etc. Often proteins from a less commonly consumed animal are called novel proteins. These can be alligator, kangaroo, ostrich, and many more. Novel proteins offer a range of solutionsdintegrating easily into food products and adding nutrients. But novel proteins are not just plant-based, e.g., cell-based meat (taking and growing meats from plant or animal cells) is both sustainable and has minimal ecological impact (Figure 2.9). Novel proteins may help

5.4. Novel EmulsifiersdYeast Food emulsifiers are created by alcoholysis, or direct esterification of edible fatty acids taken from animal or vegetable sources containing polyols (i.e., glycerol, propylene glycol, and sorbitol). Some examples with promising prospects include wet-milled apple pomace particles, starch, and yeast-based emulsifiers.

FIGURE 2.9 What our meat may look like in the future. Getty Images-1249262197-be4a76b.jpg with permission.

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feed the growing population, although many are still in the early stage of development.

with other xylo-oligosaccharides have been associated with the consumption of other nondigestible carbohydrates probably due to effects on the microbiome.

5.7. Novel OilsdOlestra Olestra, sucrose esterified with fatty acids, was to be used as a replacement for conventional fats. The petitioner later agreed to restrict its use to savory snacks. FDA determined that olestra was safe for use in savory snacks (e.g., potato chips, corn chips). Olestra is not toxic, carcinogenic, genotoxic, or teratogenic and is neither absorbed nor metabolized but is associated with gastrointestinal tract symptoms such as cramping and loose fatty stools (steatorrhea). In addition, olestra affects the absorption of fatsoluble vitamins but does not affect the absorption of water-soluble nutrients. Studies concluded that when olestra was consumed with foods containing vitamins A, D, E, or K, the fat substitute could have an effect on the absorption of these nutrients. Therefore, FDA required replacement of fat-soluble vitamins lost through absorption. Olestra was never approved in Canada, and although the United States had initially approved its use in chips, it was withdrawn from the market due to consumer complaints of steatorrhea.

5.8. Novel CarbohydratesdPrecticX The novel food PrecticX is obtained from corncobs (Zea mays subsp. mays) via enzymecatalyzed hydrolysis and subsequent purification. The main components of the novel food, the oligosaccharides, are resistant to human digestive enzymes and are fermented by colonic bacteria. These oligosaccharides can be added to a variety of foods such as bakery and dairy products, fruit jelly, chocolates, and soy-drinks. The information provided on composition, specifications, production process, and stability of the novel carbohydrates did not raise safety concerns with the regulatory authority. There were effects observed in the animal studies with the novel food or with other xylooligosaccharides that were considered by the reviewers to be expected from the intake of nondigestible carbohydrates. The acute and transient gastrointestinal observed effects in human intervention studies with the novel food or

5.9. Recombinant Bovine Somatotropin Recombinant bovine somatotropin (rBST) is an artificial growth hormone that increases milk production (see Section Antimicrobial Resistance: More than Residues). It is illegal for use in Canadian dairy cows but is legal in the United States. The reason for removing rBST was based on assessment that there was no risk to human health, but there was a risk to animal health.

5.10. CannabisdCannabidiol Cannabidiol (CBD) is a cannabinoid which can be extracted from the cannabis plant and added to foods. It has no psychoactive properties and, depending on the method of extraction, should contain little or no tetrahydrocannabinol (THC). During the past few years there has been significant growth in the number of products sold that contain CBD. These food products are classed as novel foods in the United Kingdom but are regulated as drugs in Canada and the United States. CBD may be found in a variety of food products and supplements including, but not limited to oils, drops or tinctures, capsules, sweets and confectionery, and baked goods and drinks.

5.11. Nanomaterials Nanotechnology is a rapidly developing field and nanomaterials (NMs), are of significant technological and economic interest, and have an immense impact on many industries including the food industry (see Nanoparticulates, Vol 3, Chap 13). The current use of NMs in the food sector is in food additives and food packaging. While the nanotechnology has many applications and benefits for the food sector and new NMs are increasing, there are concerns about their safety due to the lack of knowledge regarding the interactions of NMs at the molecular or physiological levels. Additionally, while the risk of particle inhalation has received much attention, there are still gaps of knowledge

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regarding possible adverse health following oral exposure to NMs.

effects

5.12. Probiotics and PrebioticsdIntelligent Labs Probiotics with Prebiotics The Food and Agriculture Organisation of the United Nations (FAO) defines a probiotic as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Prebiotic is defined as a nonviable food component that confers a health benefit on the host associated with modulation of the microbiota. A novel probiotic or prebiotic can potentially be a component of conventional foods, food supplements or foods for particular nutritional uses (PARNUTSs). PARNUTSs foods incorporating probiotics or prebiotics comprise those designed for specific dietary requirements and may include infant formulas and follow-on formulas, processed cereal-based food and baby food, food for special medical purposes and total diet replacement for weight control. A good example of a novel probiotic and prebiotic supplement is Intelligent Labs 50 billion CFU Probiotic with Prebiotics (Sanders et al., 2010).

6. ADVERSE REACTIONS TO FOOD CONSTITUENT Adverse reactions to food constituents may be an immune-mediated reaction to a food constituent or a mixture, e.g., food allergy; exposure of a susceptible population, e.g., celiac disease (CD) and lactase deficiency leading to lactose intolerance; direct or indirect, e.g., endocrine disruption; chemical toxicity, e.g., histamine in scombroid fish poisoning (see Animal Toxins, Vol 3, Chap 8); or nonallergic food hypersensitivity to the chemical/pharmacological action of food ingredients, e.g., sulphites in wine (Figure 2.10). Food allergies are central to the discussion of food additives intentional or not (Berni Canani et al., 2019) and growing evidence indicates that gut microbiota have a pivotal role in the development of food allergies (Meng et al., 2018). The dance between nutrients and gut microbiota-derived metabolites is opening the way to a postbiotic approach in the stimulation of immune tolerance through epigenetic

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regulation. Probiotics are considered generally safe to consume but may cause bacteria–host interactions and unwanted side effects in rare cases. There is some evidence that probiotics are beneficial for some conditions, but there is little evidence for many of the health benefits claimed.

6.1. Food Allergies The term “food allergy” is used to describe an adverse immune-mediated response to a food constituent, and can be either IgE mediated (e.g., urticaria and anaphylaxis) or non-IgE mediated (e.g., food protein–induced enterocolitis syndrome and eosinophilic esophagitis). Food allergies are estimated to affect approximately 8% of children and 11% of adults in North America. “Allergens” are the food constituents, usually proteins, responsible for eliciting the allergic reactions, e.g., peanuts (Pulido and Godefroy, 2010; Mueller et al., 2014; Anvari et al., 2019). Food allergies encompass a wide range of clinical disorders and can be grouped based on the immune-mediated mechanism involved: IgE, non-IgE, or mixed IgE/non-IgE (Anvari et al., 2019). The variability in the clinical manifestations of food allergies is due to differences in specific triggering proteins and the wide range of potential mechanisms, as well as factors such as host genetics, host intestinal microbiota, amount of exposure, and age. Patients with food allergies may present with common symptoms, such as GI disorders. Food allergies may be the result of a breach in oral tolerance to ingested food, or of crossreactivity between food and nonfood allergens. For example, individuals with allergies to fruits and vegetables may have been sensitized by pollen exposure. This condition is known as pollen-food allergy syndrome or oral allergy syndrome (OAS) (Carlson and Coop, 2019). Allergy to food develops when a susceptible individual is exposed to the specific food allergen for the first time, which is mistakenly identified as harmful by the human immune system. This is sensitization (Anvari et al., 2019). IgE antibodies are created specific to the protein misidentified as harmful, and these antibodies attach to the surface of basophils and mast cells.

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FIGURE 2.10

Flow diagram of adverse food reactions and their cause (Author’s diagram).

Repeat exposure to the same allergen or a cross-reactive protein results in its conjugation with the specific surface IgE antibodies, triggering mast cells and basophils to release mediators such as histamine, and promote the synthesis of prostaglandins, leukotrienes, and cytokines. Any food protein can potentially provoke an immune reaction; however, relatively few food proteins are responsible for the vast majority of significant food-induced allergy, i.e., milk, eggs, peanuts, soy, wheat, tree nuts (such as walnuts and cashews), fish and shellfish (such as shrimp) (Nance et al., 2020). Histamine is a biogenic amine released from mast cells that binds to a family of receptors on target cells in tissues inciting numerous biological reactions such as smooth muscle contraction, vascular dilatation, increased vascular permeability, mucus secretion, tachycardia, lower blood pressure, arrhythmias, and stimulation of gastric acid secretion (Borriello et al., 2017). The massive release of histamine explains the fast

onset of IgE-mediated food allergies clinical symptoms, and their ability to affect multiple systems and tissues (Kanagaratham et al., 2020). The allergic response to ingestion of a suspect food allergen via IgE, mast cells, and basophils has been recognized by the WHO and other food safety authorities. The first stage is to assess the available evidence to decide whether the allergen in question induces an IgE-mediated response (Pulido, 2010). In response to food allergies and health concerns of the consumer whose allergic reactions have a significant impact on their daily life and their families and communities, food processing technologies have been developed to eliminate food constituents that can be harmful to susceptible individuals, such as peanuts in a candy bar, and reduce the risk of unintentional allergen exposure. In addition, food labeling providing a list of ingredients allows the consumer to have information needed to make informed choices.

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6.2. Allergy-like Food Poisoning Food allergies are more common in children, can be life-threatening, and are distinct from food intolerances (Turnbull et al., 2015). IgEmediated food-allergic disease differs from non-IgE-mediated disease because the pathophysiology results from activation of the immune system, causing a T helper 2 response which results in IgE binding to Fcε receptors on effector cells such as mast cells and basophils. The activation of these cells causes release of histamine and other preformed mediators and rapid symptom onset. In contrast to the rapid release of histamine in allergic food intolerance, non-IgE-mediated food allergy is delayed (Anvari et al., 2019). Allergy-like food poisoning is the response to the consumption of food contaminated with compounds that are sometime present in food, particularly biogenic amines, such as: histamine, putrescine, tyramine, cadaverine, and phenylethylamine as these compounds can be found in fermented foods, with histamine in particular accountable for several food poisonings and outbreaks. The presence and level of biogenic amines in food material is an indicator of microbial activity (microbial decarboxylation) in stored or processed foods. Unhygienic manufacturing and storage practices are the reason for histamine poisoning in fish products. Several contaminant microorganisms, e.g., Lactobacillus sp., Bacillus sp., Bacillus subtilis strains, Staphylococcus sp., Streptococcus sp., and Enterococci sp., can produce biogenic amines in fermented foods such as sausages, wine, and Asian style foods (Sivamaruthi et al., 2018). Regardless of the mechanism of histamine release, its effects are systemic and depend on the activation and location of histamine receptors (see Immune System, Vol 5, Chap 6). Mast cells, the major producers of histamine, have a wide distribution throughout the body including the brain (Thangam et al., 2018).

6.3. Adverse Reactions to Gluten and Gluten-Related Disorders Adverse reactions to gluten are diverse and grouped as gluten-related diseases or disorders (GRDs) that include celiac disease (CD), wheatassociated allergy (WA), and nonceliac gluten/

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wheat sensitivity (NCGS) (Oxentenko and Rubio-Tapia, 2019; Cardoso-Silva et al., 2019; Cabanillas, 2020). Dermatitis herpetiformis (DH), gluten ataxia, and gluten neuropathy (Mearns et al., 2019) are clinical conditions also associated with dietary gluten and can present with or without GI manifestation of CD. Gluten and gluten-related proteins are storage proteins from wheat (Triticum aestivum) and other cereals such as rye (Secale cereale) and barley (Hordeum vulgare). Gluten-related proteins can also be found in malt, triticale, spelt, or kamut. Gluten is one of the food components most used in the food industry due to its properties; it is the cohesive wheat protein that gives elasticity to wheat dough, and structure to bread, baked goods, and other foods. Adverse reactions to gluten appear to have increased over the last decades, particularly to wheat (Cabanillas, 2020). The term “gluten” is used as a generic name for the proteins of any cereal grain (Pulido, 2010). Gluten is only partially digested by humans; after the action of gastric, duodenal, and pancreatic enzymes a 33-amino acid peptide (33mer) and other immunogenic peptides remain. These undigested peptides are key in eliciting the adverse reactions to gluten and are the causative factor to GRDs entities. However, these elements each has distinct pathophysiological pathways. In celiac disease, a T-cell-mediated immune reaction triggered by gluten ingestion is central in the pathogenesis of the enteropathy, while wheat allergy develops as a rapid IgE- or nonIgE-mediated immune response. In nonceliac wheat sensitivity, classical adaptive immune responses are not involved. Instead, recent research has revealed that an innate immune response to a yet to be defined antigen, as well as the gut microbiota, is pivotal in the development in this disorder (Cardoso-Silva et al., 2019). A full spectrum of GRDs account for more than the 1% prevalence rate estimated for CD in the population. Tissue transglutaminase 2 (TG2) is the autoantigen identified with CD, whereas TG3, an epidermal transglutaminase is identified in DH, and TG6, a neural transglutaminase, is implicated in gluten related neurological conditions (Mearns et al., 2019). Among the GRDs, CD is the best studied and has been used as a model for the understanding of autoimmunity, the interplay between genetics,

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the immune system, the gastrointestinal system and the environment. CD is the only autoimmune condition for which the genetic background and the trigger, gluten, are known (Caio et al., 2019, 2020; Husby et al., 2019; Oxentenko and Rubio-Tapia, 2019). Celiac Disease CD is a chronic immune-mediated enteropathy caused by dietary gluten in genetically predisposed individuals of various race/ethnicity, particularly Caucasians, and can present at any age, preferentially affecting women (Lebwohl and Rubio-Tapia, 2021). It is estimated that prevalence in developed countries is 0.5%–1% with 10%–20% increased risk among first-degree relatives of those diagnosed with CD, and in individuals with Down’s syndrome or type 1 diabetes (Ludvigsson and Murray, 2019). Active CD is clinically characterized by chronic inflammation of the small intestinal mucosa (Waerling-Hansen and Sams, 2018), which may result in atrophy of intestinal villi, malabsorption, and clinical manifestations due to nutrient deficiencies and/or to the associated immune-mediated adverse reactions. The immune system needs multiple specific micronutrients, which are often deficient in the general population and more likely in celiac individuals, including vitamins A, D, C, E, B6, and B12, folate, zinc, iron, copper, and selenium. These play vital roles at every stage of the immune response and for proper function of physical barriers. For diagnosis, individuals presenting with possible CD are screened for IgA antibodies to tissue transglutaminase 2 (tTG) and antiendomysial IgA antibodies (EMAs). Endoscopy aids in the diagnosis and in the identification of the best sites for biopsy sampling, since the lesions may be patchy. CD diagnosis is confirmed by duodenal biopsies demonstrating the hallmark of pathological changes summarized in detail below (Figure 2.11), which classically manifest in the proximal small intestine but can extend to the jejunum. Unfortunately, several classification systems are used for grading a CD lesion, based on the histological features of decreased enterocyte height (flattening of enterocytes, reduction or absence of brush border); crypt hyperplasia in the presence of more than one mitosis/crypt; and villous atrophy (decrease in

villous height, alteration of normal villus/crypt ratio of 3:1). Assessment includes the number and distribution of intraepithelial lymphocytes (IELs) using immunohistochemistry (Figure 2.11). CD patients can present with GI, and/or extraintestinal symptoms, which are highly variable in character and severity, and are influenced by factors such as age, immunologic status, gluten exposure (amount, duration, or timing of introduction to gluten), and the extent and severity of damage caused to the intestine (Aaron et al., 2019). Hence, the diagnosis is often missed (Pulido et al., 2013; Zarkadas et al., 2013). Under certain circumstances biopsy can be avoided. Serological testing may be sufficient to make the diagnosis of CD, e.g., pediatric cases and adults with a 10-fold increase in IgA antitissue transglutaminase (tTG) antibody levels in combination with EMA positivity are considered by some sufficient for CD diagnosis in the absence of duodenal biopsies (Husby et al., 2019). Virtually all celiac individuals express the human leukocyte antigen HLA-DQ2 or HLADQ8 haplotypes on their antigen-presenting cells (APCs). These predisposing genetic hallmarks HLA-DQ molecules bind enzymatically modified gluten peptides, and the HLA-DQ– peptide complexes trigger inflammatory T-cell responses in the small intestine. In addition, gluten induces innate immune responses that contribute to the intestinal pathology. Continued gluten exposure makes the adverse immune reactions self-perpetuating. In contrast, many people with similar risk factors do not develop CD, suggesting a multifactorial etiology. The only treatment for CD is glutenfree diet (GFD) for life (Sparks et al., 2021; Pulido et al., 2013; Tye-Din et al., 2018; Valitutti and Fasano, 2019). The food industry is responding to the consumer demand for gluten-free food by designing and producing new technologydriven ad hoc products (Caio et al., 2020; Lamacchia et al., 2021). The result of this demand means that GFD is reaching up to 7% in some places (Cabanillas, 2020). “Leaky Gut” Recently, increasing attention has been paid to the intestinal barrier, a dynamic system

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FIGURE 2.11 Biopsy samples from human small intestine, either normal (A–B) or with celiac disease (B–D). (A) Normal duodenal mucosa showing well-maintained architecture, finger-like villi, and 3: 1 villus (V)/crypt (Cr) ratio. H&E, 10. (B) Normal distribution of intraepithelial lymphocytes (IELs) CD3þ immunohistochemistry, 10, enclosure 20. (C) Mucosa from an untreated celiac individual showing villous atrophy characterized by flattening of the villi and loss of normal (3: 1) villus (V)/crypt (Cr), crypt hyperplasia (Cr) and dense inflammatory infiltrate of the mucosa (asterisk). 40. (D) CD3þ immunohistochemistry showing the increase in intraepithelial lymphocytes (IELs >30/100 enterocytes). 20, enclosure 40. Photographs provided by Dr. Mohsin Rashid, Dalhousie University, Canada. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 35.3, p. 1068, with permission. [Page 1068].

comprising various components, which regulate the delicate crosstalk between metabolic, motor, neuroendocrine and immunological functions, as well as the brain (Figure 2.12). Dysbiosis may disturb the brain–gut axis. Among the elements characterizing the intestinal barrier, the microbiota plays a key role, modulating gut integrity maintenance, the immune response and the inflammatory process, linked to the CD and the increased reporting of nonceliac gluten sensitivity cases (Caio et al., 2020) (see Digestive Tract, Vol 4, Chap 1).

Disruption of the intestinal barrier is being proposed as one of the mechanisms leading to autoimmune and inflammatory conditions, including CD, type 1 diabetes, inflammatory bowel disorders (IBDs) (Oligschlaeger et al., 2019), and food allergy. Increased intestinal epithelial permeability (“leaky gut”) may be a primary etiologic factor predisposing to disease development. The gut-associated lymphoid tissue (GALT), the neuroendocrine network, and the intestinal epithelial barrier with its intercellular tight junctions control the

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FIGURE 2.12 The brain–gut axis. Pathways of communication between microbiota and brain. A growing body of research is implicating different pathways of communication between the microbiome and brain in disorders of both mood and motility. Multiple direct and indirect (via systemic circulation) pathways exist through which the gut microbiota can modulate the gut–brain axis. They include endocrine (cortisol), immune (cytokines), and neural (vagus, ENS, and spinal nerves) pathways. Several gut microbes are capable of synthesizing neurotransmitters (i.e., gaminobutyric acid [GABA], noradrenaline, and dopamine) locally, which can act on target cells in the gut and act as an important avenue of communication. Neuroactive microbial metabolites can modulate brain and behavior through a number of ways that are still being elucidated. These include affecting epithelial cells to impact gut barrier function and enteroendocrine cells (EECs) to release GI hormones, as well as dendritic cells (DCs) to modulate immune function. Specialized structures on EECs and ECCs, known as neuropods, have been shown to transduce sensory signals from the intestinal milieu to the brain through forming synapse-like connections to afferent nerves, including the vagus nerve. The ENS is perfectly poised to be an integral hub for microbial signals and can communicate with the brain via vagal and spinal pathways. However, the exact molecular signaling pathways of all these pathways involved remain to be defined. Reprinted with permission from Margolis KG, Cryan JF, Mayer EA: The microbiota-gut-brain axis: from motility to mood, Gastroenterology 160(5):1486–1501, 2021. [Page 1488].

equilibrium between tolerance and immunity to non-self-antigens. The “zonulin pathway” is a physiological modulator of intercellular tight junctions involved in trafficking of

macromolecules and in tolerance/immune response balance. When this finely tuned pathway is dysregulated in genetically susceptible individuals, both intestinal and

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extraintestinal autoimmune, inflammatory disorders can occur (Aaron et al., 2019; Cardoso-Silva et al., 2019). Nonceliac Gluten Sensitivity Nonceliac gluten sensitivity (NCGS) is a term used to describe individuals who do not have the genetic or clinical hallmark of CD, but who have symptoms related to ingestion of glutencontaining grains and improve with GFD. The frequency of NCGS is unknown, owing to the lack of validated biomarkers, but it is thought to be more common than CD (Leonard et al., 2017). Nonceliac gluten sensitivity is diagnosed when CD and wheat allergy (WA) have been ruled out. The limited knowledge about the pathophysiology of NCGS and the lack of validated biomarkers are still major limitations for clinical studies, making it difficult to differentiate NCGS from other gluten-related disorders (GRDs). Several studies suggest that NCGS is an immune-mediated disease that likely activates an innate immune response. Other components of wheat may be responsible for the symptoms observed in individuals without CD. Glutencontaining cereals, particularly wheat, are also a primary source of Fermentable Oligosaccharides, Disaccharides, and Monosaccharides and Polyols (FODMAPs), a group of highly fermentable but poorly absorbed short-chain carbohydrates and polyols. The reduction of FODMAPs associated with GFD may explain in part, why some patients affected with NCGS report amelioration of their symptoms after starting a glutenfree diet. FODMAPs are also used as explanation for symptoms described with irritable bowel syndrome (IBS). Clinically the two conditions seem to overlap (Cardoso-Silva et al., 2019). Wheat Allergy Wheat allergy is defined as an adverse type-2 helper T-cell immunologic reaction to wheat proteins and typically presents soon after wheat ingestion. Clinical signs can include signs of anaphylaxis such as swelling or itching of the mouth, throat, and skin; nasal congestion; watery eyes; and difficulty breathing. Wheat allergy is more common in children, with reported prevalence between 2% and 9% in children and 0.5% and 3% in adults (Cabanillas, 2020).

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6.4. Exposure of a Susceptible Population Lactose intolerance primarily refers to a syndrome having adverse events following consumption of foods containing lactose. It is one of the most common forms of food intolerance and occurs when lactase activity is reduced in the brush border of the small bowel mucosa. Individuals may be lactose intolerant to varying degrees, with varying severity of these symptoms. When lactose is not digested, it can be fermented by gut microbiota leading to symptoms of lactose intolerance that include abdominal pain, bloating, flatulence, and diarrhea with a considerable intraindividual and interindividual variability in the severity of clinical manifestations. These gastrointestinal symptoms can be similar to cow’s milk allergy and could be wrongly labeled as symptoms of “milk allergy” (Di Costanzo and Canani, 2018).

6.5. Direct Chemical Toxicity Scombroid poisoning, also called histamine fish poisoning, is an allergy-like form of food poisoning that continues to be a major problem in seafood safety (Hungerford, 2010). Scombroid poisoning is unique among the seafood toxins since it results from product mishandling rather than contamination from other trophic levels. Inadequate cooling following harvest promotes bacterial histamine production and can result in outbreaks of scombroid poisoning. The exact role of histamine in scombroid poisoning is not straightforward and various possible mechanisms of toxicity have been proposed but none of them proven. Histamine action levels are used for regulation of the food until more is known about the mechanism of scombroid poisoning. Scombroid poisoning and histamine are correlated but complicated. Victims of scombroid poisoning respond well to antihistamines, and chemical analyses of fish implicated in scombroid poisoning generally reveal elevated levels of histamine. Fish with high levels of free histidine, the enzyme substrate converted to histamine by bacterial histidine decarboxylase, are those most often implicated in scombroid poisoning. Laboratory methods and screening methods for detecting histamine are available but need to be compared and validated to

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harmonize testing. Successful field testing, including dockside or on-board testing are needed to augment Hazard Analysis and Critical Control Points (HACCP) efforts and will have to integrate rapid and simplified detection methods with rapid sampling and extraction (Hungerford, 2010).

6.6. Nonallergic Food Hypersensitivity and Intolerance More than 20% of the population in industrialized countries suffer from food intolerance or food allergy (Zopf et al., 2009), one of which is a reaction to sulphites, particularly common in asthmatic individuals. Sulphites are widely used as preservatives and antioxidants in foods and drugs, often without specification. They can lead to severe hypersensitivity reactions, asthma being obviously the most frequent symptom, but also urticaria, angioedema, or other anaphylactoid symptoms may occur. Furthermore, allergic leukocytoclastic vasculitis and exacerbation of an atopic eczema have been observed. The mechanism of action (MOA) of sulphite hypersensitivity is incomplete. Asthmatic reactions have been attributed to activation of the parasympathetic system by the irritating effect of sulphites, possibly enhanced by a deficiency of sulphite oxidase. Besides this pseudoallergic mechanism, for at least some cases of sulphite hypersensitivity an IgE-mediated immediatetype allergic reaction has to be considered (Przybilla and Ring, 1987). Enzyme deficiencies have been proposed to cause other food sensitivities including low amine oxidase activity resulting in histamine intolerance and sucrase-isomaltase deficiency resulting in reduced tolerance to sugars and starch (Tuck et al., 2019).

6.7. Food Color and Food Allergy Most natural colors have a long history of safe use. However, a few clinically relevant IgEmediated food allergy reactions to the natural colors, annatto and saffron, and cochineal extract/carmine have been reported (Valluzzi et al., 2019). These are likely due to residual proteins eliciting an IgE mediated reaction. The risk of a reaction to cochineal food colors seems

to be higher for females, possibly due to sensitization from the use of this color in cosmetics. A clinical differential diagnosis between an immune-mediated food allergy and a chemically induced reaction with allergy-like symptoms may be difficult, as the triggering agent often remains unknown and sensitive individuals may react to more than one compound. Since synthetic food colors do not contain protein, the allergy-like reactions they may elicit are considered to be nonimmune-mediated, and due to chemical-induced release of histamine and other compounds from mast cells. The clinical symptoms linked with histamine release are similar to IgE-mediated allergy. An allergylike reaction has been suggested as a possible mechanism for the behavioral effects of Attention Deficit Hyperactive Disorder (ADHD) ascribed to synthetic food colors, mainly in susceptible children (Pelsser et al., 2017). This view is supported by some animal studies (Zhou et al., 2019). Although further research is required to clarify possible implicated mechanisms of food color-related behavioral disorders, from the clinical point of view, a color elimination diet may, in some cases, be a therapeutic option since food colors have no intrinsic nutritional value. Rarely have food additives been shown to cause true allergic reactions. Individual intolerance may fluctuate from time to time. Azo dyes, benzoic acid, and several other common food additives may aggravate or, more rarely, even cause urticaria. Spices are one of the most common causes of immunological contact urticaria. Nonimmunological contact urticaria is produced by numerous spices, benzoic acid, sorbic acid, cinnamic acid, and many essential oils. Asthma and rhinitis are the main hypersensitivity symptoms in the respiratory tract, where azo dyes, benzoic acid, and sulfidic food additives are the most common causative agents. Systemic and respiratory reactions to food colorants and benzoates have been claimed to occur more frequently in acetylsalicylic acid (ASA)sensitive patients than in nonreactors. Hypersensitivity reactions in organs other than the skin and respiratory tract are rare or poorly documented. Psychological factors play a role in both food and food additive reactions, the impact of these factors is still under debate (Hannuksela and Haahtela, 1987).

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7. MECHANISM OF ACTION OF CLINICAL DISORDERS RELATED TO FOOD 7.1. Gut Microbiota and Adverse Reactions to Food The food composition interplay with gut microbiota has been implicated in a wide range of conditions including obesity, diabetes, inflammatory bowel disorders (IBDs), e.g., Crohn’s disease, irritable bowel syndrome (IBS) which has altered function but no inflammation and altered responses to food (Hills et al., 2019; Loo et al., 2020). The terms microbiota and microbiome are often used interchangeably; however, microbiota are the microbial taxa associated with complex organisms such as humans, and the microbiome is the catalog of these microbes and their genes. The gut microbiota is a complex and diverse microbial ecosystem in the GI tract that coexists in a synergistic partnership with the host. It participates in the maturation and regulation of immune function, energy metabolism, and hormonal balance, including regulation of intestinal mucosal barriers, fermentation of undigested nutrients, and synthesis of short chain fatty acids (Margolis et al., 2021). The gut microbiota provides nutrients for the host by digesting food components otherwise indigestible by human enzymes, simultaneously producing energy, delivering nutrients and metabolites for the nutritional and biological benefit of the host. These microbiota-derived compounds promote the development and function of the host immune system, directly by activating cells of the adaptive (acquired/specific) and innate (nonspecific) immune system and indirectly by sustaining release of monosaccharides, stimulating intestinal receptors and secreting gut hormones (Waerling-Hansen and Sams, 2018). Consumption of dietary FODMAPs is implicated as mechanisms in some of the GI symptomatology observed in patients with IBS and with nonceliac gluten sensitivity (Hills et al., 2019). FODMAPs pull water into the small intestine and colon, causing luminal distension (Vandeputte and Joossens, 2020). Fermentation of FODMAPs by gut bacteria and yeast then produces hydrogen or methane gas. The pros and cons of dietary

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restriction of FODMAPs and pre/probiotic treatment on GI symptoms and disorders are discussed elsewhere (Hills et al., 2019; Gibson et al., 2020). The composition of the gut microbiota may be important in reducing the risk of contracting particular gut infections. Changes in the microbiota during an individual’s lifespan are accompanied by modifications in multiple health parameters, and such observations have prompted intense scientific efforts aiming to understand the complex interactions between the microbiota and its human host, as well as how this may be influenced by diet (Milani et al., 2016). Microbiome and Cytochrome P450 The gut microbiome contributes to the xenobiotic biotransformation of the host, and the first pass metabolism of many orally ingested chemicals. This is a joint effort between host drug metabolizing enzymes including P450s and gut microbiome. Gut microbiome contributes to the drug metabolism via two distinct mechanisms: direct mechanism refers to the metabolism of drugs by microbial enzymes, among which reduction and hydrolysis (or deconjugation) are among the most important reactions, whereas indirect mechanism refers to the influence of host receptors and signaling pathways by microbial metabolites. Many types of microbial metabolites, such as secondary bile acids (BAs), short chain fatty acids (SCFAs), and tryptophan metabolites, are known regulators of human diseases through modulating host xenobiotic-sensing receptors (Dempsey and Cui, 2019). Microbiome and Immunity The gut microbiota plays important roles in maintaining intestinal homeostasis. Dysbiosis of the gut microbiome is caused by the imbalance between the commensal and pathogenic microbes. The commensal microbiome regulates the maturation of the mucosal immune system, while the pathogenic microbiome causes immunity dysfunction, resulting in disease development (Shi et al., 2017). Inflammation, which is caused by abnormal immune responses, influences the balance of the gut microbiome, resulting in intestinal diseases and potentially others, e.g., chronic diseases ranging from

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gastrointestinal inflammatory and metabolic conditions to neurological, cardiovascular, and respiratory illnesses (Durack and Lynch, 2019). Microbiome and Food Additives The frequency of intestinal and systemic immune-inflammatory disorders has increased in previously low incidence areas. This increase is likely due to the high consumption of proteins, saturated fats and sugars as well as by a broad use of food additives (e.g., emulsifiers, bulking agents). Food additives may perturb gut homeostasis, thereby contributing to promotion of tissue-damaging inflammatory responses. Direct profiling of the gut microbiome in human cohort studies has demonstrated that individuals with food allergy have distinct gut microbiomes as compared to healthy controls, and dysbiosis precedes the development of food allergy. Mechanistic studies in mouse models of food allergy have confirmed that the composition of the intestinal microbiota can imprint susceptibility or resistance to food allergy on the host and have identified a unique population of microbial-responsive RORgt þ Foxp3 þ Tregs as critical for the maintenance of tolerance to foods (Bunyavanich and Berin, 2019). Similarly, mice given the emulsifiers carboxymethylcellulose and polysorbate 80 developed dysbiosis with overgrowth of mucus-degrading bacteria triggering colitis in animals deficient in either interleukin-10, a cytokine exerting antiinflammatory and regulatory functions, or Tolllike receptor 5, a receptor recognizing the bacterial flagellin. The polysaccharide maltodextrin induces endoplasmic reticulum stress in intestinal goblet cells, thereby impairing mucus release and increasing host susceptibility to colitis (Laudisi et al., 2019).

7.2. Neurotransmission There are a number of food-related toxicities that mediate their effect via alteration of neurotransmission. Some of these toxicants or toxins may cause serious injury and possibly death. Excitatory Amino Acids Glutamate (Glu) and aspartate (Asp) are amino acids (AAs) that act as major excitatory

neurotransmitters in the mammalian central nervous system by stimulating or exciting postsynaptic neurons (Thomas, 1995). Although these AAs are primarily involved in intermediary metabolism and other nonneuronal functions, their most important role is as neurotransmitters. It is estimated Glu mediates nearly 50% of all the synaptic transmissions in the CNS and its involvement is implicated in nearly all aspects of normal brain function including learning, memory, movement, cognition, and development (Rousseaux, 2008a,b). A number of other AAs also have neurotransmission activities (Table 2.3). Excitotoxicity of Glutamate At elevated concentrations, Glu acts as a neurotoxin capable of inducing severe neuronal damage and necrosis by causing over excitation of neurons: excitotoxicity (Pulido and Gill, 2013). Previously it was thought that hypoxia played a part in the neurotoxicity of Glu, particularly in the white matter; however, it has now been shown that hypoxic injury and GluRs over stimulation are independent of one another (Rosin et al., 2004). Regardless, neurons under various conditions can become so over stimulated by Glu that necrosis occurs through receptor-mediated depolarization and calcium influx (Gill and Pulido, 2001). There are five main factors necessary for the transition of Glu and Asp from neurotransmitters to excitotoxins. These include inadequate neuronal ATP levels, inadequate neuronal levels of magnesium; high concentrations of inflammatory prostaglandins; excessive free radicals (Singh et al., 2003) and inadequate removal of synaptic Glu (Li and Stys, 2000). Glutamate, GABA, and Glutamate Receptors Glu, glycine (Gly), and g-aminobutyric acid (GABA) are most common and betterunderstood CNS neurotransmitters. They are all metabolic intermediates and neurotransmitters, where Glu is the major excitatory neurotransmitter, and Gly and GABA are the major inhibitory neurotransmitters, in the CNS (Rousseaux, 2008a,b). Glutamate effects are mediated by a large family of GluRs (Figure 2.13): the ionotropic glutamate

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TABLE 2.3 Amino Acids and Their Roles in Neurotransmission

Amino Acid

Excitatory or Inhibitory

Neurotransmitter, Neuromodulator, or Precursor

Glutamic acid (Glu)

Excitatory

Neurotransmitter

Excitatory

Neuromodulator, neurotransmitter

Function

Ionotropic (AMPA, NMDA, and kainate) metabotropic glutamate receptors

Main excitatory neurotransmitter in CNS.

NMDA and mGluR5 (d-asp only)

l-Aspdneuromodulator (proposed neurotransmitter).

Can spill over for extrasynaptic activation. Excesses can cause excitotoxicity.

d-Aspdneuromodulator (proposed neurotransmitter); involved in hormone release, neurogenesis, learning and memory. Glutamine (Gln)

N/A

Precursor

Ionotropic glutamate receptors (but requires millimolar concentrations)

Generation of glutamate, GABA, and aspartate. Involved in regulating ammonia homeostasis. Unclear physiological relevance of glutamine-induced activation of ionotropic glutamate receptors.

Cysteine (Cys)

Excitatory

Neurotransmitter, precursor

NMDA

Physiological relevance of NMDAR activation is unclear. Excitotoxindunknown mechanism. Precursor to glutathione, taurine, Lcysteine sulphuric acid, L-cysteic acid, and hydrogen sulfide.

Methionine (Met)

N/A

Precursor

N/A

Precursor to homocysteine, which is an excitatory neuromodulator that binds to NMDA receptors.

Proline (Pro)

Excitatory

Neuromodulator

Glycine, NMDA, and AMPA/Kainate

Excess leads to hyperprolinemia (seizures, hyperlocomotion, learning, and other cognitive deficits). Stress response.

Asparagine (Asn)

N/A

Precursor

N/A

Precursor to aspartate.

(Continued)

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Deficiencies in synthesis lead to structural abnormalities in brain and cognitive deficits.

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Aspartic acid (Asp)

Receptor

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TABLE 2.3

Amino Acids and Their Roles in Neurotransmissiondcont’d

Amino Acid

Lysine (Lys)

Neurotransmitter, Neuromodulator, or Precursor

Inhibitory (adult); excitatory (developing)

Neurotransmitter

Inhibitory

Neuromodulator, precursor

Receptor

Function

Ionotropic (GABAA) and metabotropic (GABAB)

Major inhibitory neurotransmitter in the brain.

GABAA and GPRC6A

Precursor for L-glutamate.

Coreleased with glycine in some synapses.

Modulator of GABAergic transmission. Indirect regulation of D-serine. Stress response and pain.

Arginine (Arg)

N/A

Precursor

N/A

Precursor to NOx species and creatine. Reduces stress-induced anxiety.

Glycine (Gly)

Inhibitory

Neurotransmitter

Glycine receptors and NMDA

Main inhibitory neurotransmitter in the spinal cord. Coreleased with GABA in some synapses. Coagonist of (extrasynaptic) NMDA receptors. Involved in cell migration and synaptogenesis.

Serine (Ser)

Both

Precursor, neurotransmitter

NMDA and glycine (d-ser)

l-Serdprecursor to glycine and D-serine; facilitate release of glutamate and aspartate. d-Serdco-agonist for glycine and NMDA receptors; involved in Alzheimer’s disease and alcohol addiction.

Alanine (Ala)

Both

Neuromodulator

Glycine and NMDA

d-Aladweaker agonist for glycine receptors and coagonist for NMDA receptors.

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GABA

Excitatory or Inhibitory

N/A

Precursor

N/A

Precursor to glycine.

b-alanine (b-Ala)

Inhibitory

Neurotransmitter, precursor

MrgprD, NMDA, GABAA/C, and glycine

Rate-limiting precursor to carnosine. Pain modulation. Histamine-independent itch mechanisms.

Aromatic amino acids (phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), and histidine (His))

N/A

Precursors

N/A

Precursor to catecholamines, serotonin, and histamine.

BCAAs (isoleucine (Ile), leucine (Leu), and valine (Val))

N/A

Precursor

N/A

Competes with aromatic amino acid transport, indirectly modulating synthesis of catecholamines, serotonin, and histamine. Precursor for glutamate.

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Threonine (Thr)

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FIGURE 2.13 Relationship of glutamate receptors to one another. Reprinted with permissions from Rousseaux CG: A review of Glutamate receptors I: Current understanding of their biology, Japan, J Toxicol Pathol 21:21–51, 2008a. [Page 34].

receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs) (Jewett and Thapa, 2021). Neuronal cells produce and store glutamate in presynaptic vesicles, which are released into the extracellular space when required. There it binds to GluRs on the postsynaptic site of the target cells, inducing very rapid, potent excitatory neurotransmission in these cells. Multilevel regulatory mechanisms are implicated in the control of glutamate’s production, release, binding to its receptors, uptake and elimination by astrocytes (Swanson et al., 2005; Iovino et al., 2020) (Figure 2.14). When these regulatory mechanisms fail to eliminate excess glutamate in the nervous system it leads to excitotoxicity: overactivation of GluRs and subsequently massive neuronal death and tissue damage (Swanson et al., 2005; Levite, 2017; Iovino et al., 2020). The excessive activation of iGluRs is termed “excitatory excitotoxicity.” Excitotoxicity is implicated as a common central pathological factor in many neurological diseases and injuries, especially those that display a neuroinflammatory component, such as multiple sclerosis (MS), traumatic brain injury (TBI), acute brain anoxia/ischemia, epilepsy, and other neuropsychiatric disorders (Levitte 2017). Although GluRs were thought to be predominantly located in the CNS, it is now known that

they are also present in peripheral nervous and nonnervous tissues where, unprotected by the blood–brain barrier, they are more readily exposed to exogenous excitatory amino acids (EAAs) (Gill and Pulido, 2001). These receptors sites are currently an important field of medical research and drug development for the treatment of various conditions (Levite, 2017). Furthermore, GluRs in peripheral tissues are also potential targets for the effects of EAAs present in foods and could explain some of the clinical manifestations associated with these compounds, such as cardiac arrhythmia seen with the natural toxin DA (Pulido, 2008, 2014). Activation of the inotropic N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors (iGluRs) increases transmembrane calcium and sodium fluxes, and the metabotropic glutamate receptor activation (mGluRs) results in generation of inositol triphosphate and inhibition of adenylate cyclase. Numerous modulatory sites exist, especially on the NMDA receptor. Nitric oxide (NO), arachidonic acid, superoxide, and intracellular calcium overload are the ultimate mediators of neuronal death. Glutamate reuptake transporters belong to a unique family of amino acid transport systems, the malfunction of which is intricately involved in disease pathogenesis (Pulido, 2008; Pulido and Gill, 2013).

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FIGURE 2.14 The ionotropic glutamate receptors, N-methyl-D-aspartate (NMDA), kainate, and a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) subtypes, largely function to mediate fast receptor transmission, but also mediate the use dependent changes required for neuronal plasticity. The vesicular transporters (vGluT1 and vGluT2) load glutamate into vesicles presynaptically. The glial, astrocyte, and postsynaptic glutamate transporters (excitatory amino acid transporters, EAATs1–5) are thought to mediate the uptake of glutamate and therefore termination of synaptic transmission. The metabotropic glutamate receptors have a diverse synaptic localization and function pre- and postsynaptically to modulate neurotransmitter release and postsynaptic excitability, respectively. Reprinted with permission from Swanson CJ, Bures M, Johnson MP et al.: Metabotropic glutamate receptors as novel targets for anxiety and stress disorders, Nat Rev Drug Discov 4(2):131–144, 2005 [Page 132].

Monosodium Glutamate It has been suggested that monosodium glutamate (MSG) is toxic and can exert its toxicity via similar mechanisms to L-glutamate (L-Glu). Administration of MSG during the neonatal

period produces marked cognitive effects due to the loss of synaptic plasticity in rodents; however, the effect of MSG on long-term brain function in humans has been questioned. The route of administration can explain the

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differences in these findings, where human exposure is much lower via the oral route. MSG is the sodium salt of the naturally occurring amino acid glutamic acid. In addition to being a common ingredient intentionally added to foods, glutamate occurs naturally in vegetable and animal proteins and is found in 10%–25% of all food protein. Glutamate has been used as a flavoring agent for over a century. The safety of MSG as food additive has been and continues to be controversial. Regulatory approaches vary among regions and jurisdictions (Mortensen et al., 2017; Roberts et al., 2018; Zanfirescu et al., 2019; Thuy et al., 2020). Dietary management of certain conditions, e.g., fibromyalgia (Silva et al., 2019), depression, Gulf War Illness using a diet low in glutamate, is gaining popularity in clinical practice (Holton et al., 2020). MSG evokes the “umami taste.” MSG enhances the taste of food, and by the umami taste effect increases appetite; it also influences the reduction of fat mass and increases satiety (Hartley et al., 2019). Through the activation of umami receptors, MSG and foods rich in glutamate stimulate salivary secretion, gastric hydrochloric acid secretion, pancreatic exocrine secretion, and insulin release. It also enhances gastric emptying and colon motility. Umami receptor mechanisms appear to be less involved in the rapid analysis of food in the oral cavity, and more involved in increasing appetite to promote consumption (Loper et al., 2015) via the “gut–brain axis,” (Boughter and Munger, 2013; Lee and Owyang, 2017) while simultaneously assisting in regulation of protein digestion through signaling mechanisms that promote gastric secretion (Hartley et al., 2019; Raka et al., 2019). Several types of GluRs are involved in umami taste including iGluRs: NMDA and kainic receptor and mGluRs: (T1R1 (taste receptor type 1 member 1), T1R3 (taste receptor type 1 member 3) and taste-specific isoforms of mGluR, mainly mGluR4 and mGluR1, but also mGluR2 and mGluR3) (Sta nska and Krzeski, 2016; Hartley et al., 2019). A syndrome occurring after eating at Chinese restaurants was referred as the ‘Chinese restaurant syndrome,’ and was associated with the use of MSG in the food. The Federation of American Societies of Experimental Biology (FASEB, 1995) reviewed and renamed the syndrome as ‘MSG symptom complex.’ Clinical symptoms

described included burning sensation in the back of neck, forearms, and chest; facial pressure/tightness; chest pain; headache; nausea; palpitations; numbness in back of neck radiating to arms and back; tingling, warmth, weakness in face, temples, upper back, neck, and arms; bronchospasm (observed in asthmatics only); drowsiness; weakness. Affected individuals often only reported one or a few of the symptoms at any one time (Thuy et al., 2020). The safety assessment of glutamate salts, including MSG, estimated an NOAEL of 3200 mg MSG/kg bw per day and applied an uncertainty factor of 100. A recommended ADI of 30 mg/kg bw per day, expressed as glutamic acid, for glutamic acid and glutamates was developed (Mortensen et al., 2017). Still there is disagreement as to what studies should be required to ensure MSG food safety (Roberts et al., 2018; Zanfirescu et al., 2019; Thuy et al., 2020). Domoic Acid Domoic acid is structurally similar to another known toxin, kainic acid (Ka). Both are excitatory amino acid analogues of glutamate. DA induces excitotoxicity by an integrative action on iGluRs at both sides of the synapse for which it has high affinity, preferentially the Ka subtype, coupled with an effect that prevents the channel from rapid desensitization. A synergistic effect of DA with endogenous glutamate, and NMDA receptors agonists, on excitotoxicity, has been demonstrated in vitro and in vivo (Pulido, 2008; Pulido and Gill, 2013) Gulf War Illness Excitatory neurotransmission is mediated through the activation of iGluRs in the CNS and in multiple organ systems outside the CNS (Rousseaux, 2008a,b; Pulido, 2008; Pulido and Gill, 2013). Excitatory neurotoxicity through the over activation of iGluRs has been implicated in the Gulf War Illness (GWI), a disorder common to veterans of the first Gulf War (GW). It is characterized by multiple symptoms including widespread chronic pain, fatigue, cognitive dysfunction, sleep issues, and gastrointestinal problems (Joyce and Holton, 2020). There is a possibility that a diet low in glutamate may help to improve the symptomatology associated in some neuropsychiatric disorders (Kraal et al.,

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2020): chronic pain, fatigue, and sleep difficulties (Janulewicz et al., 2019) and the gut microbiome may play a role (Keating et al., 2019). Analysis of the scientific literature supports the view that multiple neurotoxic exposures, acting through a series of cellular mechanisms, lead to glutamatergic excitotoxicity and oxidative stress, and in turn are responsible for the multiple symptoms observed in GWI (Joyce and Holton, 2020). The hypothesis that reduction of glutamatergic activity ameliorates GWI symptoms has been supported by the observation that a diet low in glutamate improved pain and other symptoms in some of these GWI patients. A low glutamate diet is described as a whole food diet that restricts the consumption of free glutamate (Joyce and Holton, 2020). Glutamate occurs in the diet in bound and free forms. Free forms can be found as MSG, and naturally occurring in foods such as soy sauce, aged cheeses, seaweed, mushrooms, and tomato sauce. Free aspartate, an analogue of glutamate, capable of activating the same receptor, is also restricted in the diet. Aspartate is found in the artificial sweetener aspartame, hydrolyzed proteins, and gelatin. The diet used in the Joyce and Holton study optimized the consumption of specific nutrients known to protect against glutamate excitotoxicity and oxidative stress, including vitamin C, magnesium, vitamin D, and omega-3 fatty acids. Although further investigation is required, a diet low in glutamate with the addition of the above supplements opens new avenues to potential dietary management of GWI and other conditions where glutaminergic excitotoxicity has also been implicated, e.g., neuropsychiatric disorders (Kraal et al., 2020). Increased awareness and labeling identification of MSG, aspartame and related compounds in prepacked foods would facilitate consumers’ dietary management of potential adverse effects to these compounds. These food additives are widely used in the food industry as flavor enhancers; their potential association with the symptoms described in GWI, require awareness and further research. Serotonin PSILOCYBINdMUSHROOMS

Some contain

mushrooms Psilocybin.

(Magic Mushrooms) Psilocin is the

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pharmacologically active metabolite of psilocybin, and because of its structural similarity to serotonin, psilocin stimulates serotonin receptors in the CNS having an affinity for 5-HT1A, 5HT2A, and 5-HT2C receptors. Activation of 5HT2A receptors leads to increased cortical activity via Glu excitatory postsynaptic potentials. Activation of 5-HT1A receptors results in the inhibition of pyramidal cell activity. In addition, psilocin may have peripheral effects that involve serotonergic receptors (Dasgupta, 2019). In humans, psilocin’s psychoactive effects are similar to those produced by LSD, are observed within 20–30 min of ingestion, and include visual hallucinations, intensified hearing, and incoordination. Other autonomic-mediated effects include increased heart rate, increased blood pressure, mydriasis, tremors, and increased body temperature. The effects can last up to 8 h, but hallucinogenic activity rarely exceeds 1 h. OTHER HALLUCINOGENS THAT MAY CONTAMINATE FOOD

There is now converging evidence from biochemical, electrophysiological, and behavioral studies that the two major classes of botanical psychedelic hallucinogens, the indoleamines (e.g., psilocybin) and the phenethylamines (e.g., mescaline), have a common site of action as partial agonists at 5-HT2A and other 5-HT2 receptors in the central nervous system. The noradrenergic locus coeruleus and the cerebral cortex are among the regions where hallucinogens have prominent effects through their actions upon a 5-HT2A receptors (Aghajanian and Marek, 1999). Cannabinoids Cannabis and its use in everything from baked items to sauces has become popular (Figure 2.15). What was once thought of as far outside the mainstream has become something people are more comfortable with as the world moves to legalize the recreational use of marijuana, e.g., legalization in Canada (Health Canada, 2018a,b) and the US Regulation of Cannabis and Cannabis-Derived Products, including cannabidiol (CBD) (FDA, 2021c) in medical research. Cannabis is described as a flowering diecious plant indigenous to Central Asia. There are three

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FIGURE 2.15 Cannabis infused foodsdgummy bear candy (Kubala, 2021). Food related cannabis preparations: Edibles (https://www.nevercashed.com/edibles). Reprinted with permission (Accessed November 7, 2021).

common species of the Cannabis plant: Cannabis sativa, Cannabis indica, and Cannabis ruderalis. Cannabis contains hundreds of chemical substances, many pharmacologically active, known as ‘cannabinoids’ (see Herbal Remedies, Vol 3, Chap 4). Although Cannabis sativa L. (Cannabaceae) is the best studied source of cannabinoids, other flowering plants, liverworts, and fungi also contain them (Gu¨lck and Møller, 2020). The mammalian brain cannabinoid receptors (CBx) are the basis of the endocannabinoid system. which regulates a broad range of biological functions, including memory, mood, appetite, brain reward systems, and drug addiction, as well as metabolic processes such as lipolysis, glucose metabolism, and energy balance (Carter, 2020; Gu¨lck and Møller, 2020). Cannabis oral consumption leads to the activation of Cannabis receptors CB1 and CB2dboth coupled to a G protein (Childers et al., 1993)d located in the central and peripheral nervous

system, including the GI tract, and vagal and spinal neurons, leading to modulation of inflammation or repair and of the GI (Cohen and Neuman, 2020). CB1 cannabinoid receptors appear to mediate most, if not all of the psychoactive effects of d9-tetrahydrocannabinol [dTHC] and related cannabinoid compounds. This G-proteincoupled receptor has a characteristic distribution in the nervous system: It is particularly enriched in the cortex, hippocampus, amygdala, basal ganglia outflow tracts, and cerebellum, a distribution that corresponds to the most prominent behavioral effects of cannabis (Mackie, 2008). Cannabinoid CB2 receptors have only been detected outside the central nervous system, mostly in cells of the immune system, presumably mediating cannabinoid-induced immunosuppression and antiinflammatory effects (Patel et al., 2010). With the discovery of cannabinoid receptors for exogenous cannabinoids, endogenous cannabinoids (anandamide, 2arachidonylglycerol [2-AG]) have been described subsequently (Mackie, 2005; Nocerino et al., 2000). Interestingly, CB receptors have been found in some nematodes, onychophorans, and crustaceans (McPartland et al., 2006), indicating its importance probably from Jurassic times. The endocannabinoid system modulates the Glu system. For example, both the serotonergic and endocannabinoid systems modulate frontocortical Glu release and cannabinoid CB1 receptor antagonists, rimonabant (SR141716) and AM251, directly potentiate GABAA receptors inferring that CB1 receptor agonists may do the reverse; thus, damping the excitatory effects of Glu gives the endocannabinoids a neuroprotective property via negative signaling through the G-protein-coupled cannabinoid receptors (Rousseaux and Greene, 2016). Endocannabinoids not only act at cannabinoid receptors, but potentially at vanilloid and serotonin 5-HT3 receptors, both of which are expressed in the gastrointestinal tract. Additionally, experimental evidence suggests that endocannabinoids mediate neuron-astrocyte communication (Navarrete and Araque, 2008). The safety/risk assessment of oral use of cannabinoids is a challenge due to issues such as extract potency and type, doses, and concentration of active compounds (Rousseaux and

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Schachter, 2003). The introduction of synthetic cannabis products, the potential interaction with food compounds, alcohol, caffeine, and other extracts already added to some commercial beverages like beer and power drinks (Kubala, 2021), further complicates the issue for regulators, the public, and healthcare providers.

7.3. Channel Blockers Animal venoms (scorpions, snakes, cone snails, worms, sea anemones, frogs, wasps, etc.) are invaluable sources of pharmacologically active compounds with various molecular targets. Among venom compounds, peptide toxins target the diverse ion channels (potassium, calcium, sodium, and chloride channels). These molecules are often highly potent, somewhat selective and sometimes have a potential therapeutic value depending on their cellular targets. Some peptide toxins are structurally optimized to be developed as candidate drugs to treat pain and specific human pathologies, such as autoimmune and neurological disorders. Many marine toxins that may contaminate food are channel blockers, an understanding of which has led to the development of ion blocking cardiac medications, e.g., Nifedipine a calcium channel blocker (see Animal Toxins, Vol 3, Chap 8). Saxitoxin The phycotoxin (saxitoxin (STX)) may accumulate in filter feeding mollusks and cause toxicity following ingestion (see section 3.3). The target of STX, the agent that causes PSP, is the voltage-gated sodium channel in nerve and muscle cells, to which it binds with high affinity and can result in death via respiratory paralysis. This effect is reversible. One STX molecule per sodium channel effectively blocks the inward flow of sodium ions into the cell, with guanidinium able to act as a cationic substitute for the sodium ion. STX also binds to the human potassium channel modifying channel gating rather than blocking the channel. Finally, STX acts on voltage-gated calcium channels, though the blockage is not complete as in sodium channels (Cusick and Sayler, 2013). Tetrodotoxin Tetrodotoxin (TTX) is produced not only by puffer fish but also by bacteria and reaches

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various species of animals via the food chain (Narahashi, 2008). TTX inhibits voltage-gated sodium channels in a highly potent and selective manner without effects on any other receptor and ion channel systems. TTX blocks the sodium channel only from outside of the nerve membrane and is due to binding to the selectivity filter resulting in prevention of sodium ion flow. It does not impair the channel gating mechanism.

7.4. Endocrine Modifiers Goitrogens Goitrogens are naturally occurring substances that can interfere with the function of the thyroid gland. Thyroid enlargement is in response to inadequate T4 production cause by goitrogens. Naturally occurring goitrogens are found in legumes, cabbage, cauliflower, broccoli, turnip, and forms of root cassava. Soy, or soy enriched foods, can also aggravate thyroid problems reducing T4 absorption and interfering with thyroid hormone action, and are reported to increase auto-immune thyroid disease (see Endocrine system, Vol 4, Chap 7). Cruciferous plants (genus Brassica), as previously mentioned, are goitrogenic because they contain glucosinolates that are converted in the GI to glucose and isothiocyanates by the enzyme myrosinase derived from the plant or from the intestinal tract. Thiocyanates, perchlorates, and certain other ions compete with iodide for uptake by thyroid follicular cells. Phenobarbital, rifampin, and certain other medicinal compounds are goitrogenic because they increase the degradation of T4 and T3 (Rao and Lakahmy, 1995). Phytoestrogens Dietary phytoestrogens are bioactive compounds with estrogenic activity. With the growing popularity of plant-based diets, the intake of phytoestrogen-rich legumes (especially soy) and legume-derived foods has increased. Evidence from preclinical studies suggests these compounds may have an effect on hormones and health, although the results of human trials are unclear. The effects of dietary phytoestrogens depend on the exposure (phytoestrogen type, matrix, concentration, and bioavailability),

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ethnicity, hormone levels (related to age, sex, and physiological condition), and health status of the consumer. Sex hormone alterations have been found in the late stages of childhood but in premenopausal and postmenopausal women, the reported impacts on hormones are inconsistent. In adult men, different authors report goitrogenic effects and a reduction of insulin in nonalcoholic fatty liver patients (Domı´nguezLo´pez et al., 2020). Interestingly, phytoestrogens can be a veterinary problem. Wethers fed Yarloop clover (Trifolium subterraneum) lactated, and the ewes did not ovulate (Chamley et al., 1985). Estrogenic Mycotoxins Zearalenone is a nonsteroidal estrogenic mycotoxin, which is one of the over 400 mycotoxins produced by several species of Fusarium fungi (see Mycotoxins, Vol 3, Chap 6). Contaminated products can lead to huge economic losses in livestock and pose endocrine risks to animals and humans (Minervini and Dell’Aquila, 2008). Zearalenone, a xenoestrogen, is an exogenous compound which resembles the structure of naturally occurring estrogens. This property of zearalenone determines its ability to bind to estrogen receptors of the cell and its bioaccumulation, which leads to disorders of the hormonal balance of the body. The consequence of excessive estrogenic stimulation may lead to numerous diseases of the reproductive system such as prostate, ovarian, cervical or breast cancers (Rogowska et al., 2019). The use of implants of the widely used anabolic growth promotor zeranol (alphazearalanoldbased on zearalenoned“Ralgro”) in food animals results in very low residues of this compound in the edible tissues. In comparison, residues of myco-estrogens, specifically the beta-resorcylic acid lactones (RALs), such as zearalenone and its metabolites, are commonly found as contaminants of cereals and are greater than those for zeranol. Zeranol, the RALs and their metabolites are not carcinogenic, teratogenic, or mutagenic (Baldwin et al., 1983) and the oral rat LD50 exceeds 40 g/kg. No toxicological effects have been reported other than those attributed to the estrogenic character of these molecules. In comparison with estradiol, zeranol and the related myco-estrogens are 100–1000 times less

active hormonally. It is concluded that a likely no-observed-effect level (NOEL) for these compounds will be of the order of 0.05–0.1 mg/ kg body weight/day. This would result in a safety margin greater than 500-fold between the expected dietary exposure to these compounds (considered together) and the likely NOEL. On the basis of exposure to zeranol alone, this margin of safety would increase to more than 35,000 (Lindsay, 1985). Although the concentration of zeranol declines steadily following removal of exposure to zeranol, it can be readily detectable 120 days following cessation of treatment (Dixon et al., 1986).

8. SAFETY ASSESSMENT OF FOOD Food can be a vehicle for hazardous substances or pathogens that can negatively affect human health. Risk–benefit assessment (RBA) of foods integrates nutrition, toxicology, microbiology, chemistry, and human epidemiology for a comprehensive health impact assessment (Figure 2.16). By integrating health risks and benefits related to food consumption, RBA facilitates science-based decision-making in food-related areas and the development of policies and consumer advice. Two major approaches to food safety can be applied: the bottom-up (estimating the disease incidence due to the exposure) and the top-down (using epidemiological and incidence data to estimate the number of cases attributable to a certain exposure) (National Food Institute, 2019).

8.1. Risk/Safety Assessment in Food At the international level, the evaluation of food additives was initiated as a result of an FAO/WHO Conference on Food Additives held in Geneva, Switzerland in 1955. The Conference recommended that expert committees should be convened to address the safety of chemical additives in food. The outcome of the recommendation was the establishment of an international expert scientific committee known as the Joint FAO/WHO Expert Committee on Food Additives (JECFA), which has been meeting since 1956, to provide independent scientific expert advice on the safety of food additives. However,

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FIGURE 2.16

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Risk assessment matrix (Author’s diagram).

JECFA does not have universal jurisdiction (FAO, 2006). The Codex Alimentarius Commission (CAC) is an international body that sets standards on food products that are traded internationally based on the advice of JECFA. The CAC has established committees on various food products or food-related issues (e.g., hygiene, labeling), including a Committee on Food Additives (FAO, 2007). The Codex Committee on Food Additives (CCFA) established prioritization criteria for the reevaluation of food additives previously evaluated by JECFA. The FAO/WHO Codex Alimentarius Commission provides guidance to national governments for risk assessment, risk communication, and risk management pertaining to food safety (see also Risk Assessment, Vol 2, Chap 16; Risk Communication and Management: Building Trust and Credibility with the Public, Vol 2, Chap 17). With regard to the risk characterization of micronutrients (vitamins and minerals), adverse effects arising from low (deficiency), and high (toxicity) intakes must be considered (see Nutritional Toxicologic Pathology, Vol 3, Chap 3). In some cases, normal constituents of food can present a health hazard to susceptible populationsdfor example, peanut anaphylaxis.

8.2. Food Additives Acceptable Daily Intake The goal of the toxicological testing of a food additive is to determine the acceptable daily

intake (ADI) of the additive (Figure 2.17). The ADI is compared to the estimated daily intake (EDI) of the additive. Typically, if the EDI is less than the ADI (in the same units, such as mg/kg body weight/day), the use of the additive is considered to lie within the bounds of “reasonable certainty of no harm,” for its intended use in food (Choudhuri et al., 2019). Regulatory approaches must be followed to establish the safety of these substances, e.g., by setting the ADI level for any given substance (Lu, 1988; FAO, 2006; Choudhuri et al., 2019). ADIs are typically established after extensive testing, using a range of animal studies that generally include, but are not limited to, studies on the metabolic fate of a chemical, acute oral studies, lifetime carcinogenicity studies, and reproductive and developmental studies. Food Colors Red 3 causes cancer in animals, and there is evidence that several other dyes also are carcinogenic. Three dyes (Red 40, Yellow 5, and Yellow 6) have been found to be contaminated with benzidine or other carcinogens and at least four dyes (Blue 1, Red 40, Yellow 5, and Yellow 6) cause hypersensitivity reactions. Numerous microbiological and rodent studies of Yellow 5 were positive for genotoxicity. Toxicity tests on two dyes (Citrus Red 2 and Orange B) also suggest safety concerns, but Citrus Red 2 is used at low levels and only on some Florida oranges and Orange B has not been used for several years. The inadequacy of much of the testing and the evidence for carcinogenicity, genotoxicity, and hypersensitivity, coupled with the

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FIGURE 2.17 Food additives in food. https://www.sciencedirect.com/science/article/pii/B9780128194935000066 (Accessed November 19, 2021, with permission).

fact that dyes do not improve the safety or nutritional quality of foods, indicates that all of the currently used dyes should be removed from the food supply and replaced, if at all, by safer colorings (Kobylewski and Jacobson, 2012). Most synthetic food colors are “tar colors” originally derived from coal tar but now mostly manufactured synthetically. Tar colors can be divided into four groups based on their chemical structure: azo, triphenyl methane, xanthene, and sulfonate indigo. Most synthetic colors permitted in food are azo dyes, including allura red, tartrazine, sunset yellow, amaranth, and others; some triphenyl methanes (brilliant blue and fast green) and xanthenes (erythrosine) are also permitted. While food colors have been subjected to extensive toxicological testing

and are widely used, the scientific data on their potential for adverse effects such as hypersensitivity reaction in children are limited (Choudhuri et al., 2019).

8.3. Food Contaminants As described above, contaminants in food can come from the environment from raw material to the dining table, manufacturing and leaching of compounds from the container, toxins from biota, and animal medication. The effect of the health risk of residues must be determined for the consumer and quantified using animal dose–response and human exposure data.

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Residues

WITHDRAWAL PERIODS

MAXIMUM RESIDUE LIMIT

The withdrawal period (withholding period or for milk and eggs discard time) is the minimum period of time after the last dose that a veterinary drug has been administered that must elapse before an animal or foodstuff from an animal can enter the food chain, i.e., slaughter, taking milk, honey, or eggs for human consumption. The purpose of the withdrawal period is to ensure that foods do not contain residues of pharmacologically active substances greater than the MRL. The exact wording of the definition of the withdrawal period can vary between countries and/or regions but the principle is similar. Withdrawal periods are expressed in days (for tissues, milk, and eggs), milking’s (12h milking intervals), or degree days (fish). Withdrawal period is established through data from marker residue depletion studies that provide evidence that after a certain time the drug residues in tissues (muscle meat, fat, liver, kidney, etc.) are below the MRL (VICH GL48, 2016).

The maximum residue limit (MRL) is the maximum allowed concentration (expressed as mg/kg) of residue of a pesticide or veterinary drug in a food product that has been retained by an animalda residue. Residues are traces of pesticide or veterinary drugs in an animal. MRLs are based on safety data and primarily on the results of toxicity testing. The primary purpose of the MRLs for pesticides and veterinary drugs is to protect the health of those who consume food of animal origin (FAO, 2021c). The MRL is established by determining the ADI. The ADI is the amount of a residue that is considered safe for a person to eat every day for their lifetime considering a number of safety factors. The ADI is determined by identifying the no-observed-adverse-effect level (NOAEL) through a series of toxicology studies with the active substance. Once the NOAEL is determined, this figure is then divided by “uncertainty” or “safety” factors, e.g., 100–1000 in order to account for species extrapolation and intraspecies variability, etc. The established MRLs are such that consumers can ingest generous amounts of animal foodstuffs every day without exceeding the ADI. Factors that are considered for establishing the MRLs include: amounts of each food eaten per day and how the substance is metabolized and distributed in various tissues. An MRL is then set for each edible tissue and the product to ensure the ADI is not exceeded. In some countries/regions, MRLs are referred to as maximum residue limits and others as maximum residue levels. The term pesticide tolerance is also used by other authorities. The Global MRL database contains the maximum acceptable levels of pesticides and veterinary drugs in food and agricultural products in the United States, as well as 70 other countries, the European Union and the Codex Alimentarius Commission. The database includes fruit, vegetable and nut commodities as well as pesticides approved for use on those commodities by the USEPA. The database also includes pesticides and veterinary drug residue tolerances for hay, feed, grains, oilseeds, poultry, eggs, meat and dairy. A summary of international sources of MRLs is found in Table 2.4.

ANTIMICROBIAL RESISTANCE: MORE THAN RESIDUES

The emergence of drug resistance has been observed following the introduction of each new class of antibiotics, and the threat is compounded by a slow drug development pipeline and limited investment in the discovery and development of new antibiotic agents (Landers et al., 2012). The problem is rarely one of exceeding the MRL but rather spreading of antimicrobial resistance throughout the food chain resulting in a potentially untreatable pathogen in humans and animals. Recently, the World Health Organization called antimicrobial resistance (AMR) “an increasingly serious threat to global public health that requires action across all government sectors and society” (Martin et al., 2015; van Boeckel et al., 2017). Antimicrobial use in animals can, and has, contributed to the emergence of antimicrobial resistance in bacteria that may be transferred to humans, thereby reducing the effectiveness of antimicrobial drugs for treating human disease (FDA, 2021f). It is quite clear that interventions that restrict antibiotic use in food-producing animals are associated with a reduction in the presence of antibiotic-resistant bacteria in these animals (Tang et al., 2017). The bacterial

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TABLE 2.4 International Regulatory Documents for Maximum Residue Limits (FAO, 2021c) International Sources of MRLs International Source

Explanation

Link

Codex Alimentarius

MRLs are set by the codex committee on pesticide residues (CCPRs), based on recommendations made by the FAO/ WHO joint meeting on pesticide residues (JMPRs).

https://www.fao.org/fao-whocodexalimentarius/codex-texts/dbs/ pestres/en/

Global MRL database

The United States Department of agriculture (USDA) used to maintain an international database of MRLs. This database is now managed by Bryant christie Inc., as the global MRL database.

https://www.bryantchristie.com/

New Zealand MRL web page

The New Zealand Ministry of primary industries maintains a web page on pesticide maximum residue limit (MRL) legislation around the world.

https://www.mpi.govt.nz/ agriculture/plant-productsrequirements-and-pesticide-levels/ pesticide-maximum-residue-levelsmrls-for-plant-based-food-for-nz-andother-countries/pesticide-maximumresidue-level-legislation-around-theworld/

EU MRL database

The European commission sets its MRLs applicable in the EU (referred to as maximum residue levels), which are not always the same as codex MRLs.

https://ec.europa.eu/food/plant/ pesticides/eu-pesticides-database/ mrls/?event¼search.pr

USEPA pesticide residue tolerance

The US environmental protection agency (USEPA) sets pesticide residue tolerances applicable in the United States. The official publication of pesticide tolerances for the United States is in the e-code of federal regulations (e-CFR).

https://www.epa.gov/pesticidetolerances/how-search-tolerancespesticide-ingredients-code-federalregulations

Australian MRLs

Australian MRLs are published in the agricultural and veterinary chemicals code Instrument No. 4 (MRL Standard).

https://apvma.gov.au/node/10806

New Zealand food standards

The MRLs applicable in New Zealand are published in the New Zealand (maximum residue limits of agricultural compounds) food standards.

https://www.mpi.govt.nz/ agriculture/agricultural-compoundsvet-medicines/maximum-residuelevels-agricultural-compounds/

capability to face antimicrobial compounds has enabled bacteria to survive over time. Commonly, AMR traits are included in mobilizable genetic elements enabling the homogeneous diffusion of the AMR traits pool between the ecosystems of diverse sectors, such as human

medicine, veterinary medicine, and the environment (Palma et al., 2020), but the public are unaware of the importance of agricultural antimicrobial use as a factor in antimicrobial resistance even among experts in medicine and public health (Silbergeld et al., 2008; CDC, 2013).

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CLENBUTEROL: WITHDRAWAL BECAUSE OF RESIDUES

Clenbuterol is a selective b2 adrenergic agonist that has similar pharmacologic properties to structurally related compounds such as salbutamol and terbutaline. b2 adrenergic agonists are a class of drugs that act on the b2 adrenergic receptor. They cause smooth muscle relaxation and have effects on dilation of the bronchial passages, vasodilation in muscle and liver, relaxation of uterine muscle, and release of insulin. The primary use of this class of drugs in human medicine is to treat asthma and other pulmonary conditions. This class of drugs has several adverse effects that rise from excessive activation of the b2 adrenergic receptors. These effects include tachycardia, tremors, hypotension, and myocardial injury. In veterinary medicine clenbuterol is used for similar effects in horses with respiratory disease. It is also used as a tocolytic agent in cattle and horses to obstetrical procedures (Denooij, 1984) b2 adrenergic agonists are also repartitioning agents, which reduce fat deposition and increase muscle tissue (Bohorov et al., 1987). Therefore, they have been used as a growth promotant to obtain lean meat in food producing animals. The use of Clenbuterol as a growth enhancing agent has been prohibited in the United States, Canada, and European Union over concerns of residues in food of animal origin (Kuiper et al., 1998). Several outbreaks of food poisoning have occurred over the years including both consumption of meat and offal from illegally treated animals (CEHP, 2007). Symptoms noted in consumers included increased heart rate, muscle tremors, headaches, nausea, fever, and chills that resolved over days. As a result of its illegal use clenbuterol is a common drug included in residue surveillance plans for several countries. RBST: MINIMAL RESIDUES, WITHDRAWN BECAUSE OF ANIMAL WELFARE ISSUES

Bovine somatotropin (bST) is an anabolic growth hormone that is produced in the pituitary gland of animals and is important for normal growth and development. The anabolic and growth promoting effects of the somatotropins are mediated through insulin-like growth factor (IGF-1). rBST is recombinant bovine somatotrophin and is a synthetic version of the bovine somatotropin. bST has been used to increase

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milk production in lactating dairy cattle by preventing mammary cell death. The FDA approved the use of bST for lactating dairy cows in 1993 after concluding that it would be safe and effective. The FDA concluded that milk and meat from cows treated with bST is safe to eat. bST is a large protein and is degraded by digestive enzymes in the gastrointestinal tract and not absorbed intact. The digested breakdown fragments have no biological activity. Additionally, bST does not promote biological activity in humans. The FDA concluded that milk and meat from cows treated with bST is safe for human consumption at any time after the animal is treated (FAO/WHO, 2013). Although bST use is approved for use by the United States, FAO/WHO, and the National Institutes of Health (NIH), its use is currently banned in several countries including the European Union and Canada. According to an EU report, rBST substantially increased health problems in cows, such as mastitis, foot problems, injection site reactions, and reproductive disorders (EU, 1999). Concerns around possible health effects on humans consuming milk from cows treated with bST continue. One of the major concerns is the slightly higher concentration of IGF-1 in milk from cows treated with bST. IGF-1 in milk is not denatured by pasteurization; however, the extent of which intact active IGF-1 in absorbed through the human digestive tract remains uncertain. However, even if some of the IGF-1 in milk were absorbed, the incremental human exposure would be negligible when compared with total daily human production of IGF-1 of 10 mg/day. Functional Foods The safety of probiotics is tied to their intended use, which includes consideration of potential vulnerability of the consumer, dose and duration of consumption, and both the manner and frequency of administration. Unique to probiotics is that they are alive when administered, and unlike other food or drug ingredients, possess the potential for infectivity or in situ toxin production. Since numerous types of microbes are used as probiotics, safety is also intricately tied to the nature of the specific microbe being used. The presence of transferable antibiotic resistance genes, which comprises

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a theoretical risk of transfer to a less innocuous member of the gut microbial community, must also be considered. Genetic stability of the probiotic over time, deleterious metabolic activities, and the potential for pathogenicity or toxicity must be assessed depending on the characteristics of the genus and species of the microbe being used. Immunological effects should be considered, especially in certain vulnerable populations, including infants with undeveloped immune function. A few reports about negative probiotic effects have surfaced, the significance of which would be better understood with more complete understanding of the mechanisms of probiotic interaction with the host and colonizing microbes. Use of readily available and low-cost genomic sequencing technologies to assure the absence of genes of concern is advisable for candidate probiotic strains. Unfortunately, there is a dearth of probiotic safety studies specifically designed to assess safety contrasted with the long history of safe use of many of these microbes in foods (Sanders et al., 2010). Genetically Modified Plants and Organisms Foods derived from genetically modified (GM) plants and organisms (GMOs) are widely consumed in many countries and offer possible solutions to meet current and future challenges in food and medicine. In the field, evidence of toxicity has not been established but yields and food quality has improved. Yet, there is a strong undercurrent of anxiety that GM and GMOs are unsafe for human consumption, sometimes fueled by an irrational belief contrary to the scientific evidence. The unfortunate result is that some countries are turning away GM and GMOs destined for famine relief because of the perceived health risks. The major concerns include their possible allergenicity and toxicity despite the vigorous testing of genetically modified foods prior to marketing approval (Lee et al., 2017).

9. REGULATION OF FOOD 9.1. History of Food-Related Disease Concerns around food safety and quality have existed since the beginning of civilization. The original focus of governments was to ensure

the purity of the nation’s food supply and protect against the sale of unsafe food and economic fraud. In 375 BCE, food adulteration was discussed in the book “Arthrashastra” by Chanakya, an Indian teacher and philosopher. Indeed, there is evidence that the Egyptians, Chinese, Greeks, and Romans preserved and labeled food thousands of years ago (Mahmoud, 2020). Food preparation techniques such as cooking, drying, salting, canning, fermentation, and freezing were developed to reduce foodborne illness and food spoilage, as foodborne outbreaks have plagued humankind throughout the centuries. Historical accounts of foodborne illness date back to 323 BCE when Alexander the Great died of typhoid fever caused by Salmonella typhi. In 1692, toxic fungus (Claviceps Purpurea) in rye grain was considered a possible cause of the convulsive symptoms leading to the Salem witchcraft trials (Caporael, 1976) and the painful effects of ergotism in St Anthony’s fire (Zavaleta et al., 2001). Much later, US President Zachary Taylor was sickened and died from ingesting potato salad contaminated with Salmonella at a picnic in 1850. During the Spanish War in 1898, more than 20,000 American soldiers were sickened by contracting typhoid fever (Michell, 2014).

9.2. History of Food Regulation As food production moved from the homestead using raw locally obtained materials to manufactured products (e.g., bread), sanitation, hygiene, and food safety legislation were developed to protect commerce and the adulteration of food (Jarvie, 2014). The first English food law, Assize of Bread and Ale Law, was proclaimed in 1202 by King John (Ross, 1956). This law prohibited the adulteration of bread with ingredients such as ground peas or beans and managed the weight, quality, and price of the bread and ale. American colonists passed the first US food safety law in 1785, Massachusetts Act Against Selling Unwholesome Provision (Michelle, 2014). A great percentage of foods including tea, coffee, and milk sold in the United States and Canada were adulterated at that time (Mahmoud, 2020). The law referenced “whereas some evilly disposed persons, from motives of avarice and filthy lucre, have been induced to

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sell disease, corrupted, contagious, or unwholesome provisions, to the great nuisance of public health and peace” should be punished by fine or imprisonment (Fortin, 2017). Similarly, in Canada the Inland Revenue Act of 1875 was the earliest Canadian law to protect the public against adulteration of drinks, food and drugs. Amended in 1884, it became known as the Adulteration Act. The Adulteration Act was replaced by the 1920 Food and Drugs Act. The Food and Drugs Act and Regulations were completely revised in 1949 and given Royal Assent in 1953. A high incidence and prevalence of foodborne illnesses such as tuberculosis, typhoid fever, and botulism were associated with increased morbidity and mortality rates globally in the early 1900s. In addition, there were growing concerns around the presence of colorants and preservatives in food. In 1902, Dr. Harvey Wiley, the chief chemist of the United States Department of Agriculture (USDA)’s Bureau of Chemistry, recruited volunteers for the “poison squad.” This “poison squad” would eat foods that contained new chemical preservatives. After observing the sickness in the squad members, Dr. Wiley concluded that chemical preservatives should only be used when necessary (Twilley and Graber, 2018). The responsibility for ensuring the safety of the foods was placed on the producers, and they were to inform the consumers of the food ingredients on food labels. The “Jungle,” written by Upton Sinclair, published in 1906, was a fictional novel describing immigrants’ lives in industrialized cities. Sinclair spent approximately 9 months undercover at a meatpacking plant in Chicago researching for the book. The book raised public concerns around the health and safety of the Chicago meatpacking industry. After reading this book, Theodore Roosevelt pressed for the US Congress to pass the Pure Food and Drug Act and the Federal Meat Inspection Act of 1906 (Khomina, 1906). These were the first US food laws that addressed both the public food supply and food safety and are still in place today. Increasing regulatory attention was given to food adulteration and mislabeling. Food chemistry became a recognized field during this period, and the confirmation of the “purity” of food was based on the chemical properties of simple food composition. Chemistry, however, posed other

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problems when industrial chemicals were used to preserve or color foods or to disguise their true nature. Food preservatives such as formaldehyde and borax were added to foodstuffs to hide unsanitary production practices. The increasing and often insufficiently controlled use of food additives became a matter of public concern throughout the world. In Canada, the Inland Revenue Act of 1875 was the earliest Canadian law to protect the public against adulteration of drinks, food and drugs. Subsequently, a report on the subject of preservatives and coloring ingredients in food was presented at a meeting of the British Medical Association in 1905. The report resulted in the passage of the Adulteration Act, under which the first list of additives prohibited for use in food was published (Pugsley, 1960). This Act was replaced by the 1920 Food and Drugs Act and later the Food and Drugs Act and Regulations in 1953. In Australia, a report by a Victorian Government Analyst reviewed colors, preservatives, flavors, saccharin, and alum. This report led to the formation of the Australian Pure Food Act in 1905. By 1912 most of the other states in Australia followed with similar legislation. After World War II, it became apparent that improved agricultural trade would be important to aid reconstruction efforts and provide people with safe food (Randel, 2021). The first Food Agriculture Organization of United Nations (FAO) conference was held in Hot Springs, Virginia, in 1943. During this conference, the need for international standards for nutrient content and purity of imported foods was recognized (Figure 2.18). The FAO was founded in 1945 and the World Health Organization in 1948. After their foundation, the two organizations started to have joint expert meetings discussing nutrition and other related areas. “Food regulations in different countries are often conflicting and contradictory. Legislation governing preservation, nomenclature, and acceptable food standards often varies widely from country to country. New legislation not based on scientific knowledge is often introduced, and little account may be taken of nutritional principles in formulating regulations.” At the 1955 Joint FAO/WHO meeting on food additives (Figure 2.19) the conference recommended that one or more expert committees be convened to address the scientific aspects of chemical

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FIGURE 2.18 Food regulations timeline showing the significant events and milestones in food regulation over time (Author’s diagram).

additives and their safety in food (Figure 2.19). The first meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) was held in 1956 (FAO/WHO, 2006). In 1958, the Food Additives Amendment was passed in response to public concerns about the increased use of chemicals in foods. The amendment required that before a producer could use a new food additive, they had to demonstrate its safety. Congress recognized that many

additives should not require formal premarket review by the FDA to assure their safety because the safety had already been established or by the nature of the substances. This exemption was called Generally Recognized as Safe (GRAS). In addition to the GRAS exemption, the Food Additives Amendment included a clause called the Delaney clause that prohibits any new food additive shown to cause cancer in humans or animals from entering the food chain (FDA, 2018d).

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FIGURE 2.19 The scientific basis of the Joint FAO/WHO Expert Committee on Food Additives. The Codex Alimentarius Commission (CAC) is an international body that sets standards on food products that are traded internationally based on the advice of JECFA. Author’s diagram based on JECFA https://www.who.int/groups/joint-faowho-expert-committee-on-food-additives-(jecfa)/about. (Accessed November 22, 2021).

The government invoked the Delaney clause during the Thanksgiving Cranberry scare of 1949 (Janzen, 2010). The government issued a food warning on a cranberry batch that had traces of the carcinogen, aminotriazole, a weed killer found to cause cancer in rodents. Americans across the nation panicked, and the cranberry industry suffered significant setbacks. Even the Whitehouse served applesauce instead of cranberry sauce at the Thanksgiving table that year. In this same year the FDA published its first guidance for industry “Procedures for the Appraisal of the Toxicity of Chemicals in Food” which later became known as the “black book.” The guidance became a means for the regulatory agency to influence industry practices without mandating specific standards. Numerous children became ill in the fall of 1950 due to ingestion of Halloween candy containing the color additive 1%–2% FD&C Orange No.1. This event and concerns around the possible carcinogenicity of pesticide residues in food led to the passage of the Color Additive Amendment of 1960 (Barrows et al., 2017). The amendment required that only color additives listed as “suitable and safe” could be used in foods.

In 1960 the National Aeronautics and Space Administration (NASA) and Pillsbury Company collaborated to provide safe food for upcoming space expeditions. They developed the Hazard Analysis and Critical Control Points (HACCP), a systematic approach to hazard identification, risk assessment, and control through this collaboration. This system was successful in assuring the safety of food for space expeditions. Pillsbury also utilized this system to manage a recall on their infant cereal product that had pieces of glass in the food. In 1971 the FDA requested that Pillsbury establish and manage a training program to inspect canned foods for FDA inspectors. This training program was the first time that HACCP was used to educate food producers in the industry (Safe Food Alliance Team, 2019). Codex Alimentarius Commission, a joint commission of FAO/WHO, held its first session in Rome in 1963 (Davies, 1970). The commission developed science-based international food quality standards, in which there are numerous definitions of foodstuffs and additives, restrictions on food composition, limits on pesticide and veterinary drug residues, restrictions on contaminants, and requirements for labeling. Codex standards serve as the basis for many of

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the member nations’ food regulations to this day (Shyam, 2016). In 1978 a major infant formula manufacturer reformulated its soy protein-based formulas (Levin, 1987). However, the reformulation had an inadequate amount of the essential nutrient chloride. Infants who were fed the formula had a chloride deficiency, which resulted in hypochloremic metabolic alkalosis. Infants showed signs of poor appetite and weight gain, diarrhea, and hematuria. The formula was eventually recalled, and the event received considerable media attention. The incident led to the amendment of the FDCA by the passage of the Infant Formula Act. This act required manufacturers to follow strict safety, quality, and analytical regulations to ensure all formulas comply with nutritional requirements. In the early 1960s, there were several public hearings in Congress investigating the information on food labels and other consumer products. Congress heard numerous complaints from consumers regarding confusing or deceptive labeling and packaging. In response, the Fair Packaging and Labeling Act of 1966 was passed to ensure that the Consumer Protection Act requires that all consumer commodities be labeled to disclose contents to facilitate value comparisons between competing products (Forte, 1968). In 1970 the Center for Disease Control (CDC) began recording foodborne illness-related outbreaks and deaths in the United States. The first nationwide recall on food was in 1973 due to canned mushrooms that contained botulism toxin (NYT, 1973). This incidence of botulism contamination led to the formation of the National Botulism Surveillance System, and the system collected data on confirmed cases of botulism in the United States. The mushroom recall due to botulism led to the low-acid food processing regulations. As consumers began to use increasing amounts of processed and packaged foods and spent less time preparing home-cooked meals from basic ingredients, they requested more information to help them understand the products they purchased (Kennedy and Dwyer, 2020). In addition, there was an increase in consumer interest in nutrition due to several reports highlighting the relationship between diet and the leading causes of death. Food

manufacturers took advantage of the consumer desire for additional health and nutrition information. They often used undefined claims on product labels that implied the unique value of the food, such as “extremely low in saturated fat,” to highlight the positive nutritional attributes of food products (Taylor and Wilkening, 2008). The Surgeon General’s Report on Nutrition and Health in 1988 called for the food industry to reform products to reduce the total fat and place nutrition labels on all foods. In 1990 the Nutrition Labeling and Education Act (NLEA) was passed in response to increasing concerns about food labeling. This Act was the most significant food labeling legislation in 50 years. It required that all packaged food have nutrition labeling and all health claims for foods be consistent with terms defined by the Secretary of Health and Human Services (Wartella et al., 2010). In 2009, a multistate outbreak of Salmonella typhimurium infections that sickened over 714 Americans across 46 states was linked to peanut butter (CDC, 2009). This outbreak among increasing incidents of foodborne-related illness in the 2000s led to the passage of the Food Safety Modernization Act. US president Barak Obama signed the Food Safety Modernization Act (FSMA) into law in 2011 and is considered the first significant piece of legislation addressing food safety since 1938 when the Pure Food and Drug Act was revised. The FSMA focused more on preventing food safety issues rather than reacting to problems as they occur. Under the FSMA, the FDA was given authority to regulate how food is grown, harvested, and processed and greater regulatory authority to issue a mandatory recall if a company fails to recall unsafe food voluntarily. The FSMA calls for food facilities to implement a Food Safety Plan after conducting a Hazard Analysis of RiskBased Preventative Controls (HARPC), similar to the HACCP model (NALC, 2021). Different countries had varying approaches to managing food safety; however, a common theme throughout all regulations was establishing standards to provide a clear concept of what is and is not acceptable in the system or process being regulated. Excluding diseased, disabled, and dying animals from the food supply was necessary for the food animal industry. Ensuring the exclusion of harmful

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chemicals (both additives and residues), decreasing food contamination by microorganisms, developing food safety standards and practices, and properly labeling food have been the mainstays of food regulations over time (Figure 2.20).

9.3. Food Regulations Around the World Food may contain a wide range of chemicals from various sources that were intentionally or unintentionally added to or are part of food, thus, contaminating the food and rendering it a potential health hazard. Knowledge of the chemical properties of toxic substances in food and understanding of their biological effects, mode(s) of action, and pharmacokinetics (absorption, distribution, metabolism, bioaccumulation, and elimination) are essential in assessing chemical safety (Woteki and Buckley, 2020). Regulations apply to all stages of production, processing and distribution of food and feed. The purpose of food regulations is to minimize the risk of unsafe food (it is too costly to assure that food is safe) and that consumers have the

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information needed to make an informed decision. In most countries, they also guarantee fair practices in food trade, considering animal health and welfare, plant health, and the environment. To achieve this goal, most Acts and Regulations focus on two areas, “safety” and “sale,” and may use the Precautionary Principle in the assessment (Figure 2.21). The Precautionary Principle is a contemporary redefinition of Bradford Hill’s case for action. It gives us a commonsense rule for doing good by preventing harm to public health from delay. When in doubt about the presence of a hazard, there should be no doubt about its prevention or removal (Richter and Laster, 2004). Regulation and Approval of Foods Meant for Human Consumption UNITED STATES

In the United States, the term “food products” is governed by the Federal Food, Drug, and Cosmetic Act (FD&C Act), and the regulations issued under its authority. Any article that is intended to be used as an animal food

FIGURE 2.20 An overview of the regulatory aspects of food safety and quality, at the core of which is inspection and enforcement (Author’s diagram).

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FIGURE 2.21

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The regulatory framework and registration for select areas of the globe (Author’s diagram).

ingredient, to become part of an ingredient or food, or added to an animal’s drinking water is considered a “food” and subject to regulation (FDA, 2019b, 2021d). There are nearly a dozen federal agencies and greater than 35 statutes that govern the food safety regulations. However, four agencies play the primary role in implementing food safety regulations: the Food Safety and Inspection Service (FSIS) of United States Department of Agriculture (USDA), Food and Drug Administration (FDA), Environmental Protection Agency (EPA), and the Center for Disease Control (CDC) (Foodsafety, 2019) (Figure 2.22). FOOD AND DRUG ADMINISTRATION Except for meat and poultry products, the FDA has jurisdiction over domestic and imported foods that are marketed by interstate commerce. The FDA protects public health by assuring that foods are safe, wholesome, sanitary, and properly labeled. The US food law is not limited to human food and includes food and drink meant for pet food and animal feed. The Center for Food Safety and Applied Nutrition (CFSAN) is a subsidiary of the FDA with

jurisdiction over food processing plants and food and animal feed additives (IOM, 1998). It is responsible for approving and monitoring veterinary food-animal medications. The CFSAN also enforces tolerances for pesticide residues on food that the EPA sets. The CFSAN has jurisdiction over restaurants but cedes this responsibility to the state and local authorities. USDA FSIS monitors state inspection programs that conduct inspections of meat and poultry products sold within state boundaries where they are produced. The first regulatory body for food safety was the USDA called the “The People’s Department” founded by President Lincoln in 1862 to “provide leadership on food, agriculture, natural resources, rural development, nutrition, and related issues based on sound public policy, the best available science and efficient management.” (USDA, 2014). The Food Safety and Inspection Service (FSIS) of USDA ensures that meat and poultry products are safe, wholesome and correctly labeled and packaged. It gets its authority form the Federal Meat Inspection Act (FMIA), Poultry Products Inspection Act (PPIA), and Egg Products Inspection Act (EPIA). FSIS

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FIGURE 2.22 The United States food safety system, the core of which relies on inspection and enforcement (Author’s diagram).

shares responsibility with the FDA for safety of intact-shell eggs and processed egg products. FSIS inspectors are responsible for inspection of meat and poultry slaughtering processes, which is mandatory for animals (cattle, swine, goats, sheep, horses, chickens, turkeys, ducks, geese, and guineas) used for human consumption. FSIS inspects all raw meat and poultry sold for interstate and foreign commerce, including imported foods. It also monitors meat and poultry products once they leave federally inspected plants and tests samples of egg and meat and poultry products for microbial and chemical contaminants. ENVIRONMENTAL PROTECTION AGENCY The Environmental Protection Agency (EPA) regulates pesticide products in the United States. The Federal Insecticide, Fungicide, and Rodenticide Act of 1947 (FIRFA) regulates the registrations and use of pesticides, protection of the environment, and safety to the user. Initially, the USDA was the agency that managed FIRFA; however, in 1970, the EPA began overseeing the Act (EPA, 1996b). The EPA establishes tolerance for pesticide residues in or on food and animal feed. EPA also manages protecting the

environment from chemical and microbial contaminants that may pollute the air and water and interfere with food safety (IOM, 1998).

CENTER FOR DISEASE CONTROL The Center for Disease Control’s (CDC) primary role in food safety is surveillance and investigation into the cause and source of associated foodborne illnesses. They partner with the FDA and USDA FSIS agencies to implement programs to prevent foodborne illness and identify and stop outbreaks. The CDC has recently been using whole-genome sequencing as a tool to determine a “DNA fingerprint” of certain strains of bacteria, and they can use this “fingerprint” to trace the foodborne illness to the source of the outbreak. The CDC reports that between 2009 and 2018, the topmost common foods associated with illness were chicken, pork, beef, fruits, turkey, vegetable row crops, eggs, and seeded vegetables, respectively. Future challenges to food safety identified by the CDC include central processing and widespread distribution of food (single contaminated food could cause illness across the country and world), antimicrobial resistance,

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and unexpected sources of food-related illness (e.g., flour and onions) (CDC, 2021). Food safety surveillance at the state and local level is the responsibility of the State and local health departments. Each State and Territory have separate health and agriculture departments, with many counties and cities with separate agencies. The State and local authorities monitor food safety for retail food establishments such as restaurants; however, they do not have authority at grocery stores. The States are responsible for meat and poultry inspection of the meat and poultry sold in the State where they are produced, but the FSIS monitors the process. The 1968 Wholesome Poultry Products Act (WPPA) requires that the state inspections programs be at least as rigorous as the FSIS standard. If the State cannot maintain the inspection standard, the FSIS must assume the responsibility.

food products by providing information and support to help the industry understand the regulatory requirements associated with the FDA&R.

STATE AND LOCAL REGULATORY SYSTEMS

CANADA

Food in Canada is regulated by three federal government agencies that have complementary roles in enforcing and setting standards under (CHFA, 2021) the Food and Drug Act and Regulations (FDA&R) (IOM, 1998). The primary legislation regulating food in Canada includes Food and Drugs Act and Regulations (FDA&R), Safe Food for Canadians Act (SFCA), Consumer Packaging and Labelling Act (CPLA), and Canadian Environmental Protection Act (CEPA). Health Canada sets public health policy, conducts research and risk assessments, and sets limits on the amount of a substance that is allowed in a food product sold in Canada. The Food Directorate (FD) within the Health Products and Food Branch (HPFB) of Health Canada (HC) manages food products’ health risks and benefits. Canadian Food Inspection Agency (CFIA) reports to the Minister of Agriculture and AgriFood and enforces health and safety standards described in the Food and Drugs Act and Regulations. The CFIA mitigates risks to food safety, and the agency also regulates the packaging, labeling, and advertising of foodstuffs. Agriculture and Agri-Food Canada (AAFC) is responsible for policies regulating the production, processing, and marketing of innovative

EUROPE

The European Union General Food Law (EUGFL) is the primary food safety regulation in the European Union (EU). The law holds food businesses liable for food safety failures, requires traceability records, and makes them responsible for recalls. It established European Food Safety Authority (EFSA), the rapid alert system, and details general principles of the food safety system. The EU law regulates food in three broad classes: food of animal origin, foods of nonanimal origin (must be produced under the HACCP system), and composite food. Each member state is required to develop plans to enforce the food law. The EU’s role in food safety is to maintain trust between the member states, assist in implementing the food law, and conduct crisis management (De Waal et al., 2013). The European Commission (EC) regulates food safety in the EU. It published “The European Green Deal” in 2019, which proposed a “Farm to Fork” strategy to address food safety at every step of the food chain and bolster sustainable food and farming (BUEC, 2021). This plan seeks to increase greener food production (reduced pesticides, fertilizers, and antimicrobials in farming), healthier food environments, front-of-pack nutrition labeling, and end greenwashing (misleading green claims). The primary agency for assessing food safety risk in the EU is the EFSA. EFSA operates independently of the European legislation, Executive commissions, and EU member states. Food crises in the late 1990s led to the formation of EFSA in 2002 to provide scientific advice and communicate risks associated with food. The European Commission, European Parliament, and EU Member states reach out to EFSA for scientific advice regarding food-related risks. EFSA’s scientific work is led by a committee of 10 Scientific panels with leading scientists in their fields of work. The scientific committee advises on the scientific basis of food-related questions, and panels carry out a risk assessment. Areas covered by the scientific panel include additives and products in animal feed, biological hazards (BSE-related risks), contaminants in food, food

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additives, plant health, plant protection products, and their residues (EFSA, 2021). JAPAN

In Japan there are four major laws that regulate food and agricultural products. These laws include the Food Safety Basic Act (FSBA), Food Sanitation Act (FSAct), Health Promotion Law (HPL), and Japan Agricultural Standards Law (JASL). The Food Safety Basic Act establishes the general principles for developing food safety programs and defines the role of the Food Safety Commission, a food safety risk assessment body. The Food Sanitation Law ensures the safety and sanitation of foods through the Ministry of Health and Labor and Welfare (MHLW). The law prohibits the sale of foods containing harmful substances. It also assigns the standards for foods, milk and milk products, food additives, and food containers and packages (JETRO, 2011). The Health Promotion Law seeks to encourage public health by providing health education and services. It ensures that any health claims related to food must be scientifically substantiated and relevant. The Japan Agricultural Standards Law is established by the Minister of Agriculture, Forestry, and Fisheries (MAFF). The law applies to food, drinks, oils, fats livestock and fishery products as well as other agricultural and forestry products. It develops the criteria for the Japanese Agricultural Standards (JAS) for food and agricultural products (MAFF, 2021). OTHER COUNTRIES CHINA China has been actively working on updating its food regulations since 2010. The primary laws governing food safety in China are the Food Safety Law (CFSL), Law on Farm Product Quality and Safety (LFPQS), Consumer Right Protection Law (CRPL), and Law on the Inspection of Import and Export Commodities (LIIEC). Since 2010, China has issued over 1,300 national food safety standards establishing the food safety standard framework. In 2015 the Nationals People Congress passed a revised food safety law. The 2015 Food Safety Law integrated the food safety regulations and implementation under the China Food and Drug Administration (CFDA). The revised law emphasizes monitoring the process of food

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production rather than the end product and holds food producers accountable for food safety issues caused by unsafe food. The law refocuses the food safety strategy toward risk prevention. The Food Safety Law includes more stringent oversight on special foods such as infant formula and health foods and imposes severe disciplinary actions, including criminal penalties. In 2019 China triggered the Healthy China Initiative, which focuses on reducing oil, salt, and sugar in citizens’ diets. The Chinese regulatory agencies are also revising the labeling rules and standards to highlight the nutritional content of food. In 2020 China issued 38 new national food safety standards and revised four. China is preparing to revise the 2015 Food Safety Law with more food regulatory updates. Chinese food safety regulatory agencies include the State Administration for Market Regulation (SAMR), National Health Commission (NHC), General Administration of China (GACC), Ministry of Agriculture and Rural Affairs (MARA), Ministry of Commerce (MOFCOM), and Ministry of Public Security. The SAMR is responsible for coordinating China’s food safety system by developing major food safety laws, implementing domestic market inspections, and registering special foods. The NHC is a food-safety risk assessment organization, and it develops food safety risk monitoring programs and coordinates with the SAMR. It is responsible for approving food ingredients derived from biotechnology, and it develops the national food safety standards. The GACC is responsible for public security and border protection. It handles food and agriculture quarantine and inspection at ports of entry, and it is also responsible for registering foreign facilities that produce some food and agriculture products for export to China. MARA regulates the quality and safety of domestically produced agricultural food products but does not regulate food processors. It also oversees animal and plant disease prevention and controls and regulates livestock slaughtering and raw milk production. The MOFCOM regulates catering services and alcohol product distribution, and it manages relations with the World Trade Organization (WTO). The Ministry of Public Security is responsible for criminal investigation of food and drug violations. In

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2019 it established the Food and Drug-Related Crime Investigation Bureau (FDRCIB) to monitor food and drug-related crimes (Chung and Wong, 2013; USDA, 2021). BRAZIL In Brazil, several agencies share authority for ensuring food safety. The three primary agencies controlling food safety are the Ministry of Agriculture (MAPA), the Ministry of Health (MS), and the Ministry of Environment (MMA). MAPA has jurisdiction over inspection, processing, marketing, import, and export of food, including animal origin products, fresh fruit and vegetables, alcoholic and nonalcoholic beverages, juices, grains, seeds, and animal feed (including pet food). The Ministry of Health (MS), by its National Agency of Sanitary Surveillance (ANVISA), enforces most of the regulations regarding processed food products. This agency is also in charge of producing and registering drugs, foods, food additives, packaging, medical devices, tobacco, and tobacco products. ANVISA’S primary goal is to protect the public by assessing the food standards, safety, and contaminants. The Ministry of Environment (MMA) regulates activities that affect the environment through the Brazilian Institute for the Environment and National Resources (IBMA). The IBMA monitors and assesses the possible environmental impact of pesticides. Brazil follows the international standards set by the Codex Alimentarius for tolerance of pesticides, herbicides, and fungicides on agricultural products (De Luca et al., 2020; Fonseca, 2018). AUSTRALIA AND NEW ZEALAND Australia and New Zealand share a joint food regulation safety system. Several agreements and regulations embody this food regulation system, including the Food Regulation Agreement, Joint Food Standards Treaty between Australia and New Zealand, and Food Standards Australia New Zealand Act (FSANZ) [1991]. The FSANZ compiles standards that regulate food ingredients, processing aids, colorings, additives, vitamins, and minerals. It also governs the composition of some foods (dairy, meat, and beverages) and GMOs and labeling requirements for packaged and unpackaged foods. The Australia and New Zealand Ministerial Forum on Food Regulation set food policy for both Australia and New Zealand and has oversight regarding the

implementation of the standards (AQIS, 2021; FSANZ, 2020; DAWE, 2021). The National Food Authority (NFA) was formed at the same time as FSANZ Act in 1991 in an effort to effect economic reform and cooperation between the Commonwealth and the states and territories, with the aim of achieving uniformity in food standards across Australia. FSANZ adheres to a risk analysis approach recommended by the Codex Alimentarius Commission: risk assessment (identifying hazards in food and likely risks to human health), risk management (developing control measures that minimize the risks), and risk communication (ensuring two-way exchange of information between all stakeholders in standards development). The Australian Quarantine and Inspection Service (AQIS) aims to ensure that imported foods are fit and safe for human consumption through a program of inspection for compliance with food standards. FSANZ provides advice to AQIS on the level of public health risk posed by specific foods, but AQIS has operational responsibility for inspection and sampling of imported foods to ensure that they are compliant with food standards. FSANZ acts as the central point for the collection of food surveillance data from public health units in Australia and New Zealand. The New Zealand Food Safety Authority NZFSA is New Zealand’s controlling authority for domestic food safety; imports and exports of food and food-related products (including plant products); administration of legislation covering food sales on the domestic market, primary processing of animal products, and regulation of agricultural compounds (pesticides, fertilizers, and veterinary medicines). It also has farm-to-table responsibilities (“farm to fork”), from primary production through processing to retailers and educates consumer. NZFSA’s organization includes a verification agency, which audits animal product facilities to verify that exporters are following agreedupon processes (FSANZ, 2009). The UK’s food and feed safety and hygiene policy was regulated by the European Food Safety Authority (EFSA) and European Commission (EC); however, this stopped following Brexit (UK’s withdrawal UNITED KINGDOM

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from the European Union) on December 31, 2020. The United Kingdom’s Food Standards Agency is updating all EU references to reflect the law now in force after the Brexit Transition Period. However, in Northern Ireland, EU law will continue to apply with respect the food safety laws. The UK Food Standards Agency (FSA) regulates food and feed safety and hygiene, nutrition and health claims, and food composition, including standards on labeling. The FSA covers England, Northern Ireland, and Wales. Scotland has an independent body, Food Standards Scotland (FSS), that regulates food policy in that country. Several key legislations govern food safety in the United Kingdom. The Food Safety Act provides the legal framework of all food safety legislation in England, Wales, and Scotland. The Food Safety Order of 1991 applies to food safety regulatory requirements for Northern Ireland. The Food Standards Act established the Food Standards Agency (FSA) in 1999. The General Food Law regulates food imports and exports, food safety, labeling, traceability, and product recalls (FSA, 2021). SOUTH AFRICA Food safety in South Africa is regulated primarily by three departments: the Department of Health (DoH), Department of Agriculture Forestry and Fisheries (DAFF), and Department of Trade (DTI). The DoH is responsible for enforcing domestic food safety regulations, nutritional labeling, and the safety of all ready-to-eat food products. It ensures that the Codex Alimentarius standards are incorporated into the national food safety policies. The DAFF is in charge of exported agricultural products, registration of pesticides and veterinary medicines, and meat inspection and hygiene. The DTI regulates canned and frozen fish, canned meat products, and imports and certification of exports (Boatemaa et al., 2019; MALRRD, 2021).

Feed for Animal Consumption Most countries regulate human food, animal feed and pet food under the umbrella of “food regulation.” Some countries regulate animal feed, additives and MRLs separately. Regardless of the definition of a feed versus a food, the safety of the feed additives and feeds affects both humans and animals. Additives may harm the animal and the residue of additives

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may harm humans; hence, the reason for establishing Maximum Residue Limits (MRLs). UNITED STATES

In the United States the FDA is responsible for regulating the safety of animal food. The FDA, through the Center of Veterinary Medicine (CVM), manages this responsibility under the Animal Feed Safety System (AFSS). The AFSS covers the following aspects of animal food safety: regulatory activities, enforcement, information dissemination, and cooperation with other government agencies responsible for food safety (Choudhuri et al., 2019; FDA, 2019a). INGREDIENTS AND THE APPROVAL PROCESS The F&DC Act give the FDA the authority to regulate the ingredients and additives used in animal food. Food ingredients that are shown to be safe and effective are approved as Food Additive Petitions (FAP), which are listed in 21 CFR 573. The animal food ingredients can use the Generally Recognized as Safe (GRAS) notification procedure, then once approved are listed in 21 CFR 582 and 21 CF 584. The FDA also accepts animal food ingredients defined in the AAFCO official publications. PRODUCTION, STORAGE, AND DISTRIBUTION OF SAFE ANIMAL FEED INGREDIENTS AND MIXED FEED Animal food and ingredient procedures

must comply with Current Good Manufacturing Practices (CGMPs) that address manufacturing, processing, packing, and holding animal food. There is also a requirement that a hazard analysis and risk based preventative controls be completed for food manufacturers to prevent illness or injury to animals or humans consuming food derived from animals. The FDA has developed Guidance for Industry documents that discuss hazard analysis and preventive controls (GFI 235, GFI 245, GFI, 246, and GFI 239). Animal food and ingredient manufacturers are required to comply with the food preventative control rule if they are also required to register with FDA as food facilities under the Bioterrorism Act; however, farms are typically exempt for the Bioterrorism Act. There is a document developed by CVM to address safety of animal feed maintained and fed on the farm (GFI 203).

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REPORTING TOOLS OF ANIMAL FOOD HAZARDS AND UNSAFE ANIMAL FOOD Within the United

OTHER COUNTRIES

REGULATION OF PET FOOD AND ITS LABELING The FDA regulates the manufacture of cat

CANADA The manufacture, sale, and import of livestock feeds are regulated in Canada under the Feeds Act and Regulations and Health of Animals Act and Regulations administered by the CFIA. All feeds must be safe for livestock, for humans and for the environment. Feeds must also be fit for purpose and must be properly labeled to ensure safe and appropriate use. The Canadian Food Inspection Agency (CFIA) is in the process of reviewing all of its regulations for food safety, plant and animal health. The CFIA also has responsibility for the Agriculture and Agri-Food Administrative Monetary Penalties Act, Appropriation Act, Canada Agricultural Products Act, Canadian Food Inspection Agency Act, Consumer Packaging and Labeling Act (as it relates to food), Feeds Act, Fertilizers Act, Fish Inspection Act, Health of Animals Act, Meat Inspection Act, Plant Breeders’ Rights Act, Plant Protection Act, and the Seeds Act. The CFIA has a broad mandate as it regulates all food products for humans and animals, veterinary biologics, plant seeds, fertilizers, and crops. Health Canada is responsible for setting standards and providing advice and information on the safety and nutritional value of food. The CFIA enforces the food safety and nutritional quality standards established by Health Canada. The Veterinary Drug Directorate (VDD) in the Health Products and Foods Branch (HPFB) or Health Canada (HC) sets MRLs allowed in a food products (CFIA, 2021).

food, dog food, and treats and is similar to that of other animal food. The FD&C Act requires that all animal foods be safe to eat, produced under sanitary conditions, and contain no harmful substances. The pet food is to be truthfully labeled. Canned pet foods must be processed with the low acid canned food regulations to ensure that the food is free of microorganisms. Premarket approval by the FDA is not required for pet food. However, the FDA does ensure that the ingredients used in pet food are safe. FDA has federal labeling requirements under FD&C Act, such as net weight, Guaranteed Analysis of certain nutrients, and name and address of the manufacturer or distributor. FDA also reviews claims on pet food and has a Guideline 55 for guidance on collecting data to make specific disease state health claim (FSMA, 2021).

EUROPEAN UNION The principal aim of retained EU law Regulation (EC) 178/2002, General Food Law, is to protect human health and consumer’s interest in relation to food. It applies to all stages of production, processing and distribution of food and feed with some exceptions. Food businesses must comply with the Food and Feed Safety Law. To place safe food on the market, applicants must ensure the traceability of food, appropriate presentation of food, comply with MRLs, provide suitable food information, and undertake prompt withdrawal or recall of unsafe food placed on the market. The Food and Veterinary Office (FVO) of the EFSA acts under the Council Directive (EC) 97/ 78, General Food Law of 2002, Hygiene I (Regulation [EC] 852/2004), Hygiene II (Regulation [EC] 853/ 2004), Hygiene III (Regulation [EC] 854/2004),

States, different government agencies are responsible for protecting different segments of the food supply. The USDA regulates problems with meat, poultry, and egg products. Restaurant food safety is covered by local public health authorities, but issues surrounding food products that do not contain meat or poultry (cereal) should be reported to the FDA Consumer Complaint Coordinator by state. Complaints regarding pet food can be done electronically through the Safety Reporting Portal or by calling the state FDA Consumer Complaint Coordinator. FEED STANDARDS The Animal Feed Regulatory Program Standards (AFRPS) (feed standards) established a uniform process for the design and management of state’s programs responsible for the regulation of animal food. The AFRPS is composed of 11 standards to help standardize and improve components of the state program. These 11 standards include: regulatory foundation, training, inspection program, auditing, feed-related illness or death and emergency response, enforcement program, outreach activities, budget and planning, laboratory services, sampling program, and assessment and improvement of standard implementation.

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Hygiene IV Directive (EC) 2002/99, and Regulation (EC) 882/2004. EFSA is responsible for assuring food and feed safety, nutrition, animal health and welfare, plant protection and health. Chief veterinary officers and national food safety authorities of all 27 EU Member States (EFSA, 2009), the European Chemicals Agency, the European Centre for Disease Prevention and Control, the European Commission’s Joint Research Centre, are also involved in regulating food and feed. AUSTRALIA AND NEW ZEALAND FSANZ is responsible for developing food safety measures for the handling of food, including food production and processing in the primary industries, and coordinating national surveillance activities and a national food recall scheme. The agency is governed by a board with a wide range of expertise and experience in food matters, with members drawn from both countries. All imported feeds must comply with state and territory food legislation and other legislative requirements. The Australian Quarantine and Inspection Service (AQIS) aims to ensure that imported foods are fit and safe for human consumption and conform to the appropriate standards. FSANZ provides advice to AQIS on the level of public health risk posed by specific item or mixture. AQIS also has operational responsibility for inspection and sampling of imported foods to ensure that they are compliant with food standards. FSANZ has developed food safety standards for primary production and processing. This work extends existing food safety provisions in the Australia New Zealand Food Standards Code for the processing and retail sectors to food production. The New Zealand Food Safety Agency (NZFSA) and FSANZ are jointly responsible for shared information on food emergencies and surveillance. The Food Safety Programme (FSP) within NZFSA conducts research on food safety issues, including risk assessments, and investigates food safety incidents (FSANZ, 2009). JAPAN There are seven major laws governing food and agricultural products in Japan: Food Safety Basic Act, Food Sanitation Act, Food Labeling Act, Plant Protection Act, Domestic Animal

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Infectious Diseases Control Act, Health Promotion Act, Japanese Agricultural Standards (JAS) Act. The Ministry of Health, Labour, and Welfare (MHLW) is the head authority for food safety, including food additives and MRLs. The Ministry of Agriculture, Forestry, and Fisheries (MAFF) oversees animal and plant health, geographical indications, and organic standards enforcement. The Consumer Affairs Agency (CAA) oversees labeling. Importers are solely responsible for compliance with Japanese labeling regulations, though some may request assistance from US exporters. Commercialization of genetically engineered (GE) food crops require approvals from food, feed, and environmental regulators. New GE labeling requirements will come into effect in 2023. Quarantine officials may request additional information such as ingredient proportions and manufacturing processes prior to granting entry (MAFF, 2021). CHINA China regulates feeds via Ministry of Agriculture and Rural Affairs (MARA) General Administration of Customs (GAC), Ministry of Agriculture, and State Administration for Market Regulation (SAMAR) Feed is defined as a product made with industrialized processed ingredient(s) for animals, including single feed, premixed feed, concentrate, compound and supplementary feed. Also included are feed additives and substances added to feed during the processing, formulation and use in a small or minimum volume, including nutritional feed additives and common feed additives, including pet food. Only feed ingredients listed in the directory of feed raw materials, directory of feed additives and directory of medical feed additive can be used in feed products. The regulation of the administration of veterinary drugs was mainly used to prevent and control veterinary drug residues in China. China carries out risk assessments of the export country/region to see whether this country is capable of guaranteeing product safety. There is a directory specifying the approved countries and corresponding feed product category, and only products conforming to the list are allowed to be exported to China. Thailand, Taiwan, Philippines, Uzbekistan, Netherlands, France, Belgium, Germany, Denmark, Czech Republic, Italy, the United States, Canada, Brazil, Argentina, New Zealand, and

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Australia are allowed to export pet food products to China. The first version of this regulation was issued by the State Council on May 21, 1987, and major modifications were made in 2001 and 2004. In order to make sure that the regulation on administration of veterinary drugs was smoothly implemented, the Ministry of Agriculture of China developed some appropriate supporting regulations, such as Measures for Registration of Veterinary Drugs (Chung and Wong, 2013). SOUTH AFRICA In South Africa, the regulation of food laws is overseen by four government departments: the Department of Agriculture, Forestry and Fisheries (DAFF), the Department of Health (DoH), and the Department of Trade and Industry (DTI). The Department of Environmental Affairs (DEA) is also involved for issues such as domestication and transportation of insects and for the promotion of good practices. The government ministries operate under the following national acts: the Agricultural Product Standards Act, Animal Diseases Act, Animal Identification Act, Animal Improvement Act, Animals Protection Act, Fertilisers, Farm Feeds, Agricultural Remedies and Stock Remedies Act, Meat Safety Act, 2000 (Act No.40 of 2000), Performing Animals Protection Act, Veterinary and Para-Veterinary Professions Act, and the Liquor Products Act. The DoH requires that all foodstuffs shall be safe for human consumption in terms of the Foodstuffs, Cosmetics and Disinfectant Act. This Act addresses the manufacture, labeling, sale, and importation of foodstuffs. Matters regarding the hygiene of foodstuffs are addressed by the National Health Act, and the hygiene requirements at ports and airports including vessels and aircraft are addressed by the International Health Regulations Act. The South African Bureau of Standards (SABS) falls under the jurisdiction of the Department of Trade and Industry and controls canned meat and frozen and canned fishery products through the Standards Act (MALRRD, 2021).

10. CHALLENGES AND FUTURE DEVELOPMENTS IN FOOD SAFETY Food is a business like none other. Food is not a commodity nor is it a consumer good; it is

a human right, so defined by the United Nations (FAO, 2020a). We have one planet and as such one health (Farber, 2017). There are numerous issues surrounding food security, namely agricultural biodiversity, sustainability, accessibility, and food waste, let alone world hunger (WFP, 2020). Addressing these issues will be an ongoing process to bring high quality, safe food to all on planet earth. More acutely is the precarious nature of the food supply chain. These supply chains are long, global and highly interconnected. In addition, the current “just in time” management provides little room for disruptions of the food supply. For example, the global impact of the COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, on food security and safety, and the supply chain, emphasizes the need for collaboration at all levels, national and international (OECD, 2020; Chiwona-Karltun et al., 2021). It is currently believed that SARS-COV-2 likely originated from wild bats, snakes, or pangolins, animals sold at the Huanan market in Wuhan, a city in Hubei province (Yang et al., 2020). COVID-19 is currently influencing the food and agricultural supply chain in several ways, particularly food demand and food supply (FAO, 2020a). At the time of writing, food shortages have been patchy due to the resilience of the distribution networks. However, more serious is the potential of food supply issues right at the farm. In addition to COVID-19 with loss of migrant workers and the rapid increase in cost of energy, farmers have left fields lie fallow or have used insufficient fertilizer to maintain usual productivity. Even when the farms produced their normal output, the pandemic did not permit the producer to hire seasonal worker due to quarantine and travel ban mandates. Outbreaks of COVID-19 in meatpacking plants added to the problem, as farmers have less processing plant options. Finally, a pseudo-glut (farmers can’t sell their produce) has resulted in high quality produce being plowed back into the ground or discarded. The impact of climate change is the existential threat to food security and food safety (FAO, 2020b). The direct effects of climate change on our food supply may result in global famine as droughts and floods may lead to a dystopian existence. Already climate change is having local

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effects through fire and floods. Once the supply chain has recovered, there may not be enough food to stock the shelves. As the climate deteriorates, global food shortages may become common. Predictions of the impact of climate change on food safety include increased foodborne diseases, e.g., Salmonella sp., destruction of crops by plant pathogens such as fungi which produce mycotoxins (e.g., aflatoxin) and antimicrobial resistance (Costello et al., 2009). In addition, the food production chain has become more and more complex, providing greater opportunities for chemical and microbiological contamination of food. It is expected that the incidence of foodborne illness will continue to rise (Hall et al., 2008). Harmonization of regulatory requirements is important but always a challenge. For example, the regulatory approach to GMs and GMOs not only differs in requirements and classifications, but also in respective consumer perceptions between the United States and Europe (Frewer et al., 2013). Government regulations for GMs and GMOs range from relatively relaxed policies in the United States that focus on the final food product, to strict rules in the European Union that consider the genetic engineering process used to make the food. Despite these differences, they use evidence-based decision making to determine the potential impacts of genetically modified foods on human and environmental health, thus, ensuring the safety of the food supply (EPA, 2021; Lau, 2015; USDA, 2021a,b,c). New technologies, such as clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing muddy the water further. The global menu may or already includes algae, synthetically grown meat, plant-based meat alternatives, edible insect burgers, and protein bars (Figure 2.23), and even insect based pet food is currently available (Figure 2.24). It is yet to be seen what regulations will be developed, promulgated, and enforced in countries and the globe regarding the claims and supply of these new and novel foods (Woteki and Buckley, 2020). In fact, the FDA recently approved the marketing of the AquAdvantage Salmon, an Atlantic salmon that has been genetically modified to reach an important growth point faster. FDA determined that AquAdvantage Salmon is as safe to eat and

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FIGURE 2.23 Dining on insect protein. shutterstock_528768859.jpg. With permission (Accessed November 7, 2021).

FIGURE 2.24 Pet food containing cricket protein. https://jiminys.com/products/cricket-crave-dog-food?gclid¼EAI aIQobChMIos_X8uiG9QIV7wOzAB3MoAAeEAAYASAAEg K3TvD_BwE. (Accessed December 28, 2021).

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as nutritious as non-GM Atlantic salmon and would not harm the environment (FDA, 2020b). Regulatory changes may be needed as nanotechnology enters the market. Nanomaterials are most likely to be used in packaging but could also be used as additive to food products. The first question should be what the toxic effects of the nanomaterials are, followed by how much of the nanomaterial leaches into the food from the packaging. Advance analytical techniques will be needed to assess the concentration of nanomaterials in these food products. New uses of compounds derived from foods can be expected such as the incorporation of cannabis products in foods and beverages as well as foods used as a vehicle for ingestion of cannabis, e.g., gummy bears. Regulation of these products is a complex, state and national regulatory policy and public health challenge that requires multidisciplinary collaboration and analysis with a limited amount of data. Modified and fortified products will continue to expand. New pre- and probiotics may pose microbiological health risks and may amplify the threat of antimicrobial resistance. New mixtures of teas and other beverages with potentially toxic novel mixtures of herbs may pose an elevated health risk when compared with previous teas, e.g., comfrey. Adulteration of food will probably continue even though adulteration of beer and bread initiated modern food regulation. At present, olive oil, milk, fruit juices, spices, and sweeteners are most commonly associated with fraud (Moore et al., 2012). Meat has not been widely associated with adulteration; however, lab-derived meat and other novel products may become more attractive for fraud (Nieburg, 2013). Rising food prices, long and complex supply chains and high mark-ups on some products will continue to favor fraud, making it easier and more profitable, e.g., “mislabeling” (Oceana, 2013). Finally, new adulterants may not be detected either because the analysis was not aimed at the new adulterant, such as has been seen in the doping scandals in the athletic industry. Additives will continue to be developed and it will be interesting to see what role they have played in the ongoing and worsening world-wide obesity pandemic (Haridy, 2019). New analytical methods may need to be developed for new additives and

contaminants, and intoxication due to the intrinsic toxicity of some biota may become more prevalent due to increased numbers of the biota, e.g., blue-green algal poisoningdincreased algae due to increasing temperature, increased concentration of carbon dioxide and eutrophication. Biomonitoring of human bio-specimens (serum and urine) will be important for recording the food and cooking associated accumulation of such contaminants as acrylamides, heterocyclic amines (HCAs), phenols, and phthalates analyzed from urine, and perfluorinated compounds (PFCs) and organic chloride pesticides (OCPs) analyzed from serum samples in risk assessments. Ensuring that food is safe and nutritious is a highly complex process, requiring national and international cooperation, multidisciplinary approaches, and active participation of the food industry. Discussion of the risk and benefit of compounds that are intentionally added to food, contaminants of food and inherent toxicity of the food itself will increase due to innovations in livestock production, genetically modified organisms and animals resulting in novel foods. The novel areas are where the toxicologic pathologist will be of immense help to define adverse effects caused by these foreign foods and assist safety assessment regarding risks and benefits and support the promulgation of regulations.

11. CONCLUSIONS Assessing food safety toxicologically consists of many complex interfaceted, scientific- and nonscientific segments. The safety of food derived from biotechnological advances has potential health risks and must be carefully assessed. Although historically driven by scientific advances, the field is also heavily affected by public opinion and consumer acceptance of industrial, academic and regulatory innovations. New methods and policies need to be developed and agreed upon internationally to provide the scientific basis for decisions regarding human health. Such assessments need to weigh the health benefits against possible negative health risk implications: benefit–risk determination. Differences in global and national law pertaining to regulated food products, affect a path forward to globally

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GLOSSARY

harmonized approaches when conducting regulatory relevant research and risk assessment to ensure chemical food safety worldwide. Clear communication pertaining to safety assessment in these areas is generally lacking at national and international levels. The availability of safe and nutritious food for all is a human right, a goal still to be achieved particularly in less developed nations (FAO, 2021b).

GLOSSARY AA Amino acid AAFC Agriculture and Agri-Food Canada (Canada) AAFCO The Association of American Feed Control Officials ADI Acceptable daily intake AFRPS Animal Feed Regulatory Program Standards (feed standards) (United States) AFSS Animal Feed Safety System (United States) AMR Antimicrobial resistance APC Antigen presenting cells ASP Amnesic shellfish poisoning Asp Aspartate AZA Azaspiracid AZP Azaspiracid shellfish poisoning BA Secondary bile acids BPA Bisphenols bST Bovine somatotropin Bt Bacillus thuringiensis BTX Brevetoxin-b CAC Codex Alimentarius Commission CB Cannabinoid receptor (CB1 CB2) CBD Cannabidiol CCFA Codex Committee on Food Additives (FAO/WHO) CD Celiac disease CDC United States Center for Disease Control (United States) CEPA Canadian Environmental Protection Act (Canada) CFDA China Food and Drug Administration (China) CFIA Canadian Food Inspection Agency (Canada) CFP Ciguatera fish poisoning CFSAN Center for Food Safety and Applied Nutrition (United States) CFSL Food Safety Law (China) CGMP Current Good Manufacturing Practices CPLA Consumer Packaging and Labelling Act (Canada) CRPL Consumer Right Protection Law (China) CTX Ciguatoxins CVM Center for Veterinary Medicine (United States) DA Domoic acid DH Dermatitis herpetiformis DSP Diarrheic shellfish poisoning EAA Excitatory amino acid EC European Commission (EU) EDI Estimated daily intake EFSA European Food Safety Authority (EU) EMAs Antiendomysial IgA antibodies EPA Environmental Protection Agency (United States) EPIA Egg Products Inspection Act (United States) EU European Union (EU) EUGFL European Union General Food Law (EU)

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FAO Food and Agriculture Organisation of the United Nations (WHO) FAP Food Additive Petition (United States) FB1 Fumonisin B1 FD&C Act Federal Food, Drug, and Cosmetic Act (United States) FDA United States Food and Drug Administration FDA&R Food and Drugs Act and Regulations (Canada) FDRCIB Food and Drug-Related Crime Investigation Bureau (China) FIFRA The Federal Insecticide, Fungicide, and Rodenticide Act (United States) FMIA Federal Meat Inspection Act (United States) FODMAPs Fermentable oligosaccharides, disaccharides, monosaccharides, and Polyols FPIC Food processing–induced chemicals FSA Food Standards Agency FSAct Food Sanitation Act (Japan) FSBA Food Safety Basic Act (Japan) FSIS Food Safety and Inspection Service FSMA United States Food Safety Modernization Act (United States) GABA g-aminobutyric acid GACC General Administration of China (China) GALT Gut-associated lymphoid tissue GFD Gluten-free diet Glu Glutamate GluRs Glutamate receptors Gly Glycine GM Genetically modified food GMO Genetically modified organism GNT Guanitoxin GRAS Generally Recognized as Safe (United States) GRDs Gluten-related disorders GW Gulf War GWI Gulf War illness HAB Harmful algal blooms HACCP Hazard Analysis and Critical Control Points HARPC Hazard analysis of risk-based preventative controls HCN Hydrogen cyanide HPL Health Promotion Law (Japan) IARC International Agency for Research on Cancer IBD Inflammatory bowel disease IBS Irritable bowel syndrome iGluR Ionotropic glutamate receptors IOC Intergovernmental World Health Organization Oceanographic Commission of UNESCO IRIS Integrated Risk Information System JAS Japanese Agricultural Standards (Japan) JASL Japan Agricultural Standards Law (Japan) JECFA Joint FAO/WHO Expert Committee on Food Additives (FAO/ WHO) Ka Kainic acid LFPQS Law on Farm Product Quality and Safety (China) LIIEC Law on the Inspection of Import and Export Commodities (China) LPS Lipopolysaccharide MAFF Minister of Agriculture, Forestry, and Fisheries (Japan). MARA Ministry of Agriculture and Rural Affairs (China) mGluRs Metabotropic glutamate receptors MHLW Ministry of Health and Labor and Welfare (Japan) MOA Mechanism of action MOE Margin of exposure MOFCOM Ministry of Commerce (China) MPS Ministry of Public Security (China) MRL Maximum residue limit MS Multiple sclerosis MSG Monosodium glutamate NCGS Nonceliac gluten/wheat sensitivity

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C H A P T E R

3 Nutritional Toxicologic Pathology Matthew A. Wallig1, Amy Usborne2, Kevin P. Keenan3 1

University of Illinois at Urbana-Champaign, Urbana, IL, United States, 2Formerly Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, United States, 3Formerly Charles River Laboratories, Frederick, MD, United States

O U T L I N E 1. Introduction

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4. Macronutrients and Micronutrients 4.1. Introduction 4.2. Macronutrients 4.3. Micronutrients

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1. INTRODUCTION The appropriate combination of food consumed by animalddietdis vital for its health and wellbeing, but it is also a potential source of harmful chemicals, both natural and man-made. Further complicating this is the fact that most nutrients can be toxic when consumed in excess. Deficiencies of some of these same nutrients may also result in effects that resemble toxicosis or which enhance the toxic potential of other nutrients or chemicals in the diet. This chapter is an overview that explores the toxicological and pathological effects of both excess and deficient nutrients, including macronutrients, micronutrients (e.g., vitamins), and essential trace elements. Foods are among the most complex mixtures of exogenous organic and inorganic chemicals to which laboratory and domestic animals, as

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00003-4

5. Dietary Contaminants 5.1. Analyses for Contaminants 5.2. Pesticides 5.3. Mycotoxins, Heavy Metals, Phytoestrogens, and Other Contaminants

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well as humans, are exposed (see Food and Toxicologic Pathology, Vol 3, Chap 2). The diet needs to be optimized for the species, sex, age, and physiological status (growth, maintenance, reproduction, lactation, exercise, heat loss) because the dietary composition and feeding methods can affect the animal’s physiology and metabolism as well as response to test substances in toxicological bioassays and carcinogenicity studies. Laboratory animals are fed two types of diets: those composed of chemically defined or natural ingredients. Purified or chemically defined diets are formulated using a single nutrient or nutrient class (e.g., starch and sugars, casein and soybean protein, vegetable oils, lard, and cellulose) or using elemental ingredients (e.g., specific amino acids, sugars, fatty acids, vitamins, trace elements, and salts). These defined diets are usually used in short-term bioassays to compare with similar diets

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containing specific additives or having deficiencies for short-term, controlled experimental protocols. In contrast, natural-ingredient diets are formulated with agricultural products and by-products such as whole grains, grain meals, high protein meals, and natural mineral sources, as well as other livestock ingredients and supplements. These are typically used in toxicology studies. Formulations of natural-ingredient diets can be fixed with no change in ingredients, but these diets risk changes over time in nutritional content as specific nutrients change from batch to batch of ingredients. Natural ingredient diets may also be open formulations in which the amounts of specific ingredients vary to maintain a more consistent concentration of protein, fat, carbohydrate, and other nutrients over time from batch to batch. All-natural formulations will have low levels of natural and artificial contaminants present that can include pesticide residues, heavy metals, and natural plant and fungal toxins, carcinogens, and phytoestrogens (see Mycotoxins, Vol 3, Chap 6; Poisonous Plants, Vol 3, Chap 7; Animal Toxins, Vol 3, Chap 8; Bacterial Toxins, Vol 3, Chap 9; Metals, Vol 3, Chap 10; Agrochemicals, Vol 3, Chap 11; New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). For toxicity studies in which natural ingredients are used, the nutrients and potential contaminants should be measured by the feed manufacturer on each batch. Furthermore, these should be obtained as certified diets with the chemical and nutrient assay results on each batch informing whether contaminants are present and if they are at acceptable low levels. Special diets should also be assayed for essential nutrients such as Vitamin C content in guinea pig and primate diets, and Vitamin D levels in primate diets, along with the shelf life for those diets containing labile nutrients and additives. The most authoritative source of information on nutritional requirements for rodents is the current edition of the National Research Council (NRC), Nutrient requirements of Laboratory Animals (NRC, 1995). The NRC also publishes nutritional requirements for other terrestrial and aquatic animals used in experimental studies (see General Requirements section, Table 3.1). However, the values given in the NRC publications and most textbooks are estimates of minimal nutrient levels required, with no margins for safety. In addition, most

requirements are based on changes in the growth curves of rapidly growing weanlings fed ad libitum at varying concentrations of one nutrient while keeping all other nutrient levels constant. A typical 1- or 2-month feeding study defines optimal nutrient concentration as the level that does not statistically decrease the maximal growth curves of groups of rapidly growing weanling animals. These studies do not consider physiological age, sex, reproductive status, lactation status, strain, sex, or any variation in nutrient intake or bioavailability. Therefore, many diet formulations are educated estimates of nutrient requirements, with safety margins added to ensure minimal requirements will be met and maintained for the shelf life of the diet.

2. CALORIC EXCESS AND OBESITY Dietary energy is derived from fats, carbohydrates, and, to a lesser extent, dietary protein. Energy units are expressed as calories, or the amount of heat it takes to raise 1 g of water by 1
C. Calories are also expressed as joules (J), where 1 J ¼ 4.184 calories. One thousand calories are referred to as a kilocalorie (kcal), and this unit is used in calculations of nutrient energy content. Metabolizable energy equals the gross energy of the diet minus the energy lost in fecal matter, urine, and combustible gases, calculated as 4 kcal/g for carbohydrates and proteins, 7 kcal/g for alcohols, and 9 kcal/g for fats. Deduction of energy loss as body heat equals the net energy used for all body processes and the nonnitrogenous organic matter of tissues and secretions. Without adequate energy intake, other important organic nutrients cannot be used for normal maintenance and other functions. Dietary energy sources in excess of needs for maintenance, growth, reproduction, lactation, “work,” and heat production are stored as glycogen in the liver and as body fat stores. When fat stores are not utilized, a dietaryinduced obesity and other metabolic diseases, such as type 2 diabetes mellitus (T2DDM) in humans, may ensue. The incidence of obesity, diabetes, and related metabolic disorders due to intake of caloric excess and their related complications such as cardiovascular disease, renal disease, and cancer have become a major human healthcare issue

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worldwide and an important cause of morbidity and mortality in many developed countries. In genome-wide association studies there have been over 300 loci associated with T2DM and greater than 500 loci associated with other obesity traits such as body mass index (Ingelsson and McCarthy, 2018). Additionally, epigenetic factors, intrauterine imprinting, and environmental agents are added risk factors (see New Frontiers in Endocrine Disruptor Research, Vol 3 Chap 12). To put genetic, epigenetic, physiological, and environmental factors into a homeostatic model system for control of food intake and energy expenditure helps isolate specific mechanisms for studying obesity and allows development of effective treatments. The discovery of the hormone leptin in 1994, and the massive obesity produced in the leptin protein deficient Ob/Ob mouse, defined obesity as a problem of biology rather than willpower. These rodent models of obesity, which are due to a single mutation in the leptin gene (such as the Ob/Ob mouse) or in the leptin receptor gene (such as the db/db mouse or the Zucker, ZDF, and SHHR rats), contributed to the understanding of signaling pathways but did not model the more complex polygenetic syndromes of obesity and type 2 diabetes seen in heterozygous human populations. For this purpose, primates, pigs, dogs, and outbred rodents that develop the phenotypes of obesity and type 2 diabetes and have polygenetic disease trait loci that can interact with each other, and thus be modulated by diet, environment, and potential treatments, may better represent the clinically relevant human conditions. Small laboratory rodents are fed free-choice or ad libitum (AL), while other laboratory animals, especially monogastric species (rabbits, pigs, dogs, primates, etc.), are fed controlled amounts of food to prevent the numerous comorbidities of diabetes and obesity, and other health problems induced by overnutrition. The cause of obesity is a subject currently under active reassessment and research. Formerly believed to be simply an imbalance of ingested energy as food and expended energy for physiological functions, new possible alternative models are being investigated. The impetus for this change in thinking lies in the lack of efficacy of simply reducing caloric intake and increasing caloric expenditure to impact the obesity epidemic in humans. The carbohydrate-insulin model,

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proposed and debated within the last 10 years, does not have all tenets completely supported by currently available data and research, but has served as a basis for beginning the discussion of what are the most important facets of human overnutrition and their sequelae, such as nonalcoholic fatty liver disease (NAFLD) steatohepatitis (NASH), diabetes mellitus, insulin resistance, and the metabolic syndrome (visceral obesity, hyperinsulinemia, glucose intolerance, hypertension, hypertriglyceridemia, high LDL cholesterol, and low HDL cholesterol) (Ludwig and Ebbeling, 2018). Other nutritional factors, such as the composition of the gut microbiome (Sonnenburg and Backhed, 2016) or maternal obesity, may also impact adiposity in both humans and rodents and are currently being investigated for their contribution to obesity. Obesity has done for adipose biology what human immunodeficiency virus did for immunology (see Adipose Tissue, Vol 4, Chap 6). The need for research, the funding available for research, and the interest of the public have all increased exponentially as obesity rates in humans have climbed. The characterization of adipose tissue has been refined, with four types being classified histologically: white; brown; beige/brite; and perivascular (Grigoras et al., 2018). Adipose tissue is commonly considered one of the largest endocrine organs in the body. Of the most interest in overnutrition is white adipose tissue (WAT). Storage of triglycerides (TGs) (due to excess consumption) in the cytoplasm of WAT leads to hypertrophied and, to a lesser extent, hyperplastic adipocytes, which release fatty acids (FAs) during lipolysis. WAT and the associated endothelial and immune cells have been found to secrete over 25 adipokines so far, and are involved in steroid metabolism, insulin and immune responses, and angiogenesis and neoplasia, in addition to TG storage. Adipocytes secrete leptin, a satiety hormone that can limit appetite and increase energy expenditure. In obese animals and humans, the large adipose mass is dysfunctional as both a metabolic and endocrine tissue, including induction of a leptin resistant state in certain organs. This has complex consequences that increase morbidity and mortality in the obese, including metabolic syndrome, increased inflammation, and cardiac overload leading to cardiomyopathy and heart failure, with or

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TABLE 3.1 General References for Nutrient Requirements, Vitamins and Minerals GENERAL REQUIREMENTS

FDA, US Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (2002) additives and color additives used in food guidance for industry and other stakeholders toxicological principles for the safety assessment of food ingredients redbook (2000). Redbook II, Chapter II, Agency review of toxicology information in petitions for direct food July 2000; revised July 2007 https://www.fda.gov/regulatory-information/search-fdaguidance-documents/guidance-industry-and-other-stakeholders-redbook-2000. last accessed December, 2021 NRC, National Research Council (1977) Nutrient requirements of rabbits, 2nd rev. Ed. National Academy Press, Washington, D.C. NRC, National Research Council (1994) Nutrient Requirements of Poultry. 9th rev. Ed. National Academy Press, Washington, DC. NRC, National Research Council (1995) Nutrient Requirements of Laboratory Animals, 4th rev. Ed., National Academy Press, Washington, D.C. NRC, National Research Council (2001) Nutrient Requirements of Dairy Cattle, 7th rev. Ed., National academy Press, Washington, D.C. NRC, National Research Council (2003) Nutrient Requirements of Non-human Primates, 2nd rev. Ed., National Academy Press, Washington, D.C. NRC, National Research Council (2006) Nutrient Requirements of Dogs and Cats, 1st ed., National Academy Press, Washington, D.C. NRC, National Research Council (2011) Nutrient Requirements of Fish and Shrimp, 4th rev. Ed., National Academy Press, Washington, D.C. NRC, National Research Council (2012) Nutrient Requirements of Swine, 11th rev. Ed., National Academies Press, Washington, D.C. NRC, National Research Council (2016) Nutrient Requirements of Dairy Cattle, 8th rev. Ed., National Academy Press, Washington, D.C. CALORIC RESTRICTION

Hart, R.W., Neumann, D. A., and Robertson, R.T., (eds) (1995) Dietary Restriction, Implications for the Design and Interpretation of Toxicity and Carcinogenicity Studies, ILSI Press, Washington, D.C. MACRONUTRIENTS

Benevenga, N. J., and Steele, R. D. (1984). Adverse Effect of Excessive Consumption of Amino Acids. Ann. Rev. Nutr. 4:157e181. https://doi.org/10.1146/annurev.nu.04.070184.001105. Brosnan, J.T., and Brosnan, M.E. (2006) The sulfur-containing amino acids: An overview. J Nutr. 136,(6):1636S e1640S. https://doi.org/10.1093/jn/136.6.1636S. (1095) Munro H.M. (1978) Nutritional consequences of excess amino acid intake. Adv. Exp. Med. Biol. 105:119e29. https://doi.org/10.1007/978-1-4684-3366-1_8. PMID: 103372. NAS, National Academies of Sciences, Institute of Medicine (US) Panel on the Definition of Dietary Fiber and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes (2001) Dietary Reference Intakes Proposed Definition of Dietary Fiber. Washington (DC): II. Definitions of Dietary Fiber. National Academies Press, Washington, DC. https://www.ncbi.nlm.nih.gov/books/NBK223586/ last accessed December 3, 2022. Reeves, P.G. (1997) Components of the AIN-93 Diets as Improvements in the AIN-76A diet. J.Nutr. 127(5 Suppl): 838Se841S. https://doi.org/10.1093/jn/127.5.838S. Roehrig, K.L. (1988) The physiological effects of dietary fiberda review. Food Hydrocolloids 2(1):1e18.

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TABLE 3.1

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General References for Nutrient Requirements, Vitamins and Mineralsdcont’d

VITAMINS AND MINERALS

Shireman, R. (2003). Essential fatty acids: An overview, Encyclopedia of Food Sciences and Nutrition, ed 2nd, (Caballero, P., Tvugo, C., and Fringlas, C., editors.), Elsevier (academic press), pp. 2169e2175. Ammerman, C. B., Baker, D. H., Lewis, A. J. (1995). Bioavailability of nutrients for animals: Amino acids, minerals and vitamins. Academic press, san Diego, CA. Bobeck, E.A. (2020). Nutrition and health, companion animal applications: Functional nutrition in livestock and companion animals to modulate the immune response. J. Anim. Sci. 98(3):1e8. https://doi.org/10.1093/jas/skaa035. Chrichton, R. R. (2008). Biological inorganic chemistry: An introduction, Elsevier, amsterdam, Netherlands. Food and agriculture Organization of the United Nations (2002). Human Vitamin and mineral requirements. Report of a joint FAO/WHO expert consultation, Bangkok, Thailand. Food and Nutrition Board. (2000) Dietary reference intakes for Vitamin C, Vitamin E, selenium and carotenoids Institute of medicine. National Academies Press, DC, USA. IOM, Institute of Medicine (US) Panel on micronutrients, subcommittees on upper reference levels of nutrients and of interpretation and use of dietary reference intakes, and the standing Committee on the scientific Evaluation of dietary reference intakes. (2011). Dietary reference intakes for Vitamin A, Vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. National academy press (Washington, DC). Jones, T. C., Hunt, R. J., and King, N. W. (1996) Nutritional deficiencies. In “Veterinary pathology” (T. C. Jones, R. J. Hunt and N. W. King, eds.), 6th Ed., Chapter 16, pp. 781e815, Williams and Wilkins, Baltimore. Mertz, W. (1998). Review of the scientific basis for establishing the essentiality of trace elements. Biol. Trace Elem. Res. 66:185e191. Mertz, W. (Ed.) (1987). Trace elements in Human and animal nutrition. 5th Ed., Vol. 1 and 2, Academic Press, Inc, San Diego, CA. NAS, National Academies of Sciences Subcommittee on Mineral Tolerance of Animals (1980). Mineral tolerance of domestic animals. National Academy Press, Washington, DC. NAS, National Academy of Sciences Subcommittee on Vitamin Tolerance (1987). Vitamin tolerance of animals. National Academy Press, Washington, DC. NIH, National Institutes of Health, Office of Dietary Supplements: https://ods.od.nih.gov Last accessed December 3, 2022. Pais, I., and Benton Jones, J. (1997). The Handbook of trace elements. St., Lucie press, Boca raton, FL. Rechcigl, M. (Ed), (1978). In: “Nutritional disorders.” CRC Handbook series in nutrition and food, Vol. 3, Sec. E, CRC Press, Boca Raton, FL. Taylor, A., (1996). Detection and monitoring of disorders of essential trace elements. Ann. Clin. Biochem. 33:486 e510. Trumbo, P., Yates, A.A., Schlicker, S., and Poos, M. (2001) Dietary reference intakes: vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. J. Am. Diet. Assoc. 101(3):294e301. Ziegler, E., Filer, L. (eds) (1996) Present knowledge in nutrition, 7th Ed., ILSI press, Washington D.C.

without hypertension and atherosclerosis, depending on the species. Fat cells produce aromatase that converts adrenal androstenedione into estrone, which can bring about reproductive

senescence and lead to increased risk for pituitary and mammary gland tumors. In rodents, the excessive growth from caloric excess induces hypersecretion of pituitary prolactin and growth

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hormone that increases IGF-1 production, which induces systemic growth effects. Combined with islet cell insulin hypersecretion, this induces a potentially tumorigenic state, with increased cell proliferation and decreased apoptosis in numerous tissues in both animals and humans (Wallig amd Keena, 2013). Laboratory animal models of obesity include those produced genetically (monogenic; polygenic; transgenic) and those produced by nongenetic factors, such as diet or compound induced, and surgically manipulated (Suleiman et al., 2020). Rodent models are the most commonly employed, but large animals including dogs, pigs, and nonhuman primates and nonmammalian models such as zebra fish also have their uses (Kleinert et al., 2018). While there is no one model that completely recapitulates the human condition, using the model most associated with a specific physiologic change to answer a specific question often allows extrapolation of the issues being addressed to human health. In normal laboratory animals, the complex, systemic effects of overeating and obesity may interact and confound events that alter or mask the effects of a test article in a toxicology study, especially in chronic studies. This determinant error in nutrition should be controlled by measuring food intake to provide maintenance levels in laboratory rodents in toxicity studies, as is done in studies using rabbits, dogs, pigs, and other domestic animals. In a largescale, elegant study by Vorobyev et al. (2019), interaction between genes and diet in phenotyped and genotyped mice was demonstrated by caloric restriction to be protective against clinical manifestations of lupus. Additionally, calorie-restricted mice had beneficial changes in gastrointestinal micro- and mycobiota.

3. CALORIC RESTRICTION The classic study by McCay and colleagues at Cornell University (McCay et al., 1935) showed that controlling food intake in rats significantly ameliorated age-related diseases and increased life span. Since that time, numerous studies worldwide have confirmed those data and shown that 25%–50% reduction in maximal adult food consumption (without malnutrition) consistently increases both mean and maximum life spans of laboratory rodents and other animals by delaying the development of obesity,

diabetes, renal and cardiovascular disease, and cancer. These observations have been extended to various invertebrates, fish, birds, other small mammals, dogs, pigs, and primates. Methodologies of caloric restriction (CR), whether by food restriction, dietary restriction, or intermittent fasting, have effects that appear to be due to the decreased intake of total dietary calories, rather than the reduction of a specific macronutrient or micronutrient such as dietary protein, carbohydrate, or fat. The healthful effects of CR are different than protein-calorie malnutrition (PCM), or proteinenergy malnutrition in infants or children, a serious condition seen in chronic starvation (Guleria et al., 2017). PCM has been identified in severe renal or hepatic disease, aging, chronic infectious disease, immunosuppression, and cancer, and has parameters of muscle wasting, loss of subcutaneous fat; decreased nutritional intake, bedridden/reduced functional capacity; and defined weight loss over selected time periods (Gangadharan et al., 2017). Cancer anorexia–cachexia syndrome (CACS) results in extreme weight loss, sarcopenia, anemia, edema, and depression. Death usually results from atrophy of the diaphragm and other respiratory muscles. Lipid-mobilizing factor increases FA oxidation, and pro-inflammatory cytokines and hormones such as IL-1b, IL-6, IL-8, ghrelin, leptin, angiotensin II, and GDF-15 have all been identified to increase in patients with CACS and contribute to weight loss, anorexia, and nausea/vomiting. In 2-year chronic rodent studies, at or about week 80 (104-week study), a gradual decline is observed from maximal body weights and obesity peaks, with the advanced development of age-related terminal weight loss, decreased food intake, poor body condition, and mortality. These changes are due to multiple comorbidities (renal, cardiovascular, endocrine, and musculoskeletal diseases, as well was multiple tumors) observed in these rodent populations. Determining the specific cause of death can be challenging for the pathologist when presented with aging animals that have multiple serious organ diseases. This is because a given specific disease may not, by itself, account for the death, but the numerous comorbidities all contribute in sum to the animal’s death. In subacute and subchronic studies, body weight and food consumption loss or decrements are the most common indicators of general systemic toxicity, and test-material related effects

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often show a dose–response pattern. Different methods have been proposed to assess reduced body weight gain or loss, particularly in rodents (Hoffman et al., 2002; Van Norman, 2019) but typically percentages are calculated then compared to controls and/or prestudy values, and a statistical analysis done (see Experimental Design and Statistical Analysis for Toxicologic Pathologists, Vol 1, Chap 16). An important understanding regarding weight loss is that a decrement in weight without comorbidities may have toxicologic significance but using a number to define a humane endpoint and removal from the study needs to be accompanied by additional information. This information usually includes veterinary input, and an assessment of activity, appetite, and general appearance along with a body condition score. Significance of the percentages of weight loss should be evaluated with respect to the body condition score before the study commenced and varies with species. In long-term CR studies, the mechanisms involved in extending healthy life span and controlling body weight and obesity are well studied and complex. The most studied signaling pathways include reduction of chronic oxidative damage/oxidative stress (Circadian clock system improvement); increased AMPK cascade (fasting insulin levels), decreased TOR signaling pathway (body temperature decrease), and increased sirtuin signaling (lipid metabolisms decreased) (Hwangbo et al., 2020). This list is not all inclusive and, since almost all metabolic, inflammatory, and growth mechanisms change in CR feeding, the definitive mechanisms are likely to be multifactorial and interactive. Adult ad libitum (AL)-fed rodents take about 6 weeks to stabilize body weight and metabolism following a sudden switch to 40% CR feeding. Initially, metabolic rates drop as the animals catabolize excess body fat and adjust somatic growth to the new nutritional levels provided. However, once stabilized, the CR-fed animals have equal or slightly higher metabolic rates than AL-fed animals and consume the same amount of food per unit of body weight as their AL counterparts. What appears to differ is the manner of utilization of the nutrition in CR-fed versus AL-fed animals. After feeding, the CR-fed animals have a rapid rise in respiratory quotient (RQ) as they shift to carbohydrate metabolism from other macronutrients. Later, a lowering in RQ occurs when their glycogen reserves have been used and the animals

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metabolize dietary proteins and fats. The drop in RQ indicates that the CR-fed animals have less dependence on carbohydrates for energy and have a more diverse utilization of other macronutrients throughout the day. This shift from carbohydrate metabolism at feeding to fat and protein use after feeding maintains better glycemic control, and this prevents the development of visceral obesity in CR-fed animals. In contrast, AL-fed animals maintain a constant RQ and a constant ratio of macronutrient metabolism that does not aid glycemic control, instead favoring the storage of excess energy as visceral adipose tissue. CR-fed animals, as compared to AL-fed animals, have a lower body weight, smaller skeletal size, and decreased weight of most internal organs except for brain and testis. Reduction in body and organ growth rates correlate with the delay or prevention of degenerative disease and tumors, and the extended life span or healthy life span of the animals. As would be expected in a method that prevents endocrine disruption, moderate CR does not have adverse effects on estrous cyclicity or the onset of reproductive senescence in rats. Since pregnancy and lactation typically increase protein and energy needs by over 30% of maintenance requirements, breeding females of most species should not be CR-fed. In rats, CR feeding to control body weight to as much as 70% of AL-fed values had no adverse effects on fertility, number of fetal implants per dam, and sperm parameters. These data suggest that changes in reproductive indices in rats associated with body weight changes in these ranges in toxicity studies should not be discounted as being simply secondary to body weight loss due to reduced food consumption.

4. MACRONUTRIENTS AND MICRONUTRIENTS 4.1. Introduction Dietary components traditionally are divided into macronutrients and micronutrients. Macronutrients, which make up the bulk of the diet and provide energy and raw components for cellular and tissue growth and repair, include carbohydrates, lipids, and protein. Fiber, while originally identified as that which is indigestible in monogastric species, is now known to be digested partly

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by colonic microflora, resulting in short-chain FAs (SCFAs) that can provide an important energy source as well as alter cell signaling, gene expression, and regulate a variety of processes such as inflammation and differentiation (Tan et al., 2014). Micronutrients include both vitamins and minerals, the latter being divided into the major minerals such as calcium (Ca) and potassium (K) and trace elements such as copper (Cu). The list of elements still fluctuates as new evidence emerges, supporting or opposing their essentiality. The element fluorine (F) is a good example of this. Although F (as fluoride, F) does not appear essential for full growth, the American Dental Association supports a role for F in maintaining dental health, and F has proven to be a significant aid in preventing human oral disease and its systemic consequences (Rozier et al., 2010). Yet a report and its accompanying monograph from the National Toxicology Program (NTP) suggests controversially that F, even at less than 1 ppm in the drinking water, provided by many municipal water suppliers, has potential adverse effect on neurological and cognitive development in rodents and perhaps humans (NTP, 2016, 2019). The NTP has suggested that for perhaps a small segment of the population, the significant public health benefits of supplemental F in drinking water far exceed any theoretical risk to the overall human and animal population. Nutrients not only play an essential role in optimal growth of the organism but prevent the biochemical and pathological lesions of deficiency diseases, the characteristic nature of which can be used to diagnose specific nutritional imbalances. The dose required for optimal growth in humans is used to calculate the Recommended Daily Allowance (RDA). The Food and Nutrition Board of the NRC of the National Academies of Sciences (NAS) has set RDAs for 15 different age and gender groups, including pregnant and lactating women and domestic and laboratory animals (see above). While the endpoint for production of optimal growth appears quite clear cut, determining the dose needed to overcome a deficiency disease becomes a matter of interpretation. The NRC Food and Nutrition Board does not extend its area of concern to the potentially adverse effects of doses of nutrients that greatly exceed the RDA. However, the availability of vitamin and mineral supplements on the market today permits ready

opportunity for excess intake and resulting overdose. Some nutrients have specific biological effects at doses in excess of the RDA, and most others have nonspecific toxic effects if the dose is sufficiently large. A good example of a nutrient with a specific, pharmacologically useful effect at a higher dose is niacin, once used extensively to lower elevated plasma cholesterol levels but now only used intermittently when other drugs are contraindicated (see below). This effect is unrelated to the effect of niacin as a vitamin and requires gram (g) quantities for effect rather than the milligram (mg) quantities needed for vitamin action. Even at pharmacological doses of niacin one starts to see toxicity. For many minerals, ingestion of a great excess competitively inhibits the uptake of other minerals, precipitating a secondary nutritional deficiency. For example, molybdenum (Mo) excess produces Cu deficiency, and manganese (Mn) excess produces iron (Fe) deficiency anemia (see below). In chronic zinc (Zn) overdose, first Cu deficiency is seen, and if the excessive intake persists, Fe deficiency develops, both deficiencies due to competitive inhibition of intestinal uptake by Zn (see below). The concept of nutrient overdose, so apparent to the pharmacologist and toxicologist, rarely has been addressed through nutritional research or food regulatory channels. For nonnutrient food additives such as sodium saccharin, monosodium glutamate (MSG), butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA), the concern for potential toxicity is recognized, researched, debated, and even carefully regulated. A few dietary components that are nutrients at low dose indeed are recognized as particularly toxic or even carcinogenic at higher doses. For the noncarcinogenic nutrients that have been recognized as having significant adverse effects (i.e., toxicity) at high doses, results from dose response studies are used to calculate a noobserved-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level (LOAEL). Determination of safe and appropriate dietary levels (acceptable daily intake, ADI) is carried out by dividing the experimental value by 100 (FDA, 2008). This calculation is based upon the belief that there is a threshold below which no toxicity occurs and considers possible variation in sensitivity among humans and the species

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tested, as well as among different genders and/ or ages. An example of a nutrient that has been found to have adverse effects at higher dietary doses in humans but not necessarily nonhuman species is sodium (Na, see below). This safety calculation, however, has resulted in a dichotomy that has emerged for a number of trace elements, where the calculated ADI is of similar magnitude to the RDA. Selenium (Se) is an extreme example of this dichotomy (see below), where inclusion in the diet (at no less than 0.04 ppm) is recommended to avoid deficiency diseases, even though higher doses have proven carcinogenic. The RDA for selenium (Se) is 55 mg/day adults (ODS, 2021a), yet the EPA (2002) until 2002 set the maximum contaminant level in drinking water at 10 mg/L, even though the average daily consumption of water is only 2 L, putting the safety limit below the RDA. Thus, safety limits can be close to the RDA for certain micronutrients like Se. The current safety limit for Se is 170 mL (EPA, 2002), above the RDA but still relatively close. Keeping issues such as these in mind, the remainder of this chapter provides a brief overview of some of the biochemical and pathological changes associated with nutrient deficiencies and excesses.

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nonhuman species. Adverse changes attributable to PEM in its early stages can be subtle and difficult to assess in a qualitative fashion histologically. Atrophy of adipose can be discerned grossly as an increase in myxomatous (serous) tissue with a high proportion of water-rich ground substance in places where mature adipose tissue normally would be found, in particular the intestinal mesentery, omentum, perirenal fascia marrow cavities of appendicular bones, and epicardial grooves. Histologically atrophy of adipocytes may be hard to discern, as the adipocytes shrink with loss of triglycerides to assume a more fusiform mesenchymal morphology (Figure 3.1). Atrophy of nonadipose tissue within organs can be quite subtle in early phases and ancillary techniques such as organ weights, organ to body weight ratios, and morphometry must be utilized to confirm loss of cellular mass. EXCESS

Protein excess seldom is of direct pathological relevance in most domestic and laboratory species and is disputed in humans, except in the context of individual amino acids since, in most cases, excessive protein is degraded to its amino acid constituents, individual amino acids are deaminated, and the carbon backbones are pulled into the Krebs cycle or gluconeogenic

DEFICIENCY

Protein deficiency is usually combined with carbohydrate and fat deficiency (i.e., PEM), as covered above. The lesions of generalized wasting associated with protein deficiency and/or PEM include loss of skeletal muscle mass with gross and histologic evidence of atrophy, serous atrophy of visceral and peripheral adipose tissues, anemia, abnormal bone growth, endocrine atrophy, cerebral atrophy, bone marrow hypoplasia, fatty liver, thymic and lymphoid depletion, intercurrent infections, and skin lesions. In domestic and laboratory animals, generalized edema may not be evident unless deficiency is so severe that the liver has insufficient amino acid resources for albumin production, but serous atrophy of fat, visceral organ atrophy, anemia, poor hair coat, and predisposition to infectious disease are prominent features of both protein and PEM in

FIGURE 3.1 “Serous” atrophy of epicardial adipose tissue from an elderly starved, emaciated ewe. Shrunken, sometimes elongated adipocytes are present within a pale, mucinous type ground substance. Myocardium is in the lower portion. Hematoxylin and eosin stain.

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pathways. In rodents, however, especially rats, high protein diets can lead to glomerular damage and ultimate renal failure (Bertani et al., 1989; Gumprecht et al., 1993). Controlling protein intake is important in dietary management of animals with chronic renal failure since excessive protein intake appears to increase the progression and severity of chronic renal disease leading to the development of uremia (Hostetter et al., 1986). The basic pathophysiologic change caused by excessive dietary protein is enhancement of overall nephron growth related to increased production of growth factors (increased growth hormone and IGF-1) and chronic metabolic overload (Schutz, 2011). Increased glomerular intracapillary pressure with subsequent leakage of proteins into the mesangium occurs. The proteins accumulate as mesangial deposits inducing glomerular sclerosis that eventually compromises overall glomerular function, leading to tubular dysfunction and loss, and finally to renal failure (Wakefield et al., 2011). Therefore, protein should be limited to just that required for healthy growth and maintenance in adult rodent diets. Amino Acids DEFICIENCY

Deficiencies of specific amino acids are rare, although many diets can be relatively deficient in certain groups of amino acids. Prolonged removal of a single indispensable amino acid in a particular species often results in a reduction in food consumption as the adverse effects of deficiency set in. Many corn-based diets are limited in lysine and tryptophan content while peanut and soy-based diets are often low in methionine content. The essential amino acid, tryptophan, is not only a component of proteins, in particular membrane-anchoring proteins, it also is an important precursor for the neurotransmitter serotonin, and, as such, has an important role in mood and behavior, in humans, having antidepressant and anxiolytic actions. The hormone melatonin, important in regulating the sleep cycle, is also derived indirectly from tryptophan (via serotonin) and a precursor of niacin. Although it has been utilized extensively in treating depression and anxiety in humans a link to these disorders, a specific dietary deficiency has

not been established; however, high dietary tryptophan, as opposed to tryptophan powders or supplements, has been shown to improve mood, decrease anxiety, and alleviate depression (Lindseth et al., 2015). In animals, on the other hand, the effects of acute tryptophan deficiency on appetite and behavior are not as well defined although altered aggression and reaction to stress have been reported while little effect has been reported with chronic deficiency. A lack of dietary tryptophan can result in cataract formation, corneal vascularization and has been associated nonspecifically with hepatic lipidosis and pancreatic atrophy (Moehn et al., 2012). Teleost fish, in particular trout, are sensitive to tryptophan deficiency, with defective development of vertebrae leading to scoliosis, abnormal calcification of vertebrae and kidney, and immune impairment manifestations of a deficiency (Kloppel and Post, 1975; Machado et al., 2019). Lysine, an essential amino acid in vertebrates, is especially important in cross-linking of collagen fibrils in the maturation phase of collagen synthesis. Diets deficient in lysine in humans have been linked to dental caries, impaired bone formation, and ataxia. In animals, lysine deficiency is most often linked to decreased growth with stunting, especially in immature animals, as limited lysine availability decreases protein synthesis overall (Ball et al., 2007). Methionine is an essential amino acid in vertebrates, especially in rapidly growing birds, in particular poultry. It is necessary for the initiation of translation; for transsulfuration to produce the amino acids cysteine and taurine; for accepting methyl groups in the activation of folate and degradation of choline; and for donating methyl groups in nucleic acid, polyamine, and catecholamine synthesis as well as in DNA methylation (Brosnan and Brosnan, 2006). Uncomplicated methionine deficiency is usually reported as inducing hepatic lipidosis and even steatohepatitis in rats (Oz et al., 2008), but most often in combination with choline deficiency (see choline below). Arginine is one nonessential amino acid not only utilized in protein synthesis but as a key metabolite in the synthesis of urea, generation of nitric oxide (NO), and production of polyamines (Morris, 2016). Even though it can be

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synthesized de novo, adult cats in particular, but also dogs, rabbits, and rodents appear to be susceptible to deficiency in certain situations (Ball et al., 2007). A dietary lack of arginine in cats (e.g., exclusively plant-based diets), which do not downregulate urea metabolism during times of decreased dietary intake, causes disease in a relatively short time, due mainly to hyperammonemia. It is characterized by emesis, neurological dysfunction, hyperesthesia, and tetany followed by death (Zoran, 2002). In dogs, deficiency has similar manifestations probably due to an inability to deaminate amino acids in the urea cycle (Burns and Milner, 1981). In rats, unlike cats, dogs, and other rodents, hepatic lipidosis has been observed with arginine deficiency (Milner and Hassan, 1981). Taurine, 2-aminoethanesulfonic acid, derived from cysteine, is an important intracellular amino acid. Its main function is to serve as one of the substrates for bile acid synthesis but it has additional antioxidant functions, a role in maintenance of cardiac and skeletal muscle function and an apparent dampening effect on apolipoprotein B synthesis (Huxtable, 1992). It is synthesized in sufficient quantities in all animal species from the amino acid cysteine except in felines, which must obtain additional taurine from meat-rich diets. This additional requirement is because felines must rely exclusively on taurine for conjugation of bile acids. Taurine deficiency was once thought to be limited to cats, since dogs are less reliant on dietary taurine than cats; nevertheless, deficiency in certain breeds of dogs is being increasingly reported, particular with the advent of “grain-free” and other similar types of dog foods, which are based on legumes with little or no meat in them (Kaplan et al., 2018). In dietary deficiency, taurine stores are shunted into the taurinecholate conjugation pathway at the expense of other important metabolic pathways. The principal lesion in cats, at least in severe acute deficiency, is progressive central retinal degeneration (Leon et al., 1995). The mechanism of the degeneration is not completely understood but taurine is thought to be essential for protection against and mitigation of oxidant stress in photoreceptor membranes (Ripps and Shen, 2012). Dilative cardiomyopathy also has been linked to taurine deficiency in cats and dogs (Kaplan et al., 2018). In cats, the cardiac lesions have been linked to

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taurine’s role in n maintenance of respiratory chain function and Ca homeostasis in cardiomyocyte mitochondria and osmoregulation in the sarcomeres themselves (Schaffer et al., 2010). Although taurine deficiency disease has not been reported in nonfeline and noncanine species, taurine has been used as a supplement for humans with atherosclerosis and coronary heart disease (Wojcik et al., 2010). It is also used as a supplement for overall muscle health despite the lack of clinical data. EXCESS

Generally in mammals, the addition of excessive amounts of individual amino acids at concentrations that are disproportionate to appropriate ratios in which they are required for normal growth causes a depression in food intake and growth in addition to any toxicity specific to the particular amino acid in question (Munro, 1978). Food intake depression occurs soon after animals are fed the imbalanced diet. Inclusion of excess methionine or tryptophan in rat diets produces a severe depression of food intake and growth. Feeding excess amounts of methionine or N-acetyl-L-methionine also results in retarded growth rates in rats (Benevenga and Steele, 1984). Tyrosine, when administered to adult rats, either parenterally or orally, causes depletion of essential amino acids in the cerebral cortex leading to neurological dysfunction (e.g., decreased perceptual function) and behavioral disturbances (Garlick, 2004). Leucine (and other branched chain amino acids, valine and iso-leucine) has been used as a supplement to conserve muscle mass when losing weight, to ameliorate metabolic syndrome, to promote preservation of muscle mass in the elderly, and to enhance brain function (Fernstrom, 2005; Layman and Walker, 2006). Presumably leucine triggers b-cell insulin secretion and satiety (Millward, 2012), leading to enhanced carbohydrate utilization, decreased FA synthesis, and decreased overall consumption of food. Dietary excess of leucine may be a precipitating factor in pellagra (niacin deficiency, see below), especially when tryptophan is limiting. Leucine decreases the activities of tryptophan metabolizing pathways and is utilized as a secondary source for the synthesis of nicotinamide nucleotides when intake of nicotinic acid is only marginally adequate (Ghafoorunissa and Rao, 1973; Cook and Carpenter, 1987).

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Phenylalanine is an amino acid with high toxic potential, causing brain dysfunction. The likely mechanism of phenylalanine toxicity involves the decreased uptake of large neutral amino acids into the brain, leading to impaired protein synthesis and subsequent neurological dysfunction (de Groot et al., 2013). Phenylalanine toxicity is not a concern for most species except for humans with phenylketonuria, an inborn error of metabolism with a relatively high incidence in certain Caucasian populations due to a lack of functional phenylalanine hydroxylase. Although of low toxic potential, lysine is known to inhibit tubular reabsorption of protein, and excessively high doses of dietary lysine (4500 mg/kg/day IV) in the dog cause renal tubular necrosis of the S3 segments (see Kidney, Vol 4, Chap 2) (Asanuma et al., 2006). Lysine is known to inhibit proximal tubular reabsorption of protein, an effect that promotes the formation of obstructive tubular casts that exacerbate the nephrotoxic effect. Although not nephrotoxic in the rat (Tsubuku et al., 2004), excessive lysine is toxic to rat pancreatic acinar cells causing severe vacuolation and interstitial edema within hours, followed by widespread apoptosis and necrosis (Biczo´ et al., 2011). Besides its standard use in IV hyperalimentation solutions, lysine supplements have been touted as beneficial in the healing of cold sores, enhancing athletic performance based on its effect on growth hormone, and as an antihypertensive agent, although none of these claims has been clearly substantiated (EFSA, 2011). In similar fashion to lysine, excessive doses of dietary arginine induce acute necrotizing pancreatitis resembling lysine toxicosis not only in rats but also in mice (see Exocrine Pancreas, Vol 4, Chap 5) (Kui et al., 2014). However, in mice lesions develop more slowly and are multifocal rather than diffuse in distribution. Arginine, since it is the source substrate for NO, has been shown in short-term studies in humans to be of benefit in peripheral artery disease; the long-term benefits at 3 g/day (w30% the safety limit of 20g/day), however, have been disputed (Wilson et al., 2007). Carbohydrates Since general manifestations of carbohydrate deficiency (e.g., PEM and PCD) and excess (obesity) have been covered above, only a few

selected cases of relative carbohydrate excess and deficiency will be covered in this section. DEFICIENCY

Provided adequate dietary lipids, proteins, vitamins, minerals, and micronutrients are available, a generalized deficiency of carbohydrates may be of little consequence in some omnivorous and carnivorous species. Many carnivores, in particular members of the felid family, do not have an absolute requirement for a carbohydrate source in the diet. However, most noncarnivorous domestic animals and most rodents require carbohydrates for successful reproduction and lactation. A relative carbohydrate deficiency in ruminants is triggered by a rapidly developing metabolic need for glucose. This typically happens in an obese animal in late pregnancy with multiparous fetuses or during heavy lactation and can lead to ketosis and hepatic lipidosis. This rapid and excessive mobilization of triglycerides from peripheral adipose to meet the demand for both energy and milk production leads to the formation of ketone bodies to meet the energy demands of other maternal tissues in the body. The deficiency of readily available glucose due to the peculiarities of ruminant carbohydrate metabolism sets in motion a complicated series of events that ultimately leads to the accumulation of triglycerides in hepatocytes and severe hepatic lipidosis, even to the point of hepatic dysfunction and death (Bobe et al., 2004). EXCESS

Excessive consumption of carbohydrate-rich fermentable grains such as oats (“grain overload”) in ruminants and horses could be considered a generalized carbohydrate toxicity, with a variety of adverse outcomes (Gelberg, 2017; Hargis and Meyrs, 2017a). In ruminants, consumption of excessive grains or sudden changes in dietary composition from high fiber to high fermentable carbohydrates leads to an abrupt change in ruminal microfloral balance to gram-negative bacterial flora that ferment the carbohydrate producing excessive quantities of lactic acid and a profound decrease in pH. The acidotic environment is directly toxic to ruminal epithelium, leading to sloughing of ruminal epithelium and ultimately to ulceration, with secondary invasion by microflora into the

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vasculature and sepsis. The ulcers can also be populated by nonbacterial microflora, including Candida, Aspergillus, Mucor, and Rhizopus if the animal survives the initial bout of necrotizing rumenitis. Rarely, the systemic inflammatory response elicited by the rumenitis and secondary sepsis can lead to laminitis. In horses, consumption of excessive carbohydrate in the form of fermentable grains is more likely to lead to acute acidosis, gastric dilatation due to excessive gas production that cannot be eructated, and ultimately gastric rupture. In horses in which gastric dilatation/rupture does not occur, inflammatory laminitis is likely to occur due to the secondary systemic inflammatory response that inevitably results from the overload. In young animals, especially in ruminants but also foals and piglets, excessive carbohydrate consumption via high grain diets or overly avid nursing can lead to overgrowth of Clostridium perfringens of various types within the anaerobic core of the ingested bolus and production of toxins that can cause necrosis of the stomach/ small intestine and/or toxemia and death. It is difficult, if not impossible, to produce carbohydrate toxicity in the classical sense in humans (excluding obesity). However, in humans and species not dependent on ruminal or cecal/coli fermentation, toxic responses to specific carbohydrates under certain situations, often when there is an inherited deficiency in a carbohydrate metabolizing enzyme, have been reported. Examples of a toxicosis from feeding a particular carbohydrate are those of sucrose and fructose, both of which are toxic to neonatal piglets. Sucrose toxicity is a result of the low activity of intestinal sucrase (a disaccharidase) in the intestine of young pigs (Becker et al., 1954). Therefore, neither glucose nor fructose is available as an energy source. Although neonatal pigs absorb fructose intact during the first week of life, they are not able to phosphorylate the fructose. Consequently, this hexose is not catabolized to trioses for energy utilization. As a result, animals experience osmotic diarrhea and limited growth. Another example of a toxic response to a specific carbohydrate is the inability of the laboratory rat to efficiently metabolize high concentrations (50% or greater) of sucrose (and fructose derived from sucrose) in the diet, leading to

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hepatic lipidosis, especially in the periportal zone, as well as hypertriglyceridemia (Novikoff, 1977; Bacon et al., 1984). The lesions are typical of hepatic lipidosis or NAFLD seen in other species and may be the result of enhanced FA synthesis with a concomitant reduction in apolipoprotein synthesis, triggered by the exaggerated conversion of sucrose to acetate. In addition, high sucrose diets induce marked mineralization in the renal outer and inner stripe of rodent kidneys, leading to renal failure (Boyd et al., 1965). These findings have led to substantial changes in “purified” dietary formulations that formerly utilized sucrose or fructose as a sole carbohydrate source for laboratory rodents. Fiber This section is an overview and will focus on fiber as it impacts pathology in nonruminant (monogastric) species. Effect of fiber on the microbiome and overall gastrointestinal (GI) function as well as susceptibility and response to injury will be discussed more in depth in a later chapter (see Digestive Tract, Vol 4, Chap 1]). The definitions of dietary fiber differ from country to country and even among researchers (NAS, 2001) but for the purposes of this discussion it is the sum of plant components which are not digested by the endogenous digestive enzymes of the GI tract but may be digestible by GI microflora (Holscher, 2017). Dietary fiber includes water insoluble fibers such as b-glucans (e.g., cellulose and chitin), lignins, hemicelluloses, gums, and resistant starches but also includes water-soluble fibers such as pectins, alginates, and raffinose. Soluble fiber can be metabolized by the microflora of the lower gut in nonruminants whereas insoluble fiber, or roughage, generally is nondigestible in monogastric species. However, this nondigestible fiber serves to retain water in the lumen of the lower intestine and enhances the passage of ingesta, increases the size of the cecum and colon in rodents, and increases fecal bulk (Roehrig, 1988). The chemical constitution of fiber is not homogenous in most nonpurified diets. Cellulose, hemicelluloses, pectins, and lignin in varying percentages are the most common fibers in various types of diets, depending on the plant source. The degree of fermentation of different fiber constituents by colonic microflora (i.e., the microbiome) differs from substance to substance and species to

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species. Fermentation also depends on the relative amounts of individual components as well as the proportions of various organisms within the colonic microbiome of the individual animal. SCFAs, especially butyric acid, are released by microfloral fermentation of soluble fibers in the cecum and colon to provide not only on-site energy to colonic epithelium but also to serve as important mediators of colonocyte differentiation in this portion of the GI tract. Increased fiber intake in humans has been associated with a host of well documented and many as yet unconfirmed effects on a variety of disease conditions (Anderson et al., 2009). Positive effects of enhanced dietary fiber for overweight or obese individuals include decreased transit time of food through the GI tract, leading to decreased absorption of nutrients (especially carbohydrates) and bile acids; retention of water trapped in the insoluble fibers and increased fecal bulk, leading to laxation. The faster transit time also decreases length of exposure to luminal carcinogens and increases the elimination of bile-based conjugates before they can be metabolized by colonic microflora into potentially toxic secondary metabolites. Some of these effects may be more than just a result of decreased transit time and entrapment of harmful substances in the aqueous fiber gel, for the effects of the total proportion dietary fiber as well as its composition of dietary have substantial impacts on the microbiome (see Digestive Tract, Vol 4, Chap 1) This leads to positive effects on detoxification and a positive effect on metabolism in general. The sum total effects of enhanced dietary fiber intake have been linked to decreased serum cholesterol, lowered blood glucose, lowered blood pressure, enhanced immune function, and decreased childhood obesity (Anderson et al., 2009). However, it must be noted that individual fiber components as well as insoluble/ soluble fiber proportions have a significant impact on the efficacy of fiber enhancement in the diet. Most grain-based diets, now rarely used in research, have a mixture of insoluble and soluble fiber that may vary from batch to batch, depending on grain source. Purified diets with consistent known composition, such as AIN76/76A and AIN 93G/93M, contain only insoluble fiber (cellulose). This produces consistency and minimizes potential confounding effects due to variation in fiber content between batches of diet. It should be noted, however,

that cellulose is insoluble and only a small amount is degraded by GI microflora, leading to limited SCFA production, altered microbiota composition, and smaller ceca and colon. Inclusion of soluble fiber in diets can significantly alter cecal/colonic weights and microbiota composition (Pellizzoni and Ricci, 2018). Therefore, dietary fiber composition should be considered when performing studies investigating effects of fiber and microbiota on obesity, diabetes and hypercholesterolemia. Despite its overall beneficial effects, excessive intake of insoluble fiber may result in binding of essential dietary minerals; therefore, ingestion of large amounts of fiber may interfere with intestinal absorption of these minerals. Hypomagnesemia is the chief concern although theoretically, a loss of Ca and exacerbation of osteoporosis could be an additional concern. The reduction in absorption of minerals and vitamins could, in theory, have adverse nutritional consequences, particularly in populations fed diets inherently deficient in these nutrients, for example, in developing countries or fastidious, health food conscious communities where diets may be marginal in micronutrients but high in fiber. Lipids Dietary lipids (fats) are important not only from an energy/calorie standpoint but also as carriers for the absorption of certain other essential nutrients, especially the lipid-soluble vitamins. Also, there are certain short-chain polyunsaturated FAs (SC-PUFAs) that are essential for optimal growth and health in mammalian species and yet cannot be synthesized endogenously. In particular, the n-3 and n-6 unsaturated FAs, linolenic (n-3) and linoleic (n-6), are necessary for optimal membrane-bound enzyme function and as precursors for longer chain PUFAs (e.g., arachidonic acid [AA]) that serve as substrates for the synthesis of bioactive molecules such as the prostaglandins and leukotrienes. The requirement for n-3 and n-6 FAs can be met in most mammalian species including rodents by diets containing adequate concentrations of linoleic and linolenic acids. Prior to the advent of the AIN 93 diets, corn oil was the source of lipid in most rodent diets, in particular the standard AIN 76A formulation; however, corn oil is relatively low in linoleic and linoleic

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acids. Soybean oil has replaced corn oil as the source of lipid in the AIN 93 diets since soybean oil has a much higher proportion of linolenic acid (Reeves, 1997). Other sources of essential FAs, basically bypassing the linoleic/linoleic bottleneck, are diets containing lipids rich in longchain PUFAs such as those in fish oil. DEFICIENCY

Whereas deficiencies of certain essential fatty acids, in particular linoleic, linolenic, and arachidonic acids, can be induced under experimental conditions, naturally occurring fatty acid deficiency is rare and requires a long time to develop. For most mammalian species, linoleic acid is essential, while for linolenic and arachidonic acids there is considerable species variability in essentiality. FA deficiency may occur if biliary or pancreatic dysfunction leads to impairment of fat digestion and absorption, which also may lead to deficiencies of fat-soluble vitamins (Shireman, 2003). Dogs and cats are sensitive to dietary deficiencies of linoleic acid, since they lack the enzyme necessary to synthesize this FA (Bauer, 2006). Although uncommon, deficiency of linoleic acid is seen when dogs or cats are fed diets that are low fat and homemade, poorly formulated, overcooked, or poor-quality diets in which the lipid components have become rancid. FA deficiencies typically manifest themselves as skin abnormalities, including scaling and dermatitis with seborrhea due to abnormal keratinization, poor hair quality, and secondary bacterial infections, leading to dehydration and failure of wounds to heal (Watson, 1998). Dietary FA deficiencies are typically accompanied by vitamin deficiencies, such as Vitamin E, and commonly linked to excessively low-fat, dry, poor-quality diets. EXCESS

There are numerous references to ingestion of high-fat diets as a risk factor for carcinogenesis. However, there is confounding evidence when the effects of low carbohydrate intake are taken into account (i.e., CR, rather than fat intake per se, is the protective factor; Lv et al., 2014); therefore, the issue of separating high-fat intake from high caloric intake is yet to be resolved and beyond the scope of this chapter. Increasing dietary fat concentrations per se raises the incidence

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and multiplicity of mammary cancers in a wide variety of carcinogen induced, radiation induced, spontaneous, transplantable, and metastatic rodent cancer models. Dietary PUFA content may also play a role in carcinogenesis and other chronic diseases (Ip, 1997). Again, as with total dietary fat intake, results are conflicting in both human clinical epidemiological and nonclinical animal experimental studies (Azrad et al., 2013). In general, tumor incidence is increased when diets contain high levels of specific PUFA, in particular n-6 omega FAs, and decreased when diets are supplemented with n-3 omega FAs (Bartsch et al., 1999). Mono- and disaturated FAs (e.g., oleic and linoleic acids) have also been associated with decreased carcinogenesis. As a final consideration, experimental design in rodent carcinogenesis studies is often predicated on the addition of a particular fat or fatty acid to a purified diet which is then fed to the experimental animals of choice; however, this does not account for the variety of fats, from numerous sources, ingested by human beings.

4.3. Micronutrients (for General Background References See Table 3.1) Numerous references exist for general background information on the nutrients discussed below. References regarding vitamin requirements for humans and animals, bioavailability, general features of nutritional disorders in human and animals, and additional basic information are listed first in Table 3.1. Vitamins (see Table 3.2) The lipid-soluble vitamins (A, D, E, and K) are essential nutrients that are absorbed and transported in similar fashion to typical lipids (i.e., via bile salt–dependent bicellular pathways). Then they are stored in adipose tissue and liver. Hence, they have the potential to accumulate over time when too much of the vitamin is ingested, increasing the potential for chronic toxicosis. Water-soluble vitamins like Vitamin C and the B vitamins present a somewhat different picture toxicologically from the lipid-soluble vitamins since their toxicokinetics are not the same. Water-soluble vitamins tend not to

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TABLE 3.2 Summary of Vitamin Toxicities Typical lesions associated with toxicityb

Vitamin

Toxic dosea

A

2e5  106 IU/day (acute) 1e2  105 IU/day (chronic)

Redifferentiation of epithelium into more complex forms (general mucoid) leading to perioral dermatitis, gingivitis, alopecia Periosteal proliferation of bone with loss of cortical bone, exostosis with narrowing of vertebral canal, skeletal malformation (young) Hepatomegaly due to lipid accumulation within stellate cells

Hyperirritability, dizziness, vomiting, diarrhea, erythema (acute) Dermatitis, pruritus, double vision, headache, stiffness, pathologic fractures, secondary paralysis (chronic)

D

>5  105 IU/day

Widespread deposition of hydroxyapatite in tissues, especially the kidney, pulmonary calcinosis (acute) Calcified joint capsules, abnormal spongy bone formation, pulmonary calcinosis

Hypercalcemia and hyperphosphatemia, renal failure with polydipsia and polyuria, dyspnea, coma, death

E

300e3000 IU/day

Depression of tissue Vitamin K concentrations leading to bleeding tendencies, depression of prostaglandin, and leukotriene generation Impaired wound healing

Headache, nausea, fatigue, and double vision, with severe muscle weakness, GI disturbances

K

5e25 mg/day

Generation of free radicals via redox recycling (K3 form) leading to hemolysis

Hemolysis (human), renal tubular necrosis with mineralization (horse, rat)

C

>1 g/day

Osmotic malabsorption in GI tract Enhanced conversion to oxalic acid with increased excretion and precipitation in urine (selected individuals)

Diarrhea, nausea, cramps Oxalate urolithiasis

Thiamine

>900 mg/day

Neuromuscular dysfunction

Dizziness, flushing, headache, convulsions, paralysis, cardiac arrhythmias

Not defined

Not defined

Cutaneous vasodilation Cholestatic liver disease, duodenal ulcer

Flushing, pruritus, headache Jaundice, GI disturbances

Demyelination with secondary axonal degeneration of peripheral sensory nerves, sensory nerve tracts in CNS

Progressive ataxia, loss of sensation in digits, tongue, and lips

Riboflavin Not defined Niacin

>100 mg/day (oral) >25 mg/day (iv)

Pyridoxine >2e6 g/day (over prolonged period)

Clinical manifestationsc

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TABLE 3.2

Summary of Vitamin Toxicitiesdcont’d Typical lesions associated with toxicityb

Vitamin

Toxic dosea

Biotin

Not determined

Impaired estrogen and progesterone synthesis; impaired spermatogenesis (rats)

Impaired reproductive performance (rats)

Folic acid

>15 mg/day

Neurological abnormalities Nephrotoxicity (tubular) Neural tube and cardiac defects

Malaise, depression, irritability, altered sleep patterns (adults) Resistance to antiseizure medications and insulin resistance (children) Renal failure, spasticity, aggressiveness (rodents) Malaise, irritability, depression, sleep disturbances

Not defined

Not defined

Cobalamin Not determined

Clinical manifestationsc

a

Doses based on human data, not established for most animals. Based on both human and animal data. c Mainly based on human data. Modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Table I, Vol. 2, Chapter 36, p 1091, with permission. b

accumulate in tissues, partly because they are not lipid soluble, allowing them to be filtered out of plasma by the kidneys and excreted in the urine. For this reason, it is hard to “saturate the system” with these vitamins since no organ takes them up and stores them long-term; any excess is excreted into the urine. Therefore, toxicities of water-soluble vitamins, when they occur, tend to be acute in nature and regress rapidly if the patient survives the episode. VITAMIN A (ALL TRANS-RETINOL)

Vitamin A is found in nature in many precursor forms (see below), but the active form of Vitamin A at the cellular level is not actually retinol but retinoic acid. Retinol is metabolized after entering the cell into retinaldehyde then retinoic acid. However, retinol per se is essential for function in retinal rod cells. Retinoic acid is the ligand for a family of nuclear receptors, the retinoid acid receptor family (RARs). The RARs heterodimerize with one of the retinoid X receptors (RXRs) to bind to the appropriate hormone response element in DNA as a nonactive complex until the ligand (in this case, retinoic acid) binds and forces

dissociation of an associated repressor from RAR, allowing for transcription of the genes regulated by that transcriptional unit. The RXR associated with RAR is often associated with numerous nuclear factors, including PPAR. The exact downstream effects of retinoic acid are complex and not clearly delineated (Conaway et al., 2013). Another role for Vitamin A is its essentiality for night vision. 11-Cis-retinal, a metabolite of retinol, is a critical component of the photoreceptor protein, rhodopsin, in retinal rod cells. b-Carotene, basically a dimer of Vitamin A, is the most bioavailable and crucial source of Vitamin A in plants, especially rich in leafy green vegetables and vegetables and fruits that are yellow or orange in color (indicating a high carotenoid content). However, there are a host of other plant and vegetable carotenoids that vary widely in bioavailability, and many of these are poorly converted to active Vitamin A (Sommer, 2008). The liver stores >90% of the body’s Vitamin A, mainly in hepatic stellate cells (Chelstowska et al., 2016). Most of the remaining Vitamin A is stored in adipose tissue, with species variability

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regarding its storage in other organs, for example, in the kidney in dog and monkeys (Penniston and Tanumihardjo, 2006). In addition, the liver is responsible for producing the binding protein necessary for transport of Vitamin A to peripheral tissues. Hence, “organ meats,” in particular liver, are the primary source of Vitamin A for carnivores. The RDA for Vitamin A in humans is 2300 and 3000 IU for adult females and males, respectively (IOM, 2001). DEFICIENCY

Severe Vitamin A deficiency, although virtually unknown in Western countries, is still a disease with high morbidity in some underdeveloped countries. Throughout history, including well into the 20th century, severe Vitamin A deficiency was the second most prevalent nutritional disease in humans behind protein-calorie deficiency. Marginal deficiency is another matter, however, and may be more common than generally thought, mainly because standard methods for assessing Vitamin A status often do not consider the widely varying activities and potencies of the various provitamin forms of Vitamin A. Deficiency disease begins to manifest itself when daily intake for long periods of time is less than 70% of the RDA. Such situations can occur when there is insufficient intake of fresh vegetables and fruits for herbivores/omnivores (like humans) or insufficient Vitamin A/carotenoid rich organ meats or animal fats for carnivores. Vitamin A deficiency also can occur in cases of severe protein deficiency, where synthesis of retinol-binding protein essential for transport of Vitamin A to peripheral tissues is inadequate. In addition to poor dietary intake, prolonged GI or liver disease is a common cause of Vitamin A deficiency. Vitamin A uptake from the diet is dependent on bile, pancreatic enzymes, dietary lipids, and chylomicron formation; thus, long-standing GI disease with malabsorption or maldigestion can result in deficiency (Saeed et al., 2018). Severe liver damage (e.g., secondary to chronic toxicity and NAFLD) can also lead to Vitamin A deficiency. The most common pathophysiologic effect of Vitamin A deficiency is night blindness (nyctalopia) followed by keratinization of the conjunctival epithelium and lacrimal glands, leading to “dry eye” or xerophthalmia. Both conditions

are reversible in early stages, but if deficiency persists, perforation of the cornea and permanent loss of vision occurs. Initially retinal lesions often are not obvious but ultimately there is degeneration of rods, leading to significant retinal atrophy (Labelle, 2017). The corneal, lacrimal, and conjunctival lesions caused by Vitamin A deficiency are a manifestation of the main systemic pathophysiologic effect of Vitamin A deficiencydsquamous metaplasia of pseudostratified columnar epithelium. This reflects Vitamin A’s essential, but mechanistically complicated, role in development, differentiation, and apoptosis via effects on numerous genes containing Homeobox sequences (Balmer and Blomhoff, 2002). Aberrant keratinization is not just confined to ocular adnexae in Vitamin A deficiency. Squamous metaplasia with secondary loss of function is eventually manifested in the genitourinary, upper respiratory, and GI tracts. Barrier function is impaired due to loss of cilia and/or mucusproducing epithelial cells because of squamous metaplasia. Impaired mucociliary clearance in the respiratory tract and malabsorption/maldigestion in the GI tract due to metaplastic changes in absorptive epithelium may be responsible in large part for the increased susceptibility to respiratory infections and diarrhea noted in severely deficient individuals. Squamous metaplasia of glandular ducts, such as in the pancreas, salivary glands, and cholangioles (experimentally in rats), also is common. Direct impairment of immune function also occurs, with impaired Th2 responses. Evidence of Th1 response impairment is somewhat conflicting, with some proinflammatory cellular (macrophage and neutrophil) and acute phase responses enhanced and NK responses suppressed (Stephensen, 2001). The integument is frequently affected in domestic animals, with seborrhea and raised cutaneous plaques. Temporally, this change is reflective of an initial overproduction of basal cells in the epidermis, leading to subsequent acanthosis and orthokeratotic hyperkeratosis. If persistent and long-standing this can become metaplasia. The keratinocytes are abnormally keratinized, with decreased and abnormal tonofilaments, altered keratin proteins, and decreased cell-to-cell adhesion (Fu et al., 2007; Klein-Szanto et al., 1980). A classic example of the cutaneous effects of Vitamin A deficiency is

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the historical “X disease” in cattle which occurred after exposure to chlorinated naphthalenes, once commonly used as additives to petroleum-based lubricants and wood preservatives (Figure 3.2). These now banned lipophilic toxicants interfere with the conversion of b-carotene to retinol (Hargis and Myers, 2017b). Experimental evidence in rodents (Niles, 2000) suggests that there is an enhanced susceptibility to cancer in Vitamin A–deficient individuals. However, the link between Vitamin A deficiency and cancer is not clear and the epidemiologic data are contradictory; retinoids other than Vitamin A may be more potent. EXCESS The toxicity of Vitamin A (hypervitaminosis A) is manifested in two forms: acute and chronic (Penniston and Tanumihardjo, 2006). There are numerous case reports in the human medical literature of acute toxicity after excessive ingestion of marine fish livers or livers of animals in the food chain where marine fish are heavily consumed (e.g., seal livers and polar bear livers). Precursors of the vitamin, such as the carotenoids, are not toxic, even at very high doses, although an unpleasant yellow or orange hue may discolor the individual consuming excessively high doses. However, retinoids or retinyl esters used for control of skin conditions can be Vitamin A agonists, binding to Vitamin A receptors and activating

FIGURE 3.2 Hyperkeratosis (“X disease”) at the base of the horn in a steer exposed to chlorinated naphthalenes. Courtesy of Dr. John King. Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Fundamentals of toxicologic pathology, ed 2, Academic Press, 2010, Figure 7.22, p. 154, with permission.

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them, producing a hypervitaminosis A type condition (Conaway et al., 2013). Neonates are more sensitive to toxic effects of Vitamin A or Vitamin A agonists and can become acutely intoxicated at much lower doses. General signs common to many species are vomiting (if possible), diarrhea (in neonates), and redness of the skin. In humans, headache, hyperirritability, and bulging fontanelles (in infants) commonly are manifest. Symptoms generally subside in a few days with no permanent side effects if no further vitamin is ingested. Chronic hypervitaminosis A is much more common and insidious and generally associated with self-prescribed oversupplementation by humans or improperly mixed diets in domestic animals. Aqueous preparations of Vitamin A are most prone to overdose, since absorption is better than with traditional oil-based preparations. Symptoms are variable and may not necessarily correlate with blood levels or even dosage above a certain point. Toxicity at the cellular level is manifested by redifferentiation of simple types of epithelium into more complex forms, including mucous epithelium. Accompanying this is decreased cohesion between epithelial cells in the skin. Accordingly, most affected humans report skin changes such as pruritis, erythema, eczema, and dermatitis with bleeding and cracking of the skin, especially around lips and gums, as well as hair loss and nosebleeds. Double vision and headache are frequent, and intracranial hypertension is common. The mechanism is not fully defined but perhaps due to impaired cerebrospinal fluid absorption leading to increased cerebrospinal pressure (Lombaert and Carton, 1976). In domestic animals there can be narrowing of the spinal canal and brain case due to bony changes (Figure 3.3). These changes are the result of increased proliferation of periosteal bone and bone near the growth plate. The bone initially becomes thicker and then less dense, weaker, and more prone to fracture as the osteoblasts become dysfunctional and die (Olson and Carlson, 2017a). Enlargement of the liver has been reported due to an accumulation of vast numbers of dysfunctional Vitamin A laden hepatic stellate cells, loss of hepatocytes, and ongoing fibrosis eventually leading to liver failure (Leo and Lieber, 1988). There is evidence that Vitamin A also

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FIGURE 3.3 Vertebral column and ribs from a capybara consuming excessive quantities of sweet potatoes rich in Vitamin A. Note the severe bony exostoses along the cervical and thoracic vertebral facets and at the costochondral junctions. Courtesy of Steve Weisbrode. Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 36.2, p. 1093, with permission.

can be teratogenic, apparently because of its role in inducing programmed cell death at the appropriate time in mesenchymal and epithelial development in the fetus, in particular the face, ears, eyes, digits, and brain (see Embryo, Fetus and Placenta, Vol 5, Chap 11). Epidemiologic evidence for teratogenicity, however, is still sparse. Recent epidemiologic evidence, supported to some extent by research models in rodents, suggests that excessive Vitamin A has a prooxidant effect, actually suppressing levels of enzymes such as superoxide dismutase and catalase, with the lung being the most affected. Vitamin A toxicity in domestic animals generally is a disease of domestic cats (rarely dogs) consuming large amounts of bovine liver (Polizopoulou et al., 2005). Noncarnivorous animals consuming large amounts of plant containing Vitamin A analogs will also develop osseous lesions (Figure 3.3). The effect is mainly on bone and apparently results from weakening of the bone at points of stretching or tension where ligaments and tendons attach. Fractures are most frequent in the neck and thoracic spine and in upper forelimbs, probably due to contortions associated with grooming. Fractures lead to bony callus formation; calluses then impinge on nerves leaving the spinal canal as well as impair bending of the spine, the result being

FIGURE 3.4 Expanded cervical vertebral facet in a macaque with ankylosis of cervical vertebrae after treatment with an experimental retinoid that was a Vitamin A agonist. Greatly increased mature, dense trabecular bone surrounds the cartilaginous facets at the center of the image. Hematoxylin and eosin stain. Courtesy of Steve Weisbrode. Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 36.3, p. 1094, with permission.

a stiff necked, painful animal (Figure 3.4). In dogs, and cats as well, Vitamin A excess in young animals can result in osteoporosis and destruction of growth plates, leading to dwarfism, elongation of tuberosities on long bones, rotation of the epiphyses of long bones, and pathological fractures. Similar bony lesions occur in growing chickens and turkeys. VITAMIN D (CALCITROL)

Vitamin D (also known as 1,25-dihydroxy Vitamin D) is the “sunshine” vitamin. Severe cases of either deficiency or toxicity have not been as commonly reported in recent times due to increased awareness of the consequences of both overt deficiency and excess, which are serious, often irreversible, and even life-threatening diseases. In species where sunlight is critical for adequacy, conversion of plasma cholesterol to cholecalciferol occurs via ultraviolet-B (UVB) radiation from sunlight. The cholecalciferol then is converted in an unregulated fashion by several hepatic cytochrome P450 (CYP) enzymes. The key step in Vitamin D formation occurs in

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the kidney, in a rate-limiting step where 25monohydroxy Vitamin D is hydroxylated to its active 1,25-dihydroxy form by one CYP, CYP27B1. Active Vitamin D exerts effects both at the molecular level and the cellular level via the Vitamin D receptor (VDR). Direct binding to the VDR produces rapid effects in the enterocyte (enhances Ca and phosphate uptake from the gut lumen), chondrocytes in growth plates of bones (triggers ossification), and keratinocytes (initiates photoprotection). Vitamin D’s most important short-term effect is to enhance uptake of calcium from the GI lumen via upregulation of the calcium-binding protein, calbindin, in the enterocyte. Calbindin mediates the transfer of Ca into the endoplasmic reticulum for ultimate transport to the basal portion of the enterocyte and subsequent release into extracellular fluid. For genomic effects, activated VDR translocates and binds to the RXR in similar fashion to retinoic acid. The complex then binds to and activates the Vitamin D Response Element in the promoter regions of over 200 genes, many of the transcription factors specific to the cell type being affected, including enterocytes, colonocytes, chondroblasts, and osteoblasts. A link between Vitamin D and cell differentiation (also apoptosis) has been definitively shown for in the colon. The VDR also has epigenetic effects, many of which have been linked to maintenance and enhancement of muscular function and immunity (Bikle, 2014, for additional details). Because of the roles of sunshine exposure, pigmentation status of the skin and differences in absorption of the various nonsunshine sources of Vitamin D, establishing standards for intake and guidelines for dietary consumption, have been difficult. The RDA for Vitamin D is set at 800 IU (20 mg)/day for adults over 70 years of age and 600 IU (15 mg)/day in adults less than 70 and pregnant/lactating womein have been recommended (IOM, 2011b). Sources of Vitamin D3 include organ and muscle meats from beef, pork, lamb, and poultry; fish; egg yolks; and butter (Schmid and Walther, 2013). Cooking can decrease Vitamin D3 content 10%–30% (Jakobsen and Knuthsen, 2014); however, milk and cereals are commonly fortified with Vitamin D3 in developed countries. In the past two decades numerous trials assessing the preventative effects of Vitamin D supplementation on diseases such as

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cardiovascular disease and cancer, disease resistance, and depression/anxiety (see below) have been performed. In at least one meta-analysis, the impact of Vitamin D in reducing mortality from a variety of causes was only significant for cancer (Zhang et al., 2019) such as breast, prostate, colon, and other cancers (Engel et al., 2010; Tretli et al., 2009; Di Rosa et al., 2013; Giovannucci et al., 2006). These effects have been linked to Vitamin D’s gene regulatory roles in cell prodifferentiation, proapoptotic, and antiproliferative pathways as well as its role in stimulating antioxidant pathways (Jeon and Shin, 2018). Whether supplementation has a role above and beyond correction of physiological insufficiency (see below), restoring balance in the aforementioned pathways, or an additional supraphysiologic role is not entirely clear, but there is evidence that Vitamin D supplementation over and above that needed to correct core insufficiency can overcome the dysregulation of Vitamin D metabolism seen in many types of cancer (e.g., prostatic cancer) and actually cause tumor suppression/regression. DEFICIENCY Historically, clinical Vitamin D deficiency occurs when one or more of the following factors are present: insufficient exposure to sunlight (decreased conversion of plasma cholesterol to nonhydroxylated cholecalciferol [Vitamin D3], the first step in active synthesis); inadequate dietary intake of Vitamin D in lieu of sunlight; or chronic renal disease due to insufficient conversion by remnant renal convoluted tubules of 25-monohydroxy Vitamin D to active 1,25-dihydroxy Vitamin D. Occasionally, GI disease can cause deficiency. In humans, dietary sources of intact Vitamin D are usually inadequate and plant ergosterols (Vitamin D2), cholecalciferol analogs which can be abundant, are poorly converted, if at all, to 25-monohydroxy Vitamin D and hence do not contribute significantly to Vitamin D adequacy. Although overt clinical Vitamin D deficiency is seldom encountered today, the role of “insufficiency,” rather than deficiency in the elderly, those using excessive sunscreen, people living in high latitudes, or those who stay indoors most of the time, is becoming more commonly reported (Holick, 2017). There is a growing body of evidence that even moderate “insufficiency” (serum levels 20 ng/mL or less), as

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opposed to a deficiency causing outright signs of disease (serum levels 1000 mg, serious, physiological effects have been reported. The most critical toxic effect is depression of prothrombin levels and decreased clotting time in patients undergoing anticoagulant therapy or deficient in Vitamin K, apparently because excessive Vitamin E, which is structurally similar to Vitamin K, impairs Vitamin K utilization, possibly by enhancing its metabolism via induction of CYPs that convert Vitamin K to inactive excretable metabolites (Traber, 2008). Vitamin E toxicosis is rarely reported in animals. However, hemorrhagic tendencies have been shown experimentally in chicks and rats, associated with a suppression of Vitamin K–dependent coagulation reactions (March et al., 1973; Abdo et al., 1986). VITAMIN K

Vitamin K and its analogs are structurally quite similar to Vitamin E. Vitamin K is a cofactor in the posttranslational modification of two sets of proteins: (1) Factors II, VII, IX, and X of the clotting cascade (as well as Proteins C and S), all g-glutamyl carboxylases themselves, and (2) the bone proteins, osteocalcin (bone Gla protein), matrix Gla protein (MGP), and periostin. There

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are also vascular Gla matrix proteins which are affected by Vitamin K. The posttranslational modifications involve the selected carboxylation of specific glutamic acid residues by the requisite g-glutamyl carboxylase on the proform of the target protein, adding a second carboxylic acid (–COOH) to the g carbon. These modifications are necessary for these target proteins to function properly, usually binding Caþ2 as part of the process (Dowd et al., 1995). Vitamin K1, also known as phylloquinone, is derived mainly from leafy green vegetables, and it is primarily responsible for activation of coagulation factors. Vitamin K2, on the other hand, termed menaquinone (MK), is derived mainly from bacterial action on K1, forming the MK-7 subtype, or by tissue cells themselves, forming the MK-4 subtype form K2 (Akbulut et al., 2020). Both MK-4 and MK-7 have key roles in promoting osteoblast differentiation and preventing osteoblast apoptosis, bone mineralization by carboxylation of osteocalcin and stimulation of alkaline phosphatase production, and inhibition of osteoclast differentiation (Villa et al., 2017), especially in the elderly. There are additional if somewhat different effects on blood vessels, preventing calcification and preventing apoptosis of smooth muscle cells. Most Vitamin K is derived from dietary leafy green vegetables in the form of phylloquinone (Vitamin K1), with lesser amounts in fruits and nuts. Phylloquinone accounts for 90% of dietary Vitamin K in most cases. Vitamin K2, in the form of menaquinones (MK-4 in meats and dairy and MK-7 in fermented foods such as sauerkraut and cheese), accounts for most of the remainder. Vitamin K is metabolized in liver to menadione and transported to extrahepatic tissues, where it is converted to MK-4, then transported back to the liver for further metabolism and excretion. The RDI for adult humans is generally 60–120, increasing with age (Trumbo et al., 2001). DEFICIENCY It is difficult to achieve a deficiency of Vitamin K in adults unless an individual’s intestinal microflora’s production of menaquinones (source of Vitamin K2) is eliminated or disrupted (e.g., prolonged oral antibiotic therapy) and ingestion of fruits or vegetables (source of Vitamin K1 or phylloquinone) is inadequate. Impaired lipid absorption due to chronic intestinal disease or cholestasis

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may contribute to deficiency, as with the other lipid-soluble vitamins. Therapeutically, Vitamin K antagonism using compounds like warfarin (see below) is used short-term postsurgically and long-term in elderly patients at risk for stroke to produce a subclinical deficiency, as it were. The classic clinical syndrome of clinical deficiency in humans (and animals) is a prolonged clotting time and bleeding tendencies. There have been observational studies reporting a tendency for osteomalacia leading to hip fractures in elderly patients, who tend to be Vitamin K deficient, especially under anticoagulant therapy, but there is little clinical trial evidence to support the observations (Rodrı´guez-Olleros Rodrı´guez and Dı´az Curiel, 2019). Breast-fed infants in underdeveloped countries are also at risk for Vitamin K stores that are low at birth because breast milk has low Vitamin K concentrations (Shearer, 2009). The resultant Vitamin K deficiency bleeding (VKDB) is especially dire since it is often intracranial. Children in underdeveloped countries, where diet is poor, are also at increased risk for deficiency disease. In animals, ingestion by cattle of moldy sweet clover (Melilotus spp.) or moldy sweet vernal grass (Anthoxanthum odoratum) hay/silage leads to severe hemorrhagic tendencies, including uncontrolled bleeding from the nose and mouth, extensive subcutaneous and intramuscular bleeding from the slightest trauma, bleeding into joints and abdominal cavity, as well as ecchymotic and petechial hemorrhages in internal organs such as the endocardium (Yamini et al., 1995; Runciman et al., 2002). Both plant types contain coumarin; if the plants are improperly cured or ensiled they can become contaminated by fungi such as Fusarium, which convert coumarin to the active toxin, dicoumarol. Dicoumarol is a potent inhibitor of Vitamin K epoxide reductase, the enzyme that allows for recycling of Vitamin K within the body. Over the course of a month or so of ingestion, sufficient loss of Vitamin K occurs such that hemorrhage occurs. Dicoumarol is also the active component of warfarin, used not only as a rat poison but therapeutically in cardiac patients with thrombotic tendencies or postoperatively to prevent hypercoagulation of blood. EXCESS It is almost impossible to become intoxicated by either Vitamin K1 or K2 via the

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diet or experimentally since an individual cannot consume enough plant or animal material to achieve toxic levels. Trouble arises, however, when an individual is treated parenterally with menadione (Vitamin K3), which is a provitamin that is activated to Vitamin K2 after ingestion or parenteral administration. Toxicity is associated not with Vitamin K3’s function as a cofactor but with its biochemical structure as a quinone, making it possible to undergo redox recycling to generate free radicals, resulting in hemolysis (Ansbacher et al., 1942). Acute tubular necrosis has been reported in horses. Renal, cardiac, and hepatic necrosis with secondary mineralization and necrosis have been observed in the rat at or above 100 mg/kg ip (Chiou et al., 1997). Perhaps the greatest cautionary note for humans regarding Vitamin K is for those being treated with Vitamin K antagonists to prevent thrombosis. Ingestion of Vitamin K–rich vegetables (especially cruciferous vegetables) can lead to reversal of the Vitamin K antagonism (with subsequent thrombosis) if the dose of antagonist is not adjusted accordingly. Scientists have identified a Ca/Vitamin K interaction in pigs; high levels of dietary Ca can interfere with Vitamin K–dependent clotting mechanisms (Hall et al., 1991). This can be overcome with Vitamin K supplementation. VITAMIN C (ASCORBIC ACID)

Vitamin C has a variety of functions; the most important, from a deficiency disease standpoint, is its role in hydroxylation reactions, in particular the hydroxylation of proline in collagen to stabilize its triple helical structure. Vitamin C also serves as a crucial water-soluble antioxidant in the cytosol where it serves as a free radical scavenger, to recycle Feþ3 and Cuþ2 to their biologically active forms Feþ2 and Cuþ, respectively, and a cofactor in a variety of vitamin-and mineral-dependent metabolic pathways. Dietary Vitamin C also enhances Fe uptake from the gut. The free radical intermediate of Vitamin C is relatively stable; hence, its ability not only to scavenge free electrons but also to donate them to a variety of reactions (Smirnoff, 2018). It has been found that Vitamin C modifies vascular tone as a cofactor in the production of endothelial NO, a key vasodilator (Lykkesfeldt et al., 2014). Vitamin C is nonessential to most mammals and birds, and all reptiles and amphibians, since

they can synthesize Vitamin C from UDP-Dglucose in a multistep pathway. Vitamin C is essential only in Guinea pigs, fruit bats, primates (including humans), and certain fructivorous birds, all of which have nonfunctional gulonolactone oxidase, the terminal enzyme in the synthetic pathway. Green leafy vegetables, potatoes, tomatoes, and strawberries are especially rich sources of dietary Vitamin C, with far lesser amounts present in meat, dairy, and egg products. The RDI for humans is 75 and 90 mg per day for women and men, respectively (Food and Nutrition Board, 2000). Ingestion of 100 mg per day results in maximal plasma levels of 50 mM, which do not increase with additional intake, the excess Vitamin C being excreted in the urine. Certain conditions or factors, such as pregnancy, cigarette smoking, and cancer, may increase the need for Vitamin C, and thus 200 mg/day is the maximum daily recommended dose for healthy adults. There is considerable epidemiological evidence that doses of Vitamin C above the RDA substantially reduce the risk of cardiovascular disease, including coronary heart disease, stroke, and hypertension; however, a causal link between high doses of Vitamin C and decreased risk of cardiovascular disease has yet to be established with clinical intervention studies (Lykkesfeldt et al., 2014). Also, there is no definitive experimental evidence that megadoses of Vitamin C have any effect on healthy humans, including prevention of the common cold. The positive effects of Vitamin C supplementation in unhealthy adults with chronic diseases, especially the elderly and those whose diets may be inadequate due to the primary disease or its secondary effects, have often been touted in numerous clinical reports, but even here the evidence for additional Vitamin C above the dose needed to restore normal levels is as yet unproven (Padayattym and Levine, 2016). Vitamin C has been used therapeutically in anemic patients, particularly those being treated for cancer, to enhance Fe uptake without increasing the risk for liver damage. DEFICIENCY

Deficiency is seen in primates and guinea pigs in association with diets poor in fruits and/or leafy green vegetables. “Scurvy” (or “scorbutus”) is the hallmark feature of Vitamin C deficiency, taking 1 month (guinea

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pigs) to several months (primates) to become manifest after elimination from the diet. Lesions of deficiency are classic and related mainly to increased vascular fragility. In all species there is gingivitis with hemorrhage and loosening of teeth. There are subperiosteal hemorrhages in bones as well as periosteal proliferation without deposition of osteoid or mineral and abnormal ossification characterized by calcification without ossification at growth plates (with resultant pathologic fractures). Multiple hemorrhages can be found in joints, muscle, and fascia. Poor wound healing is another feature of Vitamin C deficiency, along with skin abnormalities characterized by epidermal and follicular hyperkeratosis, abnormal hair growth, and perifollicular hemorrhage (Hirschmann and Raugi, 1999). EXCESS After megadoses of Vitamin C, acute toxicity does not appear to occur, although long-term high doses (generally above 2 g/day in humans) have been associated with GI disturbances such as nausea, diarrhea, and abdominal cramps, probably due to the osmotic effect of having a large bolus of slowly absorbed carbohydrate present in the GI tract. Perhaps of more importance is that megadoses of Vitamin C in some individuals can cause an increase in the excretion of oxalic acid into the urine, which can precipitate as oxalate uroliths. This does not occur in most individuals, where this pathway of Vitamin C metabolism is a minor one; however, there are those who have extremely efficient enzymes in their oxalate conversion pathway. Others predisposed to adverse effects include those with glucose 6-phosphate dehydrogenase deficiency or paroxysmal nocturnal hemoglobinuria, in whom hemolysis can occur (Padayattym and Levine, 2016). Excessive Vitamin C intake is dangerous to individuals with hemochromatosis, for Vitamin C increases the uptake of Fe and therefore enhances the likelihood of liver or heart damage. Individuals with chronic renal disease may not be able to excrete the increased acid load associated with megadoses of Vitamin C and suffer from acidosis. THE B VITAMINS THIAMINE (VITAMIN B1) Thiamine is the first of the B vitamins to have been discovered and the results of its deficiency are well known. However, its role in several diseases in domestic

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animals has been at times controversial and unclear. Thiamine is an essential cofactor for several key metabolic reactions, among them reactions linking the glycolytic pathway to the tricarboxylic acid (TCA) or Krebs cycle (pyruvate dehydrogenase and branched chain a-ketoacid dehydrogenase), reactions within the TCA cycle itself (a-ketoglutarate dehydrogenase), and a key reaction in the hexose monophosphate shunt (transketolase). It also is essential for the maintenance of axonal membranes. Its active form is thiamine pyrophosphate. Whole grains, legumes, fruits, yeast, and red meats such as pork are good sources of thiamine. Thiamine is readily absorbed in the jejunum and transported in the plasma to all tissue. Its half-life in tissues is substantially less than 24 h, so frequent dietary replenishment is necessary. A daily intake of 1.1–2.2 mg/day, 1.4 mg for pregnant women, is generally considered sufficient to prevent over deficiency (Polegato et al., 2019). Milling/processing of grains, in particular polishing of rice, results in loss of thiamine, which is concentrated in the hulls and bran. Adequate dietary thiamine is especially problematic in underdeveloped countries where the infrastructure for fortification or availability of thiamine fortified foods is limited, mainly Southeast Asia, sub-Saharan Africa, and Indonesia (Whitfield et al., 2018). Deficiency Besides inadequate diets, there are other risk factors associated with thiamine deficiency. Alcoholism is a major predisposing factor, not only due to poor nutrition but because alcohol impairs thiamine uptake. Chronic diseases that result in inappetence and poor dietary intake, such as cancer and other longstanding chronic illnesses (e.g., AIDS, chronic renal disease, diabetes), GI surgery, old age, and obesity, can also predispose to thiamine deficiency (Polegato et al., 2019). Overt deficiency disease in humans can manifest itself as “beriberi.” In “wet” beriberi, which is most prevalent in neonates less than 3 months of age, the initial abnormality is peripheral vasodilatation eventually leading to cardiac insufficiency and bilateral failure with resultant edema (DiNicolantonio et al., 2018). The “dry” form on the other hand, which predominates in children and adults, leads to nonspecific peripheral neuropathy with demyelination, first of sensory nerves, then of long motor nerves,

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with loss of function (Whitfield et al., 2018). This is due to inadequate energy via ATP for production and maintenance of myelin. A unique neurological form of thiamine deficiency, Wernicke–Korsakoff syndrome, is observed most often in prolonged deficiency, especially in alcoholics, and is the result of hemorrhage and concomitant necrosis of neurons with accompanying atrophy in the paraventricular regions of the midbrain, dorsolateral thalamus, medulla, cerebellar vermis, primitive forebrain, and outer cerebral cortex, in order of severity (Arts et al., 2017). Thiamine deficiency is an ever-present concern in elderly individuals or the chronically ill, where food intake may be insufficient to meet daily thiamine requirements. The counterpart of Wernicke–Korsakoff in domestic animals has been termed Chastek paralysis, first described in cats, foxes, and mink consuming raw fish rich in thiaminase. The disease now is very rare. The lesions are similar in morphology and distribution to those in humans (Irle and Markowitsch, 1982). Myocardial lesions also have been described. In the past several years, the FDA and pet food companies themselves have recalled several types of canned meat- or fish-based cat food preparations due to the occurrence of clinical thiamine deficiency in pet cats fed diets exclusively composed of the particular preparations (Kritikos et al., 2017). Some studies have linked the occurrence of thiamine deficiency in some of these preparations to use of sulfur dioxide as a preservation agent, resulting in the unintentional inactivation of thiamine in the food. Ruminants, whose microflora normally produces ample amounts of thiamine, may suffer deficiency upon consumption of thiaminase-rich plants such as bracken fern (Pteridium aquilinum) or horsetail (Equisetum arvense) (see Poisonous Plants, Vol 3, Chap 7). Excess Thiamine has been characterized as nontoxic in both humans and domestic animals, but there have been reports of toxicity associated with megadose consumption. Dizziness and flushing are the most noteworthy signs; these regress soon after ingestion is stopped. Administration of thiamine parenterally also can lead to toxicity in humans if it is administered at doses several hundred times the RDA of 1–1.5 mg (Leitner, 1947). There are important neuromuscular effects, including headache and convulsions, paralysis, and cardiac arrhythmias.

RIBOFLAVIN (VITAMIN B2) Riboflavin is source of essential cofactors for a host of metabolic pathways, many which are key for energy production. Riboflavin is an essential component of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are cofactors in a variety of integral oxidation reactions, including those in the TCA cycle, respiratory chain electron-transfer FA oxidation, purine catabolism, B6 metabolism, and monoamine oxidation (Thakur et al., 2017). It is also an essential cofactor in the reduction of oxidized glutathione by glutathione reductase in the detoxification of H2O2. Exogenous sources of riboflavin are milk, cheese, eggs, leafy vegetables, legumes, almonds, lean meats, organ meats, and mushrooms. Foods, especially cereal-based foods, are often fortified with riboflavin in developed countries to replace the riboflavin lost in milling and polishing. The RDA for riboflavin ranges from 0.3 to 0.4 mg/ day (adequate intake) in infants to 1.0 mg/day in adults to 1.4 and 1.6 mg/day for pregnancy and lactation, respectively (IOM, 1998a). Dietary riboflavin is absorbed in the small intestine. Another source of riboflavin is from gut microflora in the colon and this appears to have a role in colonocyte health, although much of this is not available for overall use in the body (Thakur et al., 2017). Therapeutic uses of riboflavin, other than supplemental to correct dietary deficiency, have included Parkinson’s disease and multiple sclerosis, due to its putative antioxidant function in the glutathione cycle and its role in folate regeneration. However, insufficient intervention and case–control studies have been performed (Saedisomeolia and Ashoori, 2018). There is promising evidence that riboflavin may be prophylactically protective against migraine in adults (Thompson and Saluja, 2017). Deficiency Deficiency, when encountered, is usually in combination with deficiencies of other B vitamins and a result of inadequate intake of food in general, as in alcoholics, the elderly, and the chronically debilitated. In domestic animals, deficiency is rare; ruminants receive their riboflavin from ruminal microflora and therefore do not require a dietary source. In humans and in animal models of riboflavin deficiency, the most consistent lesions are conjunctivitis with corneal neovascularization and

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opacity, cataracts, oral inflammation (especially glossitis), dermatitis with alopecia and scaling, normocytic hypochromic anemia, and neuropathy (especially in children and chicks). The most noteworthy lesion at the subcellular level is typified by changes in mitochondria: in some species (e.g., the mouse) mitochondria are enlarged while in other species mitochondrial volume and internal surface area are decreased. Although the signs and symptoms of riboflavin deficiency have been deemed “classic” in the past, there is growing opinion that the signs of classic riboflavin are nonspecific and reflect a combination of riboflavin deficiency with other vitamin deficiencies (notably thiamine and niacin) that usually accompany riboflavin deficiency in cases of poor dietary intake or malabsorption (Pinto and Zempleni, 2016). Excess To date, toxicity associated with high doses of riboflavin has not been characterized, and it appears that transport from the intestine into the body is saturated easily, making it difficult to achieve excessively high doses via the oral route. Also, riboflavin is eliminated very rapidly into the urine, again making it difficult to achieve high levels by parenteral routes. There is, however, a potential for photosensitization immediately after ingestion of megadoses of riboflavin, where it may reach sufficiently high blood levels in the short term to undergo redox recycling when exposed to UV light (Pinto and Zempleni, 2016). NIACIN (NICOTINIC ACID, VITAMIN B3) Niacin is vital as a cofactor for a host of metabolic reactions, especially in the breakdown of carbohydrates, proteins, and lipids for generation of ATP. Niacin is an integral component of nicotinamide adenine dinucleotide (NAD) and nicotinamide dinucleotide phosphate (NADPH) and as such has an essential role in key cellular pathways, including but not limited to glycolysis, TCA cycle, b-oxidation, hexose monophosphate shunt, electron transport chain, CYP system reactions, the glutathione cycle, and a host of enzymatic reactions requiring reduction and dehydrogenation. NAD is also involved in ADP ribosylation of proteins and PARP synthesis for DNA repair. Both NAD and NADP can be synthesized from tryptophan in the liver with varying degrees of efficiency (Yang and Sauve, 2018). The rat can derive its niacin needs

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exclusively from this pathway, so pure niacin deficiency does not occur in this species; most other species, which have the capacity to synthetize niacin endogenously, need an additional dietary source, either directly from plants or from gut microflora to meet their needs. Niacin is found in a variety of foods, both plant and animal. Foods with the most abundant amounts of niacin are yeast, tuna, and peanuts/ peanut products, but other fish, poultry, beef and pork, as well as sunflower and almonds also contain substantial amounts. Dairy and eggs are low in niacin, as are leafy green vegetables and grains, especially corn and polished rice (USDA, 2018). In developed countries, processed foods are often well fortified with niacin. The RDA for niacin is 14, 16 and 18 mg/day for adult females, adult males, and pregnant females, respectively (IOM, 1998b). Niacin is readily absorbed from the stomach and small intestine, although niacin in whole plant sources is less bioavailable than from other sources. Cooking, especially boiling, also results in substantial loss of highly soluble niacin in the cooking water. Niacin in fortified foods is the most readily absorbed. Megadoses (3–10 mg/day) of niacin in the form of nicotinic acid or one of several synthetic analogs have been used in the past to treat certain disease states in humans, in particular schizophrenia (Xu and Jiang, 2015) and hypercholesterolemia (MacKay et al., 2012). The nicotinic acid form of niacin has been shown to elevate HDL. The success of these interventions has been mixed and there have been numerous reports of adverse effects and even outright toxicity (see below). Niacin or its derivatives is also a component of many skin lotions, although evidence for beneficial effect in conditions other than deficiency is lacking. Deficiency Deficiency disease, termed “pellagra,” occurs in humans and pigs when diets rich in corn are consumed, corn being the one grain containing low concentrations of this vitamin. Chronic alcoholics also may be deficient. Niacin deficiency generally occurs in combination with other B-vitamin deficiencies, so that sorting out the various effects and relating them to deficiencies of the various vitamins may be difficult. In both humans (Pique-Duran et al., 2012) and pigs (Blair and Newsome, 1985), deficiency (pellagra) is characterized by a variety of signs,

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including anemia, bilaterally symmetrical plaquelike dermatitis of distal limbs and face with hyperkeratosis and epidermal atrophy, erythema, dermal fibrosis and alopecia, smooth tongue, diarrhea due to atrophy of GI epithelium, and nervous disorders characterized by degenerations of neurons in the brain and corresponding degeneration in associated tracts in the spinal cord. In dogs, ulcerative oral lesions have been reported, leading to the common name for the disease, “blacktongue” (Madhavan et al., 1968). Excess Niacin toxicity has become a concern recently in Western countries as reports of side effects have become common when the nicotinic acid form of niacin is used (MacKay et al., 2012). Administration of niacin but not nicotinamide will result in flushing of skin due to vasodilation in most patients, even at doses as low as 100 mg orally or 20 mg intravenously. This is accompanied by pruritis, headache, and GI disturbances. Use of nicotinamide and other forms of niacin for neurological issues prevent deficiency but have no impact on blood cholesterol and no flushing effects. Long-term, high doses of timerelease niacin preparations have been linked to abnormal liver function and even to chronic cholestatic liver disease with jaundice at doses of 750 mg per day (Rader et al., 1992). Experimentally, it has been difficult to induce niacin toxicity (Cosmetic Ingredient Review Expert Panel, 2005); however, in poultry there is growth retardation with shorter than normal legs and coarse feathering. PYRIDOXINE (VITAMIN B6) Pyridoxine is the dietary precursor molecule for pyridoxine-50 -phosphate (PLP), an essential cofactor for a huge number of metabolic reactions. PLP’s biggest role is in protein and amino acid metabolism, including transamination, decarboxylation, deamination, racemization, phosphorylation, and cleavage of cystathionine to cysteine in the methionine pathway. In addtion, it is a key factor in the synthesis of amino acid–derived neurotransmitters, conversion of tryptophan to niacin, and hemoglobin synthesis. It also has a role in lipid and carbohydrate metabolism, particularly in sphingolipid synthesis and glycogenolysis (Xu and Jiang, 2015). Clinical as well as biochemical evidence is accumulating that PLP has a key role in immunity and inflammation at least in part due to its effects on sphingolipid and the

methionine–cystathionine–cysteine pathways (Ueland et al., 2017). Sources of pyridoxine are similar to those for niacin, foods with the highest concentrations found being in liver, tuna, salmon, chicken, pork, beef, chickpeas, hazelnuts, and bananas (ODS, 2020a). Pyridoxine is readily absorbed from jejunum and ileum where it is immediately converted to PLP. The RDA for pyridoxine for adults is 1.20–1.4 mg/day and 1.6–1.7 mg/day for men (IOM, 1998g). Large doses of pyridoxine have been used to treat autism, schizophrenia, and even Down’s syndrome, and there has been some evidence that high doses are helpful in carpal tunnel syndrome, although none of these have been conclusively proven. Pyridoxine has also been used to alleviate symptoms of premenstrual syndrome. There is stronger evidence that treatment of patients with PLP in combination with L-methylfolate and methylcobalamin for dysesthesia associated with diabetic neuropathy is helpful. Recently, the FDA has approved a doxylamine–pyridoxine combination therapeutic for alleviation of nausea and vomiting in early pregnancy (Stover and Field, 2015). Deficiency In humans, subclinical pyridoxine deficiency is a common occurrence in alcoholics, chronically ill patients, or infants fed poorly formulated milk products. Pyridoxine deficiency is similar in presentation to niacin deficiency in both humans and animals, with chronic dermatitis, oral inflammation, and peripheral neuropathy. In some case convulsions can occur, especially in young calves, puppies, piglets, and rats. Cats may develop renal tubular necrosis with oxalate accumulation. An additional feature of deficiency is a hypochromic microcytic anemia. Pyridoxine can be depleted in individuals being treated with certain drugs such as isoniazid, estrogens, and penicillamine. Excess Pyridoxine is considered nontoxic, even at doses 10–500 times greater than the RDA. The toxicity of pyridoxine in humans is neurologic in nature at doses above 2–6 g/day during a 5–40 month period and slowly reversible over time (5–40 months) with cessation of therapy. Typically, there is a symmetrical, progressive ataxia, loss of sensation in the distal extremities, lips, and tongue, and a loss of sense of joint position and vibration (Schaumburg et al., 1983). The central nervous system and

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motor function are not affected. The loss of sensation may not be reversible. Pyridoxine neurotoxicity has been confirmed in dogs (Phillips et al., 1978) and rats (Perry et al., 2004) at doses of 50–300 mg/kg/day and is characterized by demyelination of peripheral sensory nerves and nerve roots, with demyelination and eventual axon degeneration in sensory tracts in the spinal cord. Falcons are sensitive to pyridoxine toxicosis at doses less (75 mg orally) than those tolerated by pigeons (200 mg orally) while chickens are much more resistant. The most prominent lesions in birds are acute hepatic necrosis, splenic lymphoid depletion, and arterial wall necrosis with hemorrhage (Samour et al., 2016). The rodenticide, Castrix (2-chloro-4dimethylamino-6-methylpyrimidine), toxic to mice, rats, Guinea pigs, rabbits, dogs, and chickens, is a chemical analog of pyridoxine, causing seizures and death. Castrix appears to inhibit the synthesis of PLP, in effect producing a severe PLP deficiency; this is further supported by the successful use of pyridoxine as an antidote (Rosen et al., 1964). BIOTIN (VITAMIN B7) Biotin is a covalently bound cofactor and essential in key carboxylation reactions. It is required by four key carboxylase enzymes in mammals, all involved in transferring CO2 between molecules in lipid synthesis or gluconeogenesis. Holocarboxylase synthetase binds biotin to its various carboxylases for them to function and biotinidase removes it for reuse (Zempleni and Kuroishi, 2012). Biotin has an additional important role as a gene regulator/modifier via biotinylation of histones, which alters DNA configuration. As such biotin has a role in gene repression, DNA repair, and chromatin stability (Zempleni et al., 2009). Biotin is distributed widely in plant and animal foods, being abundant in chicken and beef liver, eggs, and peanuts, with substantial concentrations in pork, fish, peanuts, sunflower seeds, and almonds. Most vegetables, grains, and milk have low concentrations (Staggs et al., 2004). Recommended daily allowance for adults is 30 mg/ day (IOM, 1998c). Dietary biotin is bound to ingested proteins and must be cleaved from them in the jejunum and ileum before absorption. Gut microflora also produces free biotin.

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Biotin supplementation has in the past been touted as a treatment for multiple sclerosis but recent studies have disputed the initial findings and the use of this vitamin for treatment has fallen out of favor (Motte and Gold, 2020). Biotin has also been added to supplements and claimed to improve skin and fingernail health, but studies are few and very weak with no recent research having been published (Cashman and Sloan, 2010). Dietary levels of biotin which exceed those required for optimal growth have improved hoof health in cattle, pigs, and horses and milk production in dairy cattle (Chen et al., 2011; Webb et al., 1984; Geyer and Schulze, 1994); higher than required dietary biotin also has enhanced reproductive performance in poultry (Taniguchi and Watanabe, 2007). Deficiency Biotin deficiency is difficult to attain and seldom encountered, but has been associated with diets excessively rich in raw eggs, where the avidin in egg white complexes to biotin to make it nonabsorbable. The most common cause of biotin deficiency is related to inherited defects in biotinidase and holocarboxylase synthetase, and usually manifests in the early neonatal period. When biotin deficiency occurs, under natural or experimental conditions, there is reproductive failure and marked teratogenesis in the young (Wolf and Heard, 1989). Dermatitis with alopecia, hypopigmentation, and hyperkeratosis has been reported. Low biotin status in chicks is the primary factor in fatty liver and kidney syndrome in chicks (Whitehead, 1985). Excess Toxicity due to biotin is virtually nonexistent. However, experimental evidence in rats is suggestive that high doses inhibit spermatogenesis in young rats and (Sawamura et al., 2015) adversely affect pregnancy outcomes by interfering with estrogen and progesterone biosynthesis (Paul and Duttagupta, 1976). FOLIC ACID (FOLATE, VITAMIN B9, FOLACIN) Folic acid, generally known as folate, in its bioactive form as tetrahydrofolate is necessary for methyl transfer reactions, which in turn are essential for purine and pyrimidine synthesis. Folate’s role in metabolism is the transfer of one-carbon groups (e.g., methyl, methylene, and formyl groups) between molecules in biosynthetic reactions, most importantly in the synthesis of GMP

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and AMP via transfer of two formyl groups to inosine, via transfer of a methylene group to UMP to form TMP and transfer of a methyl group, via cobalamin, to homocysteine to form methionine (Naderi and House, 2018). Thus, folate is essential for the proper development or maintenance of rapidly growing tissue or high-turnover tissues with a high physiologic rate of cell division, especially bone marrow and intestinal mucosa. Folate also has an indirect role in neurotransmitter synthesis by recycling dihydropterin (BH2) to tetrahydropterin (BH4), a necessary cofactor for monoamine synthesis. Folate also has a complex interrelationship with cobalamin, which is necessary for conversion of tetrahydrofolate to methyltetrahydrofolate, the cofactor that serves as the methyl donor in the conversion of homocysteine to methionine. In the process inactive hydroxycobalamin acquires the methyl group it needs to become the functional moiety, methylcobalamin, necessary for conversion of homocysteine to methionine. Additional folate above RDA can compensate for a relative lack of cobalamin by conserving methionine, preserving the methyl donor pathway for use in DNA and RNA synthesis, but not without potential adverse side effects (see below). Peanuts, sunflower seeds, chicken liver, and calf liver contain especially high concentrations of folic acid, and many leafy vegetables (e.g., spinach and lettuce) as well as legumes (e.g., chickpeas and lentils) and nuts have relatively high levels. Meat is generally quite low as are nonfortified grains (wheat germ being the exception). Folate is susceptible to high heat and to losses through boiling of folate-rich foods. Because of the risk of neural tube defects in developing fetuses in folic acid–deficient women, many countries fortify grains and grain-based products with folate (Shlobin et al., 2020). Recommended daily allowances for folate are 400 mg/day for adults and 600 mg/day for pregnant women. Due to extensive and high levels of fortification in many countries, daily intake often exceeds the daily allowance in adults, although not necessarily for pregnant women. Only about 50% of dietary folate is absorbed whereas about 85% of folate in fortified foods is absorbed (IOM, 1998d). Consumption of folate in supplements has greatly increased in recent decades and it is often

prescribed for the prevention of neural tube defects in pregnant women (see below). Fortification alone has been linked to a 35% decrease in spina bifida and other neural tube defects globally. In the Boston terrier, the incidence of cleft palate, a common feature in that breed, fell dramatically after dietary supplementation with folate was begun (Elwood and Colquhoun, 1997). Deficiency Although not as common as Fe deficiency, folate deficiency is of concern for the elderly, those with chronic digestive tract disease, epileptics on anticonvulsant drugs, women taking contraceptives, alcoholics, and pregnant women in whom occult folate deficiency is frequent during the second trimester of pregnancy. In overt folate deficiency, there is a macrocytic anemia and neutrophil hypersegmentation followed eventually by a decrease in circulating granulocytes and platelets. This is due to the inability of DNA synthesis (and hence cell division) in hematopoietic stem cells to keep up with RNA translation and protein synthesis in the cytoplasm, leading to enlarged hematopoietic blast cells and the release of larger than normal red cells into the circulation (Green and Datta Mitra, 2017). Neurological signs occur late in the deficiency, and are characterized by dementia/loss of cognition, affective disorders (especially depression), and peripheral neuropathy. There is considerable overlap with the manifestations of cobalamin deficiency although peripheral neuropathy is rare in folate deficiency and optic atrophy is absent (Reynolds, 2014). Pregnant women with poor initial folate status often are overtly deficient by the time of parturition. An association has been made between low folate in early pregnancy and neural tube defects, in particular spina bifida (see Embryo, fetus and placenta, Vol 4, Chap 11). This led to recommendations that women use folate supplementation during early pregnancy to prevent these defects (Williams et al., 2015). Folate status is key in maintaining homocysteine at normal levels. There is strong evidence the hyperhomocysteinemia that results from low maternal folate status may be the cause of the neural tube defects (Czeizel et al., 2013). The high homocysteine levels in plasma and tissues in adults with folate deficiency have been a wellpublicized risk factor for coronary heart disease, atherosclerosis, and stroke, but this has

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recently become controversial (Chrysant and Chrysant, 2018). Anemia associated with folate deficiency can occur in dogs but macrocytic anemia is generally not a clinical feature (Stanley et al., 2019). Subtler folate deficiencies also are suspected to occur in animals with chronic GI disease with malabsorption, dogs and cats being treated with antifolate chemotherapeutic drugs like methotrexate and with chronic debilitation due to decreased food intake. Folate deficiency combined with cobalamin (Vitamin B12) deficiency is more likely to cause macrocytic anemia in domestic animals like dogs and cats. As with most vitamin deficiencies, folate deficiency due to poor diet is often accompanied by other dietary deficiencies, making it difficult to sort out specific deficiencies. A high proportion of Greyhounds are hyperhomocysteinemic, often with concomitant hypofolatemia and often in combination with low cobalamin. Megaloblastic bone marrow precursors without macrocytes in blood typify folate deficiency in the cat (Thenen and Rasmussen, 1978). Excess Folate toxicity is very rare, but with the recent advent of megadose therapies using B vitamins, plus the high level of folate fortification already present in the diets of developed countries, many Americans excrete large amounts of unmetabolized folic acid, indicating saturation of absorption. Thus, adverse effects to folate have been reported and may likely continue to be reported. Concerns over an enhanced risk for colorectal cancer, effects on fetal development leading to insulin resistance in children, resistance to antiseizure medication, and hepatotoxicity have arisen (Patel and Sobczy nska-Malefora, 2017). In humans, 15 mg folate/day or more can cause acute toxicity characterized by malaise, irritability, depression, and altered sleep patterns. In mice, folic acid overdose is used as a model to investigate the cellular mechanisms of nephrotoxic injury, characterized by ferroptosis, a form of regulated necrosis similar to necroptosis (Martin-Sanchez et al., 2017). Adverse effects on embryonic development, including neural tube and cardiac defects, have been reported with moderate folate excess in rodents (Mikael et al., 2013). Neurotoxicity has been observed as well, with spasms, rotating movements, and increased aggressiveness.

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Excess folate can reverse anemia but not neurodegeneration associated with Vitamin B12 deficiency. Folate, as noted above, can also affect pregnancy outcomes because of the additional biological effects on cobalamin metabolism (Paul and Selhub, 2017). Similarly, high folate levels may mask the contribution of cobalamin deficiency on impaired cognition in the elderly (Stanley et al., 2019). COBALAMIN (VITAMIN B12) Cobalamin refers to a group of complex, chemically related cofactors that require cobalt (Co) for function (see below). After uptake and transport to tissues, cobalamin is metabolized to form the active moieties, adenosylcobalamin and methylcobalamin. Adenosylcobalamin is an essential cofactor for the mitochondrial enzyme, methylmalonyl CoA mutase, converting L-methylmalonyl CoA derived from proprionyl CoA produced by b-oxidation of FAs and breakdown of isoleucine, valine, methionine, and threonine into succinyl CoA for utilization in the TCA cycle. Methylcobalamin is an essential cofactor for methionine synthase in a complex cycle with folate, in which methyl-THF methylates cobalamin tightly bound to the methionine synthase, with the resultant methylcobalamin providing the methyl group for conversion of homocysteine to methionine. The unmethylated cobalamin is then ready for remethylation by methyl-THF. Hence folate and cobalamin are intricately linked to one another in the methionine/1-carbon transfer cycles (Shane, 2008). Once ingested, cobalamin must be cleaved from the dietary proteins to which it is bound by gastric hydrochloric acid and pepsin, after which it is ultimately absorbed in the ileum. Cobalamin, in the form of cyanocobalamin, is often supplemented in populations predisposed to deficiency, as discussed below. Before absorption, however, both forms must be bound to a protein, intrinsic factor (IF), produced by gastric parietal cells in stomach and to a much lesser extent (in humans) by the pancreas, which protects the newly released cobalamin forms from gastric breakdown and serves as the ligand for receptors on ileal enterocytes for uptake of the IF–cobalamin complex. Cobalamin is also produced by microbiota in the caudal ileum, but this form (hydroxycobalamin) is poorly absorbed and is insufficient to meet the

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metabolic needs of carnivores and omnivores. Ruminants and most other herbivores, which usually practice some sort of coprophagia, can usually absorb enough microbial cobalamin to avoid deficiency. Despite this complex process, absorption, even in healthy individuals, is at most 50% under ideal conditions. Cobalamin can be obtained from the diet from various animal sources, including fish, meats of all kinds, dairy, cheese, and eggs (ODS, 2019). Plant sources are noticeably lacking. The RDAs for cobalamin are 2.4–2.8 mg/day (IOM, 1998e). Most healthy nonvegan individuals obtain sufficient cobalamin from animal sources to avoid deficiency, with some exceptions. Products such as soy and almond milk can be readily supplemented with cobalamin to meet the needs of vegans. Deficiency Humans and monogastric animals with chronic gastric disease, particularly when there is loss of gastric parietal cells, are prone to acquired deficiency disease. Chronic gastritis secondary to Helicobacter pylori infection, atrophy of parietal cells typical in the elderly, excessive antacid use, chronic small intestinal disease with malabsorption, and decreased dietary intake associated with chronic diseases such as cancer are among the more common causes (Kumar, 2014). Dogs and cats, however, are more prone to deficiency disease when there is severe pancreatic atrophy or severe pancreatic disease with loss of functional tissue mass. This is because most IF is produced in the exocrine pancreas of these species (Batt et al., 1989). Cobalamin deficiency has two forms, which may not occur togetherda hematological form that is identical to folate deficiency (i.e., macrocytic/megaloblastic anemia, see above) and reflects cobalamin’s role in the methionine/1-carbon cycle, and a neurological form. Both deficiencies are often found together in countries where folate supplemented grains are not available. The neurological form is often irreversible and hence more serious; it is observed in humans naturally or in monkeys and pigs experimentally using nitrous oxide to bind to and inactivate cobalamin (Metz, 1992). Clinically there is spastic paraparesis of the lower limbs with impaired proprioception, sometimes involving other peripheral nerves and optic nerves as well. Memory loss, personality change, and cognitive impairment have

also been described (Kumar, 2014). This appears to be related to loss of adenosylcobalamin and its essentiality in the reaction converting methylmalonyl CoA to succinyl CoA. The exact biochemical mechanisms/pathways of how this leads to demyelination are not yet understood but postulated to be the accumulation of unusual FAs within myelin lipids. A role for lack of methionine due to loss of methionine synthase activity has also been postulated (Metz, 1992). Lesions are characterized by myelinic vacuolation around axons in ascending tracts and later descending spinal cord tracts (especially of the pyramidal tracts). The combination of bilaterally symmetrical lesions in both ascending and descending tracts is a characteristic feature of cobalamin deficiency. In domestic animals, in particular dogs but also cats, chronic intestinal disease and chronic pancreatitis leading to atrophy and small intestinal bacterial overgrowth can lead to decreased cobalamin uptake, resulting in cobalamin deficiency; however, in the case of bacterial overgrowth leading to inflammatory bowel disease the anemia may be masked by the enhanced production of bacterial folate, which is more readily absorbed. In addition, the breakdown of the excess methylmalonic acid inhibits the formation of carbamoyl phosphate, the main sink for excess nitrogen, resulting in hyperammonemia in dogs that can lead to neurologic deficits and seizures (Kather et al., 2020). Excess The toxicity of cobalamin has not been described in depth although studies and case reports have identified an association of hypercobalaminemia with alcoholism, solid malignant neoplasms, and liver disease in humans (Brah et al., 2014), an association also noted in cats with liver disease and malignant neoplasms (Trehy et al., 2014). However, there is little experimental evidence that cobalamin per se is toxic. CHOLINE

Choline is an essential nutrient that can be synthesized de novo by most animal species and was once thought to be nonessential in humans (Kohlmeier et al., 2005). Animals fed choline-deficient diets have adverse effects, in particular hepatic lipidosis and even steatohepatitis. Choline in some respects behaves biochemically like a vitamin but is metabolized like an amino acid.

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Choline is a component of cell membrane phospholipids in the form of phosphatidyl choline. It is also a component of lecithin, sphingomyelin, and the neurotransmitter acetylcholine. It is also essential for normal bile salt and VLDL formation. Endogenous choline is acquired as phosphatidyl choline from the donation of a methyl group from each of three Sadenosylmethionines to the ethanolamine moiety of phosphatidyl ethanolamine via the enzyme phosphatidyl ethanolamine Nmethyltransferase (PEMT). PEMT expression is induced by estrogen, hence the lower requirements for dietary choline in premenopausal females (Corbin and Zeisel, 2012). Sphingomyelin is derived from the phosphatidyl choline generated in this manner. Choline utilized in other pathways must come from the diet, although dietary choline can also be utilized in the synthesis of phosphatidyl choline via a different biosynthetic pathway. Metabolically, choline is vitally important in one-carbon metabolism and thus has extensive interactions with the folate and cobalamin pathways. In liver and kidney, choline is metabolized to betaine, an important methyl donor for regeneration of methionine from homocysteine in the liver and an important osmolyte in the kidney. In the nervous system, choline is used to form acetylcholine. In the colon, choline is metabolized to trimethylamine which is then absorbed and metabolized to trimethylamine-N-oxide (TMAO), which imparts a “fishy” odor when produced to excess (Wiedeman et al., 2018). Choline can be readily obtained from the diet, with especially high amounts found in organ meats and eggs, nonorgan meat, fish, wheat germ, wheat and oat bran, cruciferous vegetables, navy beans, peanuts, peas, spinach, and tofu (IOM, 1998f). Adequate intakes (AIs) for choline are 550 and 425 mg/day for adult males and females, respectively, and 450 and 500 mg/ day for pregnancy and lactation, respectively (ODS, 2020b). There is substantial evidence that certain subpopulations in the United States (e.g., infants, children, elderly), as well as many populations in developing countries, fail to meet these requirements. The lipid-soluble sources of choline (phosphatidyl choline and sphingomyelin) as well as soluble forms (free choline and phospholipid breakdown products) are readily absorbed from the small intestine

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and are transported to the liver, which is the location of most choline metabolism in the body, the kidney being the second most active organ. As with virtually all vitamins, associations and claims have been made for a therapeutic role for choline in improving dementia and other neurological disorders, and in preventing or ameliorating cardiovascular disease. As with most vitamins, causation has yet to be confirmed with the exception of NAFLD/nonalcoholic steatohepatitis (NASH, see below), for which firmer causal evidence associated with overt or relative choline deficiency has accumulated (Corbin and Zeisel, 2012), and for which supplementation appears to be effective. Choline is a common component of multivitamin supplements but few if any studies exist regarding its bioavailability when included in supplements. DEFICIENCY

Populations at risk for deficiency, overt or relative, with adverse effects include pregnant women and infants, where low choline status has, in some studies, been linked to neural tube defects and impaired cognition in offspring; impaired cognition in the elderly, especially postmenopausal women with poor diets; and impaired cognition in alcoholics with liver damage and poor dietary intake. None of these observed associations have been experimentally or clinically confirmed (ODS, 2020b). It is likely that choline deficiency has a role in the development of NAFLD and even NASH in humans (Sherriff et al., 2016). In experimental rodent models, where diets can be readily manipulated as to choline and overall fat content, choline deficiency alone causes lipidosis, mainly macrovesicular and centrilobular; with the addition of high concentrations of fat to choline-deficient diets, the lesions progress from lipidosis to lipidosis with hepatocellular degeneration and cell death, chronic active inflammation, fibrosis, and ultimately endstage liver, a pattern very similar to that seen in NAFLD/NASH in humans (Wei et al., 2019; Van Herck et al., 2017). Initially, it appears due in part to loss of phospholipid function in lipoprotein micelles with massive accumulation of triglycerides and abnormal membrane formation in hepatocytes. In addition, choline deficiency has severe, necrotizing effects in the renal tubules, brain, and myocardium of young

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rats, and can be fatal in just a few weeks (Repetto et al., 2010). Partial supplementation of choline can protect rodents from fatal kidney and heart lesions, but will not prevent hepatic lipidosis. EXCESS

Choline toxicity has been reported to cause hypotension, nausea, and diarrhea, as well as a fishy smell in individuals with the appropriate microbiota for producing large amounts of TMA, which is absorbed and metabolized in the liver. Excess choline has also been linked to an increased risk for cardiovascular disease, atherosclerosis, neurological disorders, and teratogenesis (EFSA, 2016). The margin of safety between required and toxic doses is small for choline, so caution is needed in dietary choline supplementation. Minerals MAJOR MINERALS

Ca, as Caþ2, is the most abundant divalent cation in the body, constituting 1%–2% of body weight. Its essential intra- and extracellular functions are myriad and vital for life. It is not only a cofactor in many critical enzymatic reactions and processes such as cytoskeletal maintenance, secretion, and intra- and extracellular locomotion, but also a vital regulatory and signaling molecule (Carafoli and Krebs, 2016). This section will deal with the nutritional aspects of Ca homeostasis only as it relates to nutritional intake. While 99% of total body Ca is found in the bone, maintenance of extracellular (103 M) and intracellular (107 M) Ca is so vital that, if intake of Ca is compromised, bone will demineralize to prevent hypocalcemia (Bronner, 2001). Even a small, short-term decrease in serum Ca triggers parathyroid hormone (PTH) secretion and Vitamin D–dependent upregulation of absorption from the gut as well as decreased urinary excretion of Ca and increased excretion of urinary P in the form of phosphate. Dietary sources high in Ca include milk and milk products (cheeses, yogurts), tahini, nuts such as almonds and hazelnuts, molasses, leafy green vegetables (especially crucifers and spinach), tofu, sesame seeds, and lentils (Food Data Central, 2021). Cereals and orange juice are often fortified with Ca as well. The RDA for Ca is 1000 mg/day for adults, somewhat CALCIUM

higher (1200 mg/day) for adolescents (IOM, 2011b). Typically, only about 30% of ingested Ca is absorbed. Ca supplements are common, and unlike for most supplements, the FDA has authorized health claims for Ca and Ca/Vitamin D supplements for the prevention or treatment of osteoporosis. It has been found that the Vitamin D effect is enhanced in the presence of high Ca. The recommended dose must contain at least 20% of the recommended daily intake for Ca (FDA, 2008). Nevertheless, the efficacy of Ca only or Vitamin D only supplementation on decreasing fracture risk (as opposed to osteomalacia/osteoporosis) has been disputed due to lack of convincing results in randomized clinical trials (Bolland et al., 2015). There are paradoxical findings regarding increased dietary Ca and/or supplements with regard to cardiovascular disease, colorectal cancer, and kidney stones (Li et al., 2018). Deficiency If the diet remains abnormally low in Ca over a prolonged period, especially if dietary P is high, chronic hyperparathyroidism stimulated by persistently low serum Ca leads to loss of bone mineral density with fibrous osteodystrophy in adults and rickets in growing individuals. This “nutritional” type of hyperparathyroidism is very rare in humans and increasingly rare in domestic animals but is still found in horses fed diets rich in bran, dogs and cats fed exclusively meat diets, and pigs fed unsupplemented cereal grains. All three types of diets are notoriously low in Ca but high in P (Olson and Carlson, 2017b). The lesions are very similar to those of Vitamin D deficiency (see above). Osteomalacia is most pronounced in the mandible, scapula, proximal, and distal ends of the long bones and vertebrae, boney areas rich in trabecular bone. Trabecular bone turns over far more rapidly than cortical bone of long bone shafts. Diets excessively high in protein or fiber also can cause chronic Ca loss, the former through increased renal clearance of Ca, and the latter through luminal chelation of Ca and subsequent decreased absorption from the gut. Unless severe, these conditions are compensated for by increased uptake and decreased clearance, respectively. Ca, P, and Mg metabolism are related intimately, in that high dietary phosphate and low dietary Mg can lead to hypocalcemia. Thus, all three conditions result in the same pathological condition:

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renal failure with loss of bone Ca and osteomalacia. Excess In humans, Ca intake >1000 mg/day, from supplements rather than dietary calcium, has been associated with an increased risk of myocardial infarction, especially in women; diarrhea or constipation; and an increase in renal Ca oxalate lithiasis, primarily in women (Yang et al., 2020). Excess dietary Ca can have an adverse effect on growing poultry ingesting high protein diets, with renal gout (urate lithiasis) caused by metabolic alkalosis due to Ca’s divalent cation status (Julian, 2005; Guo et al., 2008). In pigs, if Ca is excessive, food intake decreases, fecal weight increases, and growth is retarded (Merriman et al., 2017). In dairy bulls fed diets with Ca content appropriate to lactating dairy cows, there is persistent hypercalcitonemia in response to chronic low-grade hypercalcemia. Since calcitonin inhibits bone resorption and promotes bone production, osteosclerosis results with thickening of bones, stiffness, and lameness (Krook et al., 1969). Nephrocalcinosis, especially in female rats, is a common occurrence if the molar ratio of Ca:P is less than 1.0 or if magnesium (Mg) and/or chloride are deficient in the diet, illustrating the complex interactions between these minerals (Rao, 2002). Mineralization occurs in the straight segments of tubules at the corticomedullary junction and eventually involves the entire inner cortex. In the absence of concomitant increases in P, a high Ca diet may lead to a relative phosphate deficiency, due to chelation and precipitation in the lumen of the GI tract (see below). High dietary Ca also aggravates Mg deficiency (see below). PHOSPHORUS

Much like Ca, P in the form of phosphate has a myriad of critical roles in metabolism. It is vital for energy metabolism, membrane composition, enzyme activation/ inactivation, and transcriptional regulation and intracellular signaling pathways. This section will deal with the nutritional aspects of P homeostasis only as it relates to nutrition and interactions with other nutrients. Whole body P (mainly as PO3 4 ) is 80% distributed to teeth and bone, the rest being in soft tissues, both extracellular and intracellular. Under normal circumstances, most (70%–80%) dietary P (as phosphate) is absorbed passively

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regardless of the dose, and regulation is by excretion in urine or feces, which in most cases is extremely efficient, especially by the kidney. Dietary phosphate tends to be relatively abundant, so pure dietary deficiencies without concomitant deficiencies (e.g., potassium, manganese, or zinc) or complicating factors (e.g., starvation) are rare. Sources of P are abundant in foods, especially meat, dairy products, legumes, peanuts, and nuts. The RDA for P (as phosphate) is 700 mg/ day, but no adverse effects are observed even when P intake is raised to 2000 mg/day. This large safety margin is fortunate, since many grains and multivitamins are supplemented with inorganic phosphate, which is over 90% absorbed. Even though P intake at the 2000 mg/day level decreases Ca uptake from the gut, renal excretion of Ca decreases to compensate (IOM, 2001b). Much of the P in plant products (especially legumes, nuts, and grains) is phytate P, which is largely unavailable for absorption by animals, especially humans (Schlemmer et al., 2005). Hence, absorption of P from plant sources is about 50%. Recommended nutritional P requirements are based on highly available P, so when evaluating P levels in the diet, one should consider nonphytate P rather than the total dietary P concentrations. As with Ca, P uptake from the gut is enhanced by Vitamin D. Deficiency In P deficiency, Vitamin D production is increased, activating an active transport pathway for uptake (Reinhardt et al., 1988). This is associated with development of a mild hypercalcemia, which triggers a compensatory decrease in renal P excretion. If uncompensated, hypophosphatemia results in osteomalacia with pathologic fractures, although overt cases of hypophosphatemic osteomalacia are rarely reported (Rico et al., 1985). A subtler form of phosphorus deficiency has been reported in patients who are malnourished, alcoholic, or suffering from chronic GI disease. These are usually associated with other vitamin and mineral deficiencies as well. However, excess use of Ca-, Mg-, or aluminum-based antacids or improper use of oral phosphate binders (for treatment of hyperphosphatemia during renal failure) may develop into a purer phosphorus deficiency (Kraft, 2015). Decreased respiratory function (associated with abnormal diaphragmatic contractions), altered myocardial contractility,

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and neurologic dysfunction are additional clinical manifestations. Dietary P deficiency in domestic animals is most commonly seen in ruminants grazing on substandard pastures with acidic soil, soil leached by excessive rain, or soils with a high clay content, leading to low phosphorus content in the forage. Pica or abnormal appetite, especially osteophagia, is a common feature of phosphorus deficiency in both cattle and sheep (Gordon et al., 1954). Initially, in less severe cases clinical signs can include poor coat quality, lethargy, poor growth, and poor reproductive performance, accompanied by lameness and stiff gait. Prolonged deficiency results in severe osteomalacia with pathologic fractures (Shupe et al., 1988). Subtle, long-standing P deficiency has become a concern in pig and poultry production facilities, as well as in aquaculture, as efforts have been made by these industries to eliminate P-rich effluent into groundwater, necessitating large-scale reduction in P content in diets. In poultry and pigs, decreased growth rates can be a problem, but most importantly a decrease in bone health, with bone abnormalities and osteomalacia (Misiura et al., 2020; Li et al., 2020). Fish raised in aquaculture conditions, where P effluent is a major environmental concern, are sometimes fed formulated diets that are lower in P than required to sustain good reproductive, scale, and immunological health. On occasion this has produced skeletal abnormalities consistent with rickets in birds and mammals (Sugiura et al., 2004). Excess Although hyperphosphatemia and the risk of tissue calcification are of high concern in cases of renal failure, dietary phosphate toxicity in otherwise normal humans is becoming a concern given the already high phosphate consumption by many populations worldwide (generally twice the RDA) and extensive use of polyphosphate and orthophosphate preservatives in processed meat products, processed cheeses, frozen bakery good, sodas, snack bars, and other processed nonplant food items (Erem and Razzaque, 2018). High phosphate consumption has been linked to enhanced risk for cardiovascular disease. Effects of long-term excess dietary phosphate consumption are as yet unclear and as yet relatively unexplored. As noted above dietary phosphorus does not have to be excessively high to cause adverse

effects in domestic animals if dietary Ca is normal or low. If the dietary P (as phosphate) to Ca ratio is raised above 2:1 or even less for an extended time, secondary hyperparathyroidism results in osteomalacia and fibrous osteodystrophy, most obvious in the mandible and other flat bones of the skull (Figure 3.7). Mg (as Mgþ2) has an enormous but sometimes overlooked variety of functions in the body. It is bound to ATP, Mg-ATP being the actual bioactive molecule in ATPdependent reactions. Mg also functions as an essential divalent cofactor in numerous reactions, especially in nucleic acid metabolism, being a cofactor for many endonucleases, exonucleases, ribonucleases, and polymerases. It also is key for phosphorylation/dephosphorylation, phosphoryl transfer reactions, isocitrate lyase and L-aspartase, mitochondrial ATP synthase, Naþ/Kþ -ATPase, hexokinase, creatine kinase, adenylate cyclase, phosphofructokinase, and insulin receptor tyrosine kinase, to name just MAGNESIUM

FIGURE 3.7 Fibrous osteodystrophy in a capuchin monkey with nutritional secondary hyperparathyroidism due to excessive phosphorus in the diet. Note the thickened gingiva and displaced teeth reflecting the underlying osteodystrophy. Courtesy of Cynthia Courtney. Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 36.6, p. 1105, with permission.

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some Mg-dependent enzymes. Mg often works in coordination with a second Mg or another divalent cation, such as Zn, Mn, or Fe (Cowan, 2002). Mg is also a necessary cofactor for Vitamin D hydroxylation and binding of Vitamin D to Vitamin D binding protein (Gro¨ber et al., 2015). Whole body Mg is distributed 60%–65% in bone, w30% in muscle, and only 1% in the extracellular fluid. Magnesium is absorbed throughout the GI tract, but predominantly in the ileum, mainly by passive transport, although a transcellular, active transport pathway is also utilized. The body load of Mg is regulated by excretion/reabsorption in the renal proximal tubule and the ascending arm of the loop of Henle. It appears to be both filtered at the glomerulus and possibly secreted into the proximal tubule. The kidney is highly efficient at regulating Mg uptake to maintain normal serum concentrations within a narrow range (Houillier, 2014). Food sources that are rich in Mg include seeds, nuts, peanuts, leafy green vegetables (especially crucifers, chard, and spinach), and unprocessed whole grain products. Processing or cooking with water will result in substantial loss of Mg from food because of its high solubility in water. The RDA is w420 and 320 mg/day, for adult males and females, respectively, with about 30% of this being absorbed (IOM, 1997b). Therapeutically, iv Mg has been used as part of the treatment for preeclampsia and eclampsia (De Baaij et al., 2015). Oral Mg supplementation has been linked to improvement of a variety of conditions, including migraines, hypertension, cardiovascular disease, stroke, dementia, and diabetes, but causation and mechanisms are as yet unclear (Volpe, 2013). Deficiency High dietary levels of Ca and/or P (as phosphate) depress Mg absorption, as does fluoride (F). Although overt Mg deficiency in humans is rare, dietary magnesium “insufficiency” (consumption of less than the RDA for Mg over time) has been identified in a substantial portion (60%) of the human population, due not only to increased consumption of boiled and processed foods but also to decreasing Mg concentrations within plants grown by modern farming methods (Workinger et al., 2018). Soda beverages, with their high phosphoric acid content, are another culprit. Mg insufficiency has been linked clinically or anecdotally to

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a variety of signs. Early signs include hypokalemia, hypocalcemia, anorexia, lethargy and fatigue, nausea and vomiting, and fatigue, as well as night time muscle cramps and spasms, tension headaches, insomnia, and anxiety. More profound Mg deficiency may lead to enhanced neuromuscular excitability, with tremors, tetany, and generalized seizures. Severe Mg deficiency can cause cardiac arrhythmias including atrial and ventricular tachycardia (Gro¨ber et al., 2015; De Baaij et al., 2015). The role of low Mg in cancer is controversial and even contradictory. Numerous epidemiological, experimental, and in vitro studies have associated low Mg with increased risk/growth of primary tumors of liver, esophagus, breast, ovary, and colon. However, low Mg seems to protect against the early and rapid growth of metastasis. This has been postulated to be due to enhanced oxidant stress at various stages of cancer development (Leidi et al., 2011). Ruminants are particularly sensitive to acute Mg deficiency, manifested as hypomagnesemic tetany, which also is known as winter tetany, grass staggers, or wheat pasture poisoning. It generally occurs when serum concentrations are less than 0.7 mg/dL and is most common in early lactation and in later¼ winter/early spring in nonlactating individuals in areas where animals are grazed extensively on cool grass or winter grain pastures, which are relatively low in Mg. Fertilizing practices using heavy potassium (K) or nitrogen fertilizers can also contribute to low plant Mg. Hypomagnesemic tetany is due to abnormal neuromuscular function inducing spasticity, convulsions, tremor and tetany, hypokalemia, and a hypocalcemia that does not respond to Ca feeding (Smith and Edwards, 1988). Mg deficiency also is a major predisposing factor for “milk fever” in cattle, due to its vital role in the interaction of PTH with the PTH receptor (Goff, 2014). Rats, on the other hand, respond differently to Mg deficiency, with hyperactivity, growth retardation, vasodilatation, cardiac arrhythmias (tachycardias), convulsions, and sudden death. Pathologic changes include skin ulcers, multifocal myocardial mineralization, and fibrosis (Fiset et al., 1996; Robeson et al., 1980). Hypercalcemia, not hypocalcemia, occurs in rats, unlike other species. Rats also rapidly develop nephrocalcinosis (Bunce et al., 1974).

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Excess In humans, hypermagnesemia typically is associated with excessive use of laxatives and cathartics or pharmacological use of Mg salts rather than dietary excess. It should be noted, however, that renal insufficiency can also predispose to hypermagnesemia since serum levels are regulated by renal tubular excretion. In hypermagnesemia, Mg successfully competes with Ca, producing hypocalcemia followed by vasodilation for which it is used clinically to treat eclampsia. Lethargy progressing to fatigue then somnolence, nausea and vomiting, loss of reflexes, hypotension, cardiac block, respiratory depression, and finally death is the usual outcome of severe hypermagnesemia (de Baaij et al., 2015). In animals, effects of excess dietary Mg are very rarely reported. Dietary Mg has complex interactions with dietary Ca, P, Na, K, and Zn (discussed above and below), which complicates the interpretation of what effects seen are truly due to Mg. SODIUM/POTASSIUM Extracellular fluid has Na as its major cation, while K (as Kþ) is the major cation in intracellular fluid. Na is necessary for water balance in the cell, with water following Na as it moves into the cell and following it back out of the cell when it is actively transported out of the cell in exchange for K. Na is involved in a myriad of active and facilitated transport processes, and its entry into the cell must be counterbalanced by exchanging it for K in the efflux process; otherwise, water balance is lost. K is essential for the proper function of most cellular processes as the major buffer for proteins. Both are important in systemic water balance, with Na playing the key role in maintaining blood pressure as well as extracellular volume. Cl is the balancing counteranion, generally accompanying both cations as they move in and out of cells. The relationship between intra- and extracellular K dictates the potential difference across the cell membrane, and thus controls the threshold for depolarization and repolarization of the membrane, with Na as the secondary contributor. If hyper- or hypokalemia develops acutely, the relationship between intra- and extracellular K is disrupted. In hyperkalemia the resting membrane potential may increase even to the normal threshold for depolarization,

so that depolarization is permanent, and repolarization cannot occur. Alternatively, in hypokalemia the potential difference is so great across the cell membrane that normal stimuli may not raise the resting potential to threshold, and depolarization is blocked. Ingested Na and K are absorbed fully, mainly from the upper GI tract, and regulated primarily by urinary excretion, although excessive sweating and especially diarrhea can lead to excessive losses from the body. Homeostasis is most often lost in renal disease leading to insufficiency, when body Na, Cl, and water content increase. The pathophysiology of Na/K/Cl disruptions in homeostasis is complex and the consequences even more so in many cases. This chapter will only deal briefly with diet-related conditions associated with improper intake of these elements. Diets composed solely of plants generally do not provide sufficient Na (as Naþ) for optimal health. Consequently, from early times, humans as well as many mammals have sought environments where not only food and water are in abundance, but where there is a ready source of NaCl, i.e., salt. This can be a particular problem in winter and early spring months in temperate climates where fresh or digestible plant material may be limiting. Humans have dealt with the issue by using salt as a condiment and preservative for nonplant dietary sources. In modern times, with the development of abundant processed/preserved foods of all sorts plus the availability of Na-laden beverages (e.g., sodas), the problem of too much dietary Na and its adverse effects have come to dominate the challenge of appropriate dietary Na. However, in animals, salts often must be added to animal diets, especially for animals in confinement or in restricted environmental locations (i.e., pasture); accordingly, deficiencies and toxicoses have been associated with incorrect mixing of confinement diets or dietary supplements. K intake in humans is generally inadequate (see below) and has presented less of a problem; in rare cases of overuse of dietary supplements, excess K can have serious consequences. No RDA has been established for Na. Adequate daily intake of Na for those over age 9 is 1500 mg but the average intake for most Americans is 3400 mg (USDA and HHS, 2010), with the vast majority coming from processed

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foods, not only in the form of NaCl but also in the form of preservativesdNa nitrite/nitrate, Na acetate, Na ascorbate, Na benzoate, Na bicarbonate, and monosodium glutamate, to name just a few. Na is virtually ubiquitous in any packaged food, even, in many cases, “natural” foods, due to the widespread use of preservatives and antioxidants that are Na salts. As with Na, no RDA for K exists. Adequate daily intake is 4100 mg/day (5100 mg/day for lactation) (NASEM, 2019). Foods particularly rich in K are potatoes, bananas, beans, fruits/ fruit juices, nuts, milk, yogurt, meats, and seafood, although leafy vegetables and cereal grains also contain ample K. However, most Americans and most populations elsewhere do not meet the average daily intake requirements for K, and very little is known about the bioavailability of K from various food sources (Stone et al., 2016). In addition, 90% of absorbed K is excreted in the urine, with kidney efficiently adjusting K reabsorption/secretion to maintain stable serum K concentrations. Deficiency Dietary Na (NaCl) deficiency is accompanied by salt craving (licking of rocks by animals) followed by loss of appetite and signs of dehydration. Carnivores usually consume more than enough Na via viscera and muscle and as a rule do not exhibit signs of Na deficiency such as thirst and salt craving. Herbivores and omnivores, however, whose diets are or can be more limiting in bioavailable Na, do exhibit profound cravings for salt and water and are neurologically programmed to do so (Hurley and Johnson, 2015). Long-term Na deficiency without remediation leads to global volume contraction, hypotension, shock, collapse, and death, signs similar to Addison’s disease. Modern agricultural practices have led to a steady decrease in dietary K in most human populations (Sun and Weaver, 2020). This is of especial concern in patients on loop diuretics for hypertension, idiopathic edema, asthma, or hypertension. Numerous epidemiological and observational studies have linked increased dietary potassium intake/supplementation to suppression of hypertension and decreased risk of stroke (Aburto et al., 2015), although clinical trials have mixed results. The protection is supposedly due to K-mediated effects on vascular endothelium and vascular smooth

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muscle, resulting in vasodilation and appearing to counteract the effects of excessive Na intake (Sun and Weaver, 2020). Signs of deficiency may not be overtly apparent in mild cases, but when severe, include weakness, lethargy, and cardiac arrhythmia that could lead to death. Excess Given Na’s role in vascular dynamics and water regulation and the fact that Na consumption by Americans and many other populations is much higher than needed for optimal health, it is perhaps no surprise that there has been a “pandemic” of hypertension in humans. In numerous epidemiological and clinical trials, salt restriction alone has been found to substantially lower blood pressure (especially systolic) in many hypertensive patients (Sacks et al., 2001; Cook et al., 2014). Organ damage that may be somewhat independent of Na’s effect on blood pressure includes direct adverse effects on vascular endothelium, left ventricular myocardium, renal handling of sodium, and skin health (Robinson et al., 2019). Animals can withstand very large doses of Na as NaCl, provided adequate water is made available. Lack of water greatly enhances the chances for disease to occur. When manifest it is known as salt poisoning, sodium ion toxicity, or water deprivation syndrome. This disease can occur even when dietary Na is not be in excess: upon rapid rehydration of animals deprived of water for a period of time, such as when an outside water source freezes in winter or an automatic watering system fails and goes unnoticed for one to several days. The greatest risk for toxicity is in pigs, poultry, and cattle, but nonhuman primates, dogs, and sheep can also be intoxicated under the right conditions. The initial trigger is hypernatremia via excess consumption of Na and/or water deprivation. Initially the brain loses water and Na; this is followed by an influx of Na, K, and Cl back into the brain, followed later by an influx of organic osmolytes to restore appropriate electrolyte and osmotic balance. Upon restoration of water consumption there is a rapid shift to hyponatremia and a resultant osmotic “pull” of water into the hyperosmotic brain that is too rapid for appropriate compensation. Cerebral edema with brain swelling and leptomeningeal vascular engorgement ensue. In cases of acute Na ingestion there are gastric irritation, vomiting, and diarrhea followed by trembling, ataxia, and seizures. Cases involving

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water deprivation are manifested by blindness, ataxia, partial paralysis (“dog-sitting” in pigs and fetlock knuckling in cattle), tonic-clonic seizures, and death. Histologic lesions are typified by evidence of edema (pale neuropil around blood vessels), deep laminar cortical necrosis and necrosis of Purkinje cells, and often accumulation of eosinophils (pigs) (Figure 3.8) or neutrophils (ruminants) in Virchow–Robins spaces (Miller and Zachary, 2017a). Dietary excess of K producing disease is rarely reported in humans, but problems associated with overconsumption of supplements have become an issue, prompting limitations imposed on the amount of K allowed in a single dose of supplement. Even so, hyperkalemia is rarely reported in those with normal renal function. When it does occur, in cases of prolonged hyperkalemia, glucose intolerance and Type II diabetes have been reported.

FIGURE 3.8 Hypo-osmotic edema in the cerebrum of a pig with sodium chloride toxicity associated with initial water deprivation. Note the region of vacuolation (spongy change) in the neuropil. This lesion is often laminar (middle to deep gray lamina) and accompanied by neuronal necrosis (eosinophilic neurons) (arrows) and astrocytic swelling (evident as vacuolation). Unique to pigs is a perivascular infiltrate of eosinophils and with longer survival, an influx of macrophages (gitter cells). Inset: Note the eosinophils in the perivascular space of this postcapillary venule. Hematoxylin and eosin stain. Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Fundamentals of toxicologic pathology, ed 2, Academic Press, 2010, Figure 13.19, p. 402, with permission.

In cattle, addition of K (as KCl) to the diet (normally 0.5%–1% K on a dry matter basis) improves weight gain, and if paired with high dietary Na (0.7% on a dry matter basis), can improve milk production in dairy cattle. However, chronic dietary intake of high K, at even mildly elevated K levels (above 2% KCl), can lead to Mg depletion and depressed weight gain and susceptibility to tetany (Beede, 1991). As noted above, heavy us of high K (and nitrogen) on pastures can contribute to hypomagnesemic tetany. SULFUR Unlike plants, animals have no direct requirement for inorganic sulfur (S), either as 2 elemental S, sulfate (SO2 4 ), or sulfite (SO3 ). Animals must obtain almost all their sulfur as organic sulfur from either plants or prey in the form of S-containing metabolic intermediates such as methionine, cysteine, biotin, thiamine, or taurine (in the case of carnivores, especially felines). Animals use inorganic S produced in the body (primarily SO2 4 ) for sulfation reactions (e.g., phase II detoxification) and in the synthesis of glucosamine and glycosaminoglycans (e.g., chondroitin sulfate). Since SO2 is poorly 4 absorbed from the colon (15% or less), most SO2 4 in the body is probably derived from the oxidation of excess cysteine (Florin et al., 1991; Stipanuk and Ueki, 2011). Ruminants, but not nonruminants, can utilize inorganic S in place of methionine since the ruminal flora can synthesize methionine. Therapeutically, sulfur has been used for many years as an ingredient in therapeutic topical skin and shampoo preparations and as a wound healing promoter as well as a radioprotective agent in cancer treatment. Sulfur in the form of dimethylsulfoxide (DMSO) and methylsulfonylmethane (MSM) has also been used extensively for arthritis and joint health (Parcell, 2002). Organic sulfur containing metabolic intermediates (e.g., methionine) are used extensively in supplements (see above). Deficiency True sulfur deficiency is not encountered in humans or animals except as part of an overall deficiency of organosulfur compounds associated with dietary protein deficiency. Since animals cannot “fix” inorganic S metabolically into their various metabolic pathways, supplementation with inorganic sulfur such as SO2 is not useful therapeutically to 4

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correct deficiencies in important specific organosulfur compounds. Excess Sulfur intoxication in the nonnutrient setting during industrial exposure is well documented, as are respiratory toxicities associated with gaseous oxides of S in air pollution (see Respiratory Tract, Vol 4, Chap 4). In contrast, dietary S excess has been ignored until recently, probably because of the low toxicity of ingested S and its oxidized inorganic forms sulfate and sulfite. Ruminants, on the other hand, are more sensitive due to the ability of certain ruminal microflora to metabolize S (as SO2 4 ) to toxic hydrogen sulfide (H2S), especially when there is low dietary fiber. The problem in cattle, once confined to grazing areas where surface water has high sulfate content, has become more substantial with the use of distiller’s grains (DGS) left over from ethanol production in feedlot diets. DGS can contain high concentrations of S (up to 1.7%, well above the NRC recommendation of 0.15%). Cattle fed excess S at dietary levels above 0.4%, with neutral digestible fiber less than 7 or 8%, are prone to H2S-induced polioencephalomalacia, or PEM (Drewnoski et al., 2014). This process may take several weeks as populations of H2Sproducing bacteria proliferate, especially if low fiber is present. The H2S produced is eructated, inhaled, and absorbed from the lungs to produce the classic lesions of PEM due to inhibition of the electron-transport chain in mitochondria (Gould, 1998). Sheep can also be affected in the same manner (Gooneratne et al., 1989; Rousseaux et al., 1991). PEM due to H2S is manifested clinically as stupor and lethargy; blindness and ataxia (“blind staggers”); and head pressing, opisthotonos, paddling, and death. Histologically S-induced PEM resembles other neurotoxicoses such as lead poisoning and sodium toxicosis and is characterized by deep laminar cerebrocortical necrosis and liquefaction with initial sparing of the white matter (Figure 3.9). Pathological changes associated with S excess are not limited to CNS, but include inappetence, constipation or diarrhea, emphysema, muscle and renal damage, and hepatic necrosis (Beede, 1991). Metabolism of other minerals, including Ca, P, Mo, Mn, Mg, and Zn, may be affected adversely at very high dietary S. This is due to interaction

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of S as sulfate with other metals to produce insoluble precipitates. For example, excess S can precipitate Cu deficiency, due to Cu–S complex formation. High S also has been reported to interfere with Se uptake, and can replace P in bone, leading to sulfur rickets (Beede, 1991; Drewnoski et al., 2014). TRACE MINERALS CHROMIUM A role for Cr (in its nonmutagenic Crþ3 form) in metabolism was first suggested in the 1950s as a “glucose tolerance factor” present in porcine kidney. This has since been shown to be an artifact (Vincent, 2017). Although there is some evidence that Cr has some role in carbohydrate and lipid metabolism as a cofactor in autoamplifying insulin signaling via the Crbinding protein, chromodulin, data are insufficient as yet to define a clear-cut role for this metal in other carbohydrate and lipid metabolic pathways (Vincent, 2000). It has been difficult and controversial to define RDA and ADI for Cr (Trumbo et al., 2001; EFSA, 2014). An ADI of 35 and 25 mg/day for men and women, respectively, has been suggested. The lack of clarity regarding Cr requirements is in large part due to the difficulty in feeding diets completely deficient in the metal, but also due to a lack of a suitable biomarker for identifying activity (Vincent and Lukaski, 2018). In addition, there is a lack of evidence for its functionality as an essential nutrient. Trace levels of Cr are ubiquitous in almost all foods and 10 mg/kg in the rat, for example (Kessabi et al., 1980, 1985). As in humans acute toxicosis affects cardiovascular function by altering plasma Ca concentrations as well as causing GI irritation, liver damage, and renal tubular necrosis. Chronic fluoride toxicity, also known as fluorosis, is more common than acute toxicity. This is common in humans as well as cattle in India, Bangladesh, Sri Lanka, Scandinavia, western United States, west Africa, and parts of China, wherever ground and well water can be contaminated naturally with high concentrations of fluoride. Of major concern in human populations is dental fluorosis in children, acquired when F intake is excessive during tooth development. Although the mechanism is not fully elucidated, F appears to affect ameloblasts during the early stages of enamel production, with most F being deposited in a crystalline array different than that in deeper enamel. This is followed by defective protein lysis of matrix amelogen proteins during the later phases of enamel development, leading to increased matrix and decreased mineral composition, resulting in a harder surface but less mineralized deeper enamel (DenBesten and Thariani, 1992). The critical age for developing dental fluorosis is roughly 15–30 months in children, after which point the risk decreases substantially (Browne et al., 2004). Clinical signs of osseous fluorosis in human children and adults can take a long time, even years, to develop, depending on the dose to which the individuals or animals are exposed. Clinical manifestations include long-term

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stiffness and joint pain, calcification of ligaments, osteosclerosis, and bony deformities. Osteosclerosis is the initial lesion, followed by periosteal bone deposition, osteophyte formation, and bone and joint deformation (Srivastava and Flora, 2020). Despite some initial studies and claims that chronic low-grade F exposure might lead to increased risk of cancer, solid evidence has not been reported (Whitford, 1992). Likewise chronic F exposure has been linked to aberrant neurological development and cognitive dysfunction including autism, cardiovascular diseases, impaired insulin metabolism, impaired thyroid metabolism due to interference with iodide uptake, and impaired male reproductive function (Wei et al., 2020), but these associations have not been linked to causation. In animals, horses are reported to be much more sensitive and will exhibit clinical signs at lower doses (but over longer periods of exposure), although cattle are more frequently affected (NAS, 1974). Since fluoride deposited in bone has very low turnover, bone fluoride concentrations are used to assess toxicity in herd situations. There is considerable variation, however, between reports as to what constitutes a toxic concentration, generally considered to be somewhere around 5000 ppm dry weight. Although little research has been done or data collected, fluorosis has been anecdotally implicated or suspected in dogs with degenerative bone diseases after long-term consumption of pet foods containing bone meal derived from animals grazing in high fluoride areas or drinking contaminated water (Environmental Working Group, 2009). The typical lesions of chronic toxicity are comprehensively described in Shupe et al. (1992), which also has information on experimental fluorosis in rodent models. The lesions of fluorosis are similar in all mammalian species and present mainly in bone with teeth affected only in young animals when exposure occurs during tooth development (Figure 3.11). In teeth, fluorosis is associated with discoloration, milky white at low concentration, and brown at higher concentrations. Mild dental fluorosis is mainly an aesthetic issue, but at high levels, the fluorapatite responsible for the discoloration can cause the enamel to be brittle and prone to pitting and fracturing (Krook et al., 1983).

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deformities of the phalangeal bones. When bone becomes saturated with fluorapatite, generally above 5000 ppm in the bone, fluoride concentrations in plasma can increase to produce signs of acute toxicosis. I2/I (elemental iodinedI2, and iodidedI ) is an essential cofactor for growth and regulation of metabolic rate and body temperature. I is actively taken up by the thyroid and incorporated into thyroglobulin synthesized by the follicular cell and stored as colloid in the follicular lumen. The thyroglobulin is progressively iodinated on its tyrosine residues, up to four per residue. When stimulated by the binding of thyroid stimulating hormone (TSH) secreted by the pituitary to the TSH receptor, thyroglobulin is taken up by the follicular cell and broken down, with release of the iodinated tyrosine residues 3,5,30 ,50 -tetraiodothyronine (T4), and 3,5,30 -tetraiodothyronine (T3) into the circulation. T4, also known as thyroxine, is converted at the site of action to the active metabolite T3, known as triiodothyronine. (For a more comprehensive discussion of the mechanism of thyroid hormone synthesis and release, please see Endocrine System, Vol 4, Chap 7). T3 binds to thyroid receptor a and thyroid receptor b on target cells. When bound by their T3 ligand, these receptors are translocated to the nucleus and bind to Thyroid Response Elements on target regulatory sequences in combination with other nuclear receptor complexes, most notably those of the RXR superfamily. The result is a combination of transcriptional suppression of some genes and chromatin remodeling via histone acetylation/deacetylation as well as transcriptional activation of other genes (Wu and Koenig, 2000). Thyroxine stimulation increases utilization of carbohydrates and proteins for energy, the basal metabolic rate, heart rate and cardiac output, as well as potentiating catecholamine activity and enhancing brain development. I is absorbed fully from stomach and colon and taken up almost exclusively by the thyroid, although it can be found in kidney, salivary gland, and to a lesser extent in stomach, skin, mammary gland, placenta, and ovary. When metabolized, T3 and T4 release I for excretion via urine, milk, and, to a lesser extent, feces and sweat. IODINE

FIGURE 3.11 (A) Dental fluorosis, calf born to fluoride-intoxicated cow. Brown discoloration of enamel and enamel hypoplasia are present. (B) Microradiograph of cortical bone from a pig demonstrates several osteons, with increased numbers of peripheral osteocytes, enlarged osteocyte lacunae, and hypomineralization. (A) Courtesy of Dr. Roy Krook, College of Veterinary Medicine, Cornell University. (B) Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Fundamentals of toxicologic pathology, ed 2, Academic Press, 2010, Figure 14.5, p. 424.

Osseous lesions are more serious and typified by extensive remodeling of long bones, with marked exostosis and enostosis, leading to dense, thick brittle bones with small marrow cavities. Bones impregnated with excessive fluorapatite are more readily fractured than normal bone, and lameness is the more common clinical feature in cattle, sheep, and horses, with visible



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The ability of the thyroid gland to concentrate iodine in the form of iodinated thyroglobulin within follicular colloid has both positive and negative effects. The positive effect is that I can be retained during times of dietary I deficiency to maintain baseline basal metabolic rate. The negative effect is that episodic exposure of I-deficient individuals to a high I source can lead to excessive accumulation of iodinated thyroglobulin and pathologic enlargement of the thyroid gland (goiter). This happens because the thyroid alters its metabolic baseline during times of dietary deficiency by increasing its efficiency of uptake. When sufficiency is restored, thyroglobulin synthesis incorporating iodine may continue for some time until the enzymes responsible are downregulated, leading to massive accumulations of thyroglobulin within follicles. (For additional insight see Endocrine System, Vol 4, Chap 7). I as well as I2 can be directly taken up directly by certain epithelial cell types, independent of the thyroxine pathway. These epithelia include mammary, uterine, cervical, and gastric epithelium where I- has roles as an antioxidant, a regulator of apoptosis, and a promoter of differentiation (Aceves et al., 2013). The RDA for I is 150 mg/day for adults, 220 and 260 for pregnant and lactating women, respectively. The upper intake level of I for adults in the United States is 1100 mg/day (IOM, 2001). Particularly rich dietary sources of I are kelp, cod, oysters, and yogurt, with iodized salt (containing NaI or KI), milk, eggs, fish sticks, liver, and ice cream having substantial concentrations as well. Meats, vegetables, and fruits contain little if any I (ODS, 2021b). I requirements for domestic animals and poultry can be found in the NRC publications listed at the beginning of the References section of this section. Multivitamin preparations often contain I, limited to 150 mg/day. Many supplements contain NaI or KI, although natural I- supplements made from kelp or seaweed are also available. Supplements are often used prenatally in pregnant women to minimize the impacts of low thyroxine on fetal development. The tendency of I to overaccumulate in thyroid can be advantageous since this effect permits the use of radioiodine as a successful drug in treatment of hyperthyroidism. I- has been proposed as a therapeutic agent for fibrocystic

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breast diseases and as an anticancer agent against mammary, ovarian, uterine, and gastric cancers due to its direct extrathyroidal prodifferentiation effects on these tissues (De la Vieja and Santisteban, 2018). I also is used therapeutically in human asthmatic patients to counter viscous bronchial secretions. In dogs, cats, and horses, oral NaI is used to treat sporotrichosis and other cutaneous fungal infections, and in cattle to treat actinomycosis. Dietary NaI or Ca iodate supplements are used in animal rations, especially dairy and poultry to enhance reproductive performance and improve immune function.  Deficiency Human populations at risk for I  deficiency include those living in areas with I deficient soils; those who generally don’t consume iodized salt either by choice, due to concerns about overexposure, or lack of availability (subSaharan Africa, Southeast Asia, Eastern Europe); vegans; those with low meat, seafood, and dairy in their diets; pregnant women; and those with marginally deficient I status consuming plants or plant preparations that contain goitrogens like thiocyanate (SCN). For example, endemic neurological and neuromuscular abnormalities are common in areas of the world that are low in I and depend upon cassava as a major starch source. Cassava is rich in cyanogenic glycosides, which break down when the plant is macerated or chewed to release cyanide, which is then rapidly metabolized in the body to SCN and excreted in the urine. Cigarette smokers in I-deficient areas run the same risk due to the abundance of cyanide in cigarette smoke. SCN competitively inhibits with I uptake into the thyroid and with thyroid peroxidase to impair organic iodination of thyroglobulin, producing thyroxine deficiency (Erdogan, 2003). Brassica vegetables also contain varying types and quantities of thioglycosides (as glucosinolates), some of which, depending on the particular thioglucoside and the manner in which the plant is consumed, will breakdown to yield SCN- or goitrin (l-5-vinyl-2-thioo¨xazolidine). Goitrin inhibits iodination of thyroglobulin. Both livestock and the human populations are affected adversely although livestock more so, as they tend to eat or be fed parts of the plants that humans won’t eat and which contain higher glucosinolate concentrations. I deficiency is associated with decreased fertility, disruption of embryonic and postnatal

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development, and increased fetal and prenatal mortality. Fetal deficiency, reflective of maternal deficiency, results in neurological developmental damage known as cretinism in severe cases, with spastic dysphagia and severe mental retardation. Even in cases of mild to moderate deficiency, substantial impacts on cognitive function can occur (Zimmermann, 2009). The major lesion of I deficiency in both newborn and adult is hyperplasia of thyroid follicles and hypertrophy of individual follicular cells, usually with minimal colloid production. “Colloid” goiter often develops when adequate I is restored to the diet after a period of deficiency, due to the overproduction of colloid secondary to more efficient uptake by the hypertrophied, still-stimulated epithelium. Readily discernible thyroid goiters can grow to become grossly disfiguring, causing obstruction to nearby organs including the trachea, larynx, and esophagus. Histologic lesions in both domestic animals and humans are very similar (Miller, 2017).  Excess I -replete individuals are tolerant of high levels of I; however, individuals who are marginally or overtly I deficient are very sensitive to even moderate doses of I during repletion and can develop thyrotoxicosis, particularly those who already have goiter (Leung and Braverman, 2014). In replete individuals, excess I may cause hypothyroidism because I will inhibit release of TSH. Because overt signs of thyrotoxicosis, such as exophthalmus, may not always be evident, slowly evolving weight loss, muscle weakness, and tachycardia may go undetected until the situation is life threatening, particularly for individuals with underlying cardiac heart disease. Iodism can occur, especially calves, when the dams are fed iodine at the high end of the "safe” dietary limit over long periods of time, the iodine being readily excreted in milk. The lesions, including weight loss, anorexia, lethargy, lacrimation, scaly skin, and susceptibility to respiratory infection, mimic those of hypothyroidism. The usual lesion is thyroid atrophy but, paradoxically, a combination of thyroid atrophy and nodular hyperplastic goiter may occur in some animals as iodine-rich thyroglobulin accumulates in the follicles (Ong et al., 2014). In cats, a link has been made between excess dietary iodine and hyperthyroidism, in which thyroid hyperplasia and thyroid adenomas frequently

are present (van der Kooij et al., 2014). Maximum tolerable doses appear to be 50 ppm for most species, although horses are more sensitive, and 5 ppm is suggested as a maximum. Fe (Feþ2dbiologically active form, Feþ3dstorage form) is essential for life and serves as an electron donor (as Feþ2) and as an electron acceptor (as Feþ3). In its active form, Fe generally resides either within a heme molecule attached to the requisite enzyme or as part of an Fe–S cluster within the protein. Heme proteins containing iron include hemoglobin, myoglobin, various cytochromes in the mitochondrial electron transport chain, CYPs, and some peroxidases (e.g., catalase). Since electrons are transferred to the oxygen to “activate” it, there is potential for these enzymes to generate free radicals. Enzymes with Fe–S clusters include many of the electron transport proteins, NADH dehydrogenase and dehydrogenases in general, enzymes in the SAM methylation pathways, aconitase, ferredoxins, and lipoic acid and biotin synthetic pathways. There is a propensity for Fe-containing enzymes to generate free radicals and reactive oxygen species when conditions for enzymatic activity are not ideal or Fe is inappropriately released from cells or stores; hence iron metabolism is tightly regulated. The propensity for Fe to toggle between Feþ2 and Feþ3 generates free radicals of superoxide anion or hydrogen peroxide that are utilized by inflammatory cells for microbiocidal killing (see Biochemical and Molecular Basis of Toxicity, Vol 1, Chap 2). Healthy adult humans typically contain 3–5 g Fe, 60% of which is in hemoglobin, 10% within myoglobin, and the remainder mostly in hepatocytes and cells of the fixed macrophage (reticuloendothelial) system, such as bone marrow nurse cells, splenic red pulp macrophages, and Kupffer cells. Men store 30%–40% of absorbed Fe, while women only store 10%–15%, due to loss of blood during menses and utilization of Fe during pregnancy and lactation, putting them at risk for Fe deficiency. Infants and young children have high Fe requirements because they are growing rapidly, and, if nursing, at risk for deficiency since milk is a poor source of Fe. Fe is not excreted but 1– 2 mg/day is lost in adults through desquamation of enterocytes, sweat, and hemorrhage; hence 1– 2 mg/day must be absorbed from the diet. The IRON

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body Fe load is regulated by Fe uptake, which depends upon dietary content and the form of Fe as well as by body Fe status and erythrocyte synthetic rate. Fe absorption occurs in the duodenal enterocyte. Inorganic Feþ2 is transported across the enterocyte membrane, converted to Feþ3 and transported out of the enterocyte by Cu-containing hephaestin after which it binds avidly to plasma transferrin, which carries it to other tissues, losing the iron after binding to a transferring receptor on the target cell surface. Organic (heme) Feþ2 is absorbed by an as yet undefined process, released from the heme and exported by the same process as inorganic Fe. Heme Fe is 3–4X more bioavailable than inorganic Fe. Excess Feþ3 in enterocytes and other target tissue cells is stored by binding to ferritin, which releases Feþ3 with the appropriate stimulus. Most Fe not in erythrocytes and muscle is stored in liver and macrophages. Hemosiderin is a form of ferritin that is lysosomal, with partially degraded ferritin and trapped Fe that is not readily bioavailable. Hepcidin is the key regulatory hormone in Fe regulation, acting to inhibit the efflux of Feþ3 from enterocytes, hepatocytes, and macrophages. When hepatic Fe stores increase above a certain point, the transcription factor Bone Morphogenic Protein 6 (BMP6) is induced, which triggers hepicidin synthesis by the hepatocyte. Another factor that can induce hepcidin synthesis is the key inflammatory cytokine, interleukin-6 (IL-6), hence the decreased Fe absorption associated with ongoing inflammation. For a more detailed and in-depth discussion of Fe metabolism, see Chifman et al. (2014). The RDA for men and postmenopausal women is 8 mg/day, and 28 mg/day for menopausal women. Absorption (both heme and nonheme) is around 15% and affected by stomach acidity (acidity promotes uptake by converting Feþ2 to Feþ3), Vitamin C (promotes uptake), phytate intake (inhibits uptake), and polyphenol consumption (inhibits uptake). Cu also competes with Fe for uptake (IOM, 2001c). Food rich in heme Fe include organ meats, meats of all kinds (including seafood), and eggs. Vegetables, rich in nonheme Fe, include spinach, sweet potatoes, kale, collards, broccoli, and chard. However, many grains in developed countries are fortified with Fe, making breads and cereals good sources of Fe in these countries.

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Deficiency Fe deficiency is the most common nutritional deficiency in humans, both within the United States and worldwide, producing hypochromic, poorly regenerative, or nonregenerative microcytic anemia with concomitant low energy, impaired intellectual performance, and failure to maintain body temperature. Even in Fe-adequate areas, however, vegans and those consuming unfortified cereal-based diets are at risk for deficiency anemia. Infants born prematurely are at particular risk for Fedeficiency anemia, since neonates normally accumulate Fe stored during the third trimester. Correspondingly, pregnant women are prone to Fe deficiency in the third trimester. Chronic inflammation from a variety of causes with prolonged hepicidin release can lead to Fe deficiency due to decreased absorption and sequestration of Fe stored in liver and macrophages. Chronic GI disease can also lead to decreased absorption, and prolonged blood loss due to GI bleeding as well as chronic external hemorrhage are common secondary causes of Fe deficiency (Cappellini et al., 2020). Long-term use of proton pump inhibitors such as omeprazole to control gastrointestinal reflux disease can interfere with Fe uptake and cause iron deficiency by augmenting the expression of hepcidin (Hamano et al., 2020). Environmental lead (Pb) toxicosis often is associated with Fe deficiency. This is due partly to enhanced uptake of Pb by the Fe uptake system, which is unregulated in Fe deficiency, but also partly environmental, with those in the lower socioeconomic bracket often being both Fe deficient and living in a Pb contaminated environment (Kordas, 2010). Primary, i.e., nutritional, Fe deficiency is rare among domestic animals, although rapidly growing neonates in confinement on an exclusive milk diet may be predisposed. Milk is notoriously low in Fe. Young pigs, often raised in confinement with no access to soil, are especially prone to Fe deficiency and may have to be supplemented via Fe injection. Secondary Fe deficiency can occur in any age group where blood loss through GI or external hemorrhage, severe GI endoparasitism (e.g., small intestinal hookworm [Ancylostoma caninum] infestations in puppies and abomasal barber’s pole worm [Hemonchus contortus] infestations in sheep) can exceed the ability of the animal to take up Fe from the diet. Lesions of Fe deficiency anemia

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are the typical poorly regenerative or nonregenerative microcytic and hypochromic anemia, with increased erythrocyte fragility and target cell erythrocytes (Boes and Durham, 2017). Excess In humans, dietary toxicity is virtually nonexistent except in portions of sub-Saharan Africa where locally brewed beer is heavily contaminated with Fe (Bothwell et al., 1964). Most cases of acute Fe toxicosis occur in young children consuming large amounts of liquid oral supplements (Anderson, 1994) at doses of 20– 60 mg/kg or accidental ingestion of adult iron supplements. GI irritation leading to bloody vomitus, gastric hemorrhage, and necrosis with black stools are characteristic pathological signs of acute oral Fe excess. This can be associated with elevated serum Fe, vascular congestion of multiple organs, anorexia, oliguria, diarrhea, hypothermia, metabolic acidosis, shock, and death. Of more concern is long-term ingestion of oral supplements. The strongest evidence is for an association between coronary heart disease risk and chronic Fe overload (Salonen et al., 1994) with more tentative links to inflammatory bowel disease. Excess Fe produces depressed feed intake and growth rate in domestic animals. Hemosiderosis in bone marrow and spleen is a prominent feature. The lowest dose with an adverse effect is 500 ppm in calves and poultry. Swine appear to have minimal effects at oral doses as high as 3000 ppm, although body weight gain and feed intake are depressed at 5000 ppm Fe, and signs of P deficiency can appear (NRC, 2012). MANGANESE Manganese (Mn), a name derived from the Greek word for magic, has long been recognized as essential for full growth and normal reproduction. Mn (mainly as Mnþ2 or Mnþ3) is an essential cofactor in several enzymes vital for carbohydrate and amino acid metabolism such as pyruvate carboxylase, phosphoenolpyruvate decarboxylase, glutamine synthetase, and arginase. It has role in secondary metabolism as an activating factor for various oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Of especial importance is Mn’s role in activation of glycosyl transferases necessary for formation of proteoglycans for normal bone and cartilage matrix. It also has a critical role in cellular antioxidant

defense as an essential cofactor in Mndependent superoxide dismutase (Mn-SOD). Absorbed mainly in the small intestine, the exact mechanism of uptake is unknown, but Mn may share the same uptake system as Fe and aluminum (Al), especially since dietary Al, Fe, and phytate slow absorption. Only 3%–5% of ingested Mn is absorbed, with females almost three times more efficient than males (Finley et al., 1994). Mn homeostasis appears to be regulated by excretion. Studies have shown that Mn shares the transferrin system with Fe and Al for transport in blood. Ethanol has been found to increase the body load of Mn, possibly through upregulation of Mn-SOD. Mn is transported in plasma via the transferrin system and excreted mainly in bile but also via milk and sweat (Davis et al., 1993). Most Mn in the body is found in liver, pancreas, kidney, bone, adrenal glands, and brain. Adequate dietary intake for adults for Mn is 2–5 mg/day (IOM, 2001d). Rich dietary sources of Mn include whole grains of all kinds as well as most nuts, but chocolate, tea, shellfish, legumes, fruit, leafy vegetables, seeds, and spices like chili powder and cloves are also rich sources. Cow’s milk also contains high concentrations of Mn, and milk formulas often contain concentrations high enough to be of concern (Horning et al., 2015). Therapeutically, Mn is found in supplements and multivitamins and may be taken for conditions such as osteoporosis and osteoarthritis (Price et al., 2012). It should be noted, however, that Mn is relatively abundant in the environment and food such that “normal” intake in some circumstances can approach the level of adverse effects (Greger, 1998). Deficiency Dietary Mn deficiency is rare in humans and mostly reported experimentally. Altered glucose metabolism, altered cholesterol metabolism, and a variety of bone abnormalities have been associated with insufficient Mn consumption (Horning et al., 2015). In domestic animals, Mn deficiency can be a problem in grazing cattle, sheep, swine, and poultry, resulting in skeletal abnormalities and defective lipid and carbohydrate metabolism in addition to adverse effects on growth and reproduction (Spears, 2019). Poultry are susceptible to the effects of deficiency, and they have much higher dietary requirements than do cattle and pigs. Mn-deficient animals in general have short,

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thick, often deformed limbs, spinal curvature, and swollen and enlarged joints, due to defective synthesis of ground substance in developing cartilage. Both osteoblast and osteoclast activities also are depressed, aggravating defective bone development. Ataxia develops in animals that are deficient in utero, due to calcification of otoliths (cartilaginous structures in the inner ear). Pancreatic abnormalities, including hypoplasia, and depressed insulin synthesis and secretion, can be corrected by feeding Mn. Excess Mn toxicity due to dietary intake in humans is rarely reported. Instead, excessive Mn exposure in humans is typically from industrial dusts or contaminated water (Aschner and Erikson, 2017) and can interfere with Fe uptake and transportation, producing Fe-deficiency anemia. CNS abnormalities include hyperirritability, violent acts, hallucinations, and incoordination. Permanent damage occurs in the extrapyramidal system, producing symptoms similar to those of Parkinsonism (O’Neal and Zheng, 2015). Also, like Al, Mn has been implicated in some cases of amyotrophic lateral sclerosis. Dietary Mn appears to have few adverse effects below 1000 mg/kg in the diet unless dietary Fe levels are low. Additional reported outcomes of Mn toxicity include impaired carbohydrate metabolism (rebound hypoglycemia), anemia, and impaired cardiovascular function. Mn toxicity is not typically reported in domestic animals, but the rat has been used extensively to investigate the neurologic effects of Mn excess (Avila et al., 2016). As in humans, the neurological abnormalities mimic in many ways those associated with Parkinson’s disease. Mo (as Mo6þ) is required for the proper function of four enzymes: sulfite oxidase, aldehyde oxidase, xanthine oxidase, and mitochondrial amidoxime reductase (Mendel, 2009). In its active form, Mo, as molybdate MoO2), is bound to two S attached to an organic 4 ring complex known as molybdopterin, to form the molybdenum cofactor (Moco), which is in turn complexed to the protein portion of the enzyme. It is essential for normal sulfite and urate metabolism. Molybdenum absorption appears to be in excess of 90%, occurring in the stomach and throughout the GI tract. Following uptake, Mo concentrates in liver and kidney being MOLYBDENUM

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transported into cells as molybdate. Mo is excreted mainly into urine. Recommended intake is 17 mg (young children) to 45 mg/day (adults) (IOM, 2001e). The amount of Mo in foods is dependent on the Mo content in the soil from which the food originated but particularly rich sources of Mo include legumes and beef, with dairy products, grains, and grain-based foods, as well as peanuts and bananas also being good sources (ODS, 2020c). It should be noted that most foods in Mo-sufficient areas contain adequate Mo to meet dietary needs. Deficiency Although Mo was shown to be essential in the 1950s, clinical deficiency is very rare, occasionally being reported in relation to prolonged parenteral nutrition. Clinical signs and abnormalities are ill defined but include high xanthinuria and hypoxanthinuria, high sulfaturia, sensitivity to sulfite, and neurological dysfunction resembling retardation. These changes can be reversed by Mo supplementation. Of greater concern, perhaps, is the tentative epidemiological link between endemic Mo deficiency (central Asia from Iran to western China and sub-Saharan Africa) and esophageal squamous cell cancer (Van Rebsber and Van Rensburg, 2019). Whereas there are strong correlations between Mo status and a much higher incidence of this type of cancer, intervention studies have been mixed and causation has yet to be established (Barch, 1989). In the laboratory, signs of deficiency include low plasma uric acid, high plasma methionine, high urinary sulfate, low urinary uric acid, and increased sensitivity to sulfite toxicity associated with abnormal S metabolism. In addition, experimentally Mo-deficient animals produce high urinary xanthine and hypoxanthine, due to low xanthine oxidase activity, interrupting uric acid production. Excess Molybdenum toxicity has not been defined in humans but occurs when ruminants graze on plants growing on high Mo soils. Signs of toxicity are in large part due to secondary Cu deficiency, with growth retardation and anemia, anorexia, diarrhea, and posterior weakness with spinal cord degeneration, as described previously. This somewhat unusual manifestation of toxicity is because molybdenum complexes with S (as sulfate) and Cu to form a complex that makes Cu unavailable for uptake.

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Untreated animals become emaciated and finally die (Schwarz and Belaidi, 2013). Sheep and goats have crimping or curling of wool and loss of wool pigmentation. Molybdenum toxicosis can be reversed by Cu supplementation. Humans exposed occupationally or in areas with high natural Mo levels, such as Armenia, have elevated plasma uric acid levels and increased incidence of gout. SELENIUM Selenium (Se) is an essential micronutrient in animals but not essential for many plants. However, some plants accumulate it, especially in semiarid, high altitude environments. The main function of Se in vertebrates is as a cofactor for glutathione peroxidases and thioredoxin reductases, key enzymes in the redox cycles that maintain appropriate thiol ratios to regulate oxidant status in cells. The glutathione peroxidases are especially important in vertebrates, where their primary function is to catalyze the reduction of hydrogen peroxide to water using reduced glutathione as the hydrogen donor, forming oxidized glutathione, which is then recycled via non-Se-dependent glutathione reductase to reduced glutathione. It performs a similar function in reducing lipid peroxides in membranes to far less reactive lipid alcohols. Thioredoxin reductase, on the other hand, reduces the disulfide linkage in oxidized thioredoxin to restore it to its active status. Se is also an essential cofactor for the enzyme, 50 -deiodinase, responsible for the conversion of thyroxine to its active form triiodothyronine (see above). There is also a host of selenoproteins, rich in selenocysteine residues, with little known about their function, although a few seem to have an important indirect role in the synthesis of deoxy ribonucleotides for DNA synthesis and a key role in proper immune function (Ying and Zhang, 2019). Se exists in the body primarily as selenocysteine, and replaces the S in at least one of the cysteine moieties at the active site of the enzymes. Selenocysteine is formed via a posttranslational modification of a cysteine bound to particular tRNA (UGA codon) at the time of translation. Se may be incorporated into the diet as inorganic selenium (selenite), selenomethionine, or selenocysteine. Absorption is primarily in the caudal small intestine and is relatively efficient, with 70%–90% uptake, for organic forms, less so for selenite unless complexed with

glutathione. Se shares many uptake mechanisms with S. Organic Se (as selonocysteine or selenomethionine) can enter and participate in the same pathways as their corresponding Scontaining counterparts and can be incorporated nonspecifically into proteins. Eventually all forms of Se are converted to selenite from whence, after conversion to selenophosphate, Se can be incorporated into proteins at specific sites during translation via the UGA-tRNA. Se is excreted in the urine as a methyl-Se form conjugated to an N-acetylated galacosamine or as trimethyl-Se (Roman et al., 2014). The RDA for Se is 55 mg/day for adults and 60–70 mg/day for pregnant and lactating women, respectively. Foods particularly rich in Se are Brazil nuts and tuna, but organ meats, muscle meats, fish, poultry, eggs, dairy, legumes, and grains/grain products contain substantial amounts as well. The amount of Se in plantbased foods is dependent on the soil Se status in which the plants were grown, for animal sources, less so (ODS, 2021a). Se supplementation has been used or advocated for four general disease states where Se may have a therapeutic effectdcancer, cardiovascular disease, cognition, and thyroid diseases. These studies have been based on the hypothesis that the antioxidant effects of Se should have a dampening effect on the oxidant stress and oxidant damage that is prevalent in the first three aforementioned disease states. For cancer, there is relatively strong epidemiologic evidence that Se may decrease overall cancer risk and mortality, especially for bladder and prostate cancers, but intervention studies have produced conflicting results. For cardiovascular disease, the epidemiologic evidence is less convincing but some intervention studies have shown promising results, but, again, intervention and placebo studies have yielded conflicting results. The case for Se supplementation for improvement of cognitive decline with age is more tenuous and further studies are needed to establish a link, whereas for thyroiditis, results are more promising. However, large-scale clinical trials are lacking (ODS, 2021a; Ying and Zhang, 2019; Roman et al., 2014). Given the narrow gap between adequate and toxic doses of Se, consumption of Se supplements is not generally recommended. Deficiency The lesions of Se deficiency in both humans and animals are identical and have been

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discussed previously (see subsection on Vitamin E above). Humans at risk for Se deficiency fall into several categories, with those living in areas of the world with Se-deficient soils and access to only locally grown food being the largest at-risk group. Humans living in low-Se districts of China exhibit a cardiomyopathy termed Keshan disease that can be corrected by increasing dietary Se. Immunosuppressed individuals (e.g., those infected with HIV) and those undergoing hemodialysis are also predisposed (Roman et al., 2014). A Se-deficient, torula yeast–based diet will precipitate hepatic necrosis and reproductive failure in rats, pancreatic dystrophy and exudative diathesis in chicks, hepatosis dietetica (acute centrilobular to massive hepatic necrosis) in swine (Figure 3.12), and white muscle disease (acute myonecrosis) in lambs and calves. While these syndromes can be reversed by feeding Se, Se requirements vary inversely with vitamin E and S amino acid status of the animal, and many Se deficiency syndromes can be diminished or even abolished by treatment with Vitamin E.

FIGURE 3.12 Acute centrilobular hepatic necrosis due to selenium deficiency (hepatosis dietetica), liver, piglet. Necrotic hepatocytes around the central vein in the upper left quadrant have lysed and sinusoids have become dilated and engorged with blood. A progression from necrotic centrilobular hepatocytes on the left to vacuolated, dying hepatocytes to viable hepatocytes on the right side of the image can be observed. Hematoxylin and eosin stain. Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 36.10, p. 1116, with permission.

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Thus, while Se deficiency uncomplicated by vitamin E status can be studied in the laboratory, it is not unraveled so easily in the clinical or farm situation. Excess While the response to severe acute Se excess varies among species, the most common outcome is hemorrhagic enteritis and myocardial hemorrhage. The latter condition progresses to myocardial degeneration, cardiac insufficiency, and subsequent congestion and edema in visceral organs (Raisbeck, 2000). Whereas acute Se toxicity in humans is rare, chronic selenosis, especially from dietary sources, does occur, with exuberant use of Se-containing supplements used to boost immune function, promote antioxidant status, and prevent cancer. Both inorganic and organic Se are toxic and excess is associated with abnormally brittle hair and nails, skin rash, GI disturbances, neurological disturbances (e.g., fatigue, hyperirritability), and a unique garlic odor to the breath (IOM, 2001f). Less overt cases of Se excess have been associated epidemiologically with Type 2 diabetes, increased high-grade prostate cancer, and neurodegenerative diseases, in particular Parkinson’s disease and ALS (Vinceti et al., 2018). Animals foraging on seleniferous plants, especially in the high plains and intermountain plateau regions of North America, is the most frequent cause of Se toxicosis, termed selenosis (see Poisonous Plants, Vol 3, Chap 7). Misformulation on dietary mineral supplements has also led to Se toxicosis. The hallmark feature of selenosis is the shedding of hair and sloughing of hoofs (Figure 3.13) (Hargis and Myers, 2017d). Some seleniferous plants also disrupt vision and produce stumbling and respiratory failure, but this syndrome of “blind staggers” probably has more to do with alkaloids that are typically abundant in seleniferous plants rather than to Se per se. However, lameness, with malformation of hoofs, loss of hair, and emaciation are reproducible under experimental conditions by feeding Se, and therefore are thought to be due to chronic exposure to increased levels of Se. In addition, reproduction can be affected adversely. Fatty change, necrosis, and hemorrhage are evident in liver, kidney, and pancreas. In pigs and horses, degeneration of gray matter of the spinal cord can develop, with necrosis of neurons and cavitation and necrosis in ventral horn areas.

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FIGURE 3.13 Selenium toxicosis in a calf, with sloughing of the hoof. Courtesy Dr. John King. Figure from Haschek WM, Rousseaux CG, Wallig MA, editors: Fundamentals of toxicologic pathology, ed 2, Academic Press, 2010, Figure 7.21, p. 154. ZINC Unlike most other divalent cations, Zn (as Znþ2) has no redox chemistry. However, Zn is a strong Lewis acid, permitting it to bind well to thiolate and amine electron donors within the cell; therefore, Zn is >95% intracellular. Zn has great binding flexibility and hence is a common catalytic cofactor at the active sites of enzymes of many classes. Zn has two major roles in the cell, as a catalytic or cocatalytic cofactor in enzymes and as a structural/regulatory component of DNA-binding domains in a variety of transcriptional factors that bind directly to DNA. The unique, folded structural conformations at these binding domains have been given the sobriquet, “Zn fingers.” In its role as a catalytic cofactor, Zn is essential for a variety of enzymes, the most well-known of which are carbonic anhydrase and carboxypeptidase. Other prominent and important Zn-dependent enzymes are the matrix metalloproteinases (MMPs), phospholipase C, and DNA-dependent RNA polymerase as well as proteins within the mitochondrial electron transport chain and a-ketoglutarate dehydrogenase. Zn is also complexed to a high degree to

cellular proteins in general, where it serves as a buffer, most often associated with glutamic acid, aspartic acid, cysteine, and histidine. Zn has an important role in ion signaling and is essential in such processes as glutaminergic signaling in the nervous system, pancreatic secretion, insulin secretion, apoptosis signaling, and signaling within the immune system (Maret, 2013). Zn homeostasis is regulated by both absorption (greatest in the jejunum in humans via the ZIP4 transporter) and excretion (pancreatic secretion and direct secretion along the entire length of the gut). There is no storage pool of Zn in the body; a small functional pool within each tissue is available for transfer to plasma and tissue turnover is the usual process for stabilizing Zn status. Plasma Zn, normally 1 mg/mL, can fluctuate greatly with intake and with stressors and is mostly bound to albumin and a-2-macroglobulin, with a small amount also bound to transferrin. Fasting is associated with hyperzincemia, due to release of Zn from muscle, while infection is associated with hypozincemia. Zn is an abundant trace mineral,

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second only to iron, and is stored by transfer to the numerous cellular metallothioneins in the body. Zn concentrations are highest in prostate, retina, brain, liver, bone, muscle, kidney, and pancreas. Zn is efficiently excreted in the bile (feces) and also in the urine (Livingstone, 2015; Maret, 2013). The RDAs for Zn are 8 and 11 mg/day for women and men, respectively, with 12 mg/day recommended for lactating women (IOM, 2001g). Zn is most abundant in foods of animal origin, including shellfish (particularly oysters), beef, pork, poultry, eggs, and dairy products. Plants also can have high Zn, but Zn content is dependent on soil Zn status as well as phytate content in the plant, phytates binding Zn and preventing absorption by the gut. Plants containing the highest levels of bioavailable Zn include grains (especially wheat), seeds, nuts, and “pulses” (e.g., dry beans, lentils, and chickpeas) (ODS, 2021d). Processed grains can be supplemented with Zn. Zn supplementation has been widely used to boost immune function during illness, since Zn levels often drop during disease. However, oversupplementation has been a problem, leading to Cu deficiency in some individuals. Zn supplementation has also been recommended for populations whose diets are inadequate in animal-based or legume-based foods. However, Zn status varies widely among populations, even in regions with poor diets, since soil Zn content is variable and high Zn can interfere with uptake of Fe and in particular Cu, both of which also tend to be lacking in poor diets (Fosmire, 1990). Therapeutically Zn has been used for treatment of acne, boosting immune function, cancer patients, patients with enteropathies, and for patients with Wilson’s disease (excess Cu accumulation in the liver) as a competitive agent to impair Cu uptake. The risk, except in Wilson’s disease, is the induction of Cu deficiency. Zn therapy has also been proposed as a therapy for Type II diabetes since patients with this disease tend to have zincuria and become Zn deficient (Maret and Sandstead, 2006). Deficiency Zn deficiency is not common, and usually is caused either by a dietary component that decreases Zn bioavailability, such as phytate, or by a GI disorder disrupting absorption, including such conditions as alcoholism,

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cirrhosis, malabsorption, and pancreatitis. Excessive consumption of soy-based foods or infant formulas, which are high in phytates, can precipitate Zn deficiency. Even before an overall decrease in tissue Zn concentration is evident, deficiency causes a reduction in growth associated with a reduction in food intake, termed Zn-related anorexia. This anorectic growth retardation has the effect of permitting Zn tissue levels to remain at near normal concentrations. Chronic Zn deficiency in humans is manifested as dwarfism and sexual immaturity in the young, especially if maternal Zn deficiency was present during pregnancy and lactation. Impaired immune function and diarrhea often precede the skin lesions, termed acrodermatitis enteropathica, but even acute deficiency rapidly produces the characteristic symmetrical necrotic (acute) or parakeratotic (chronic) skin lesions, prevalent around the facial orifices and perineal areas, but also involving feet, trunk, and inner thighs (Gehrig and Dinulos, 2010). Other abnormalities include ataxia, disorientation, skeletal deformities, impaired wound healing, and impaired reproduction (Maret and Sandstead, 2006). In contrast to humans, if a rat is force fed a Zndeficient diet, it continues to grow, plasma Zn levels fall, and the animal soon dies. Zn deficiency is characterized not only by depressed appetite and retarded growth, but by skin lesions, immune deficiency, skeletal abnormalities, and impaired reproduction. In mice dietary Zn restriction triggers apoptosis of b-cells in pancreatic islets, significantly reducing their size, which can be restored to normal after dietary Zn sufficiency is restored (Sisnande et al., 2020). In most species, the primary lesions of Zn deficiency are in the skin, manifested as macules that progress to encrusted, scaly plaques due to acanthosis with parakeratosis and accumulation of parakeratotic cellular debris. This animal version of acrodermatitis enteropathica is especially common in Zn-deficient swine, and also is typically associated with infection due to loss of immune function (Neldner et al., 1978). This condition has also been reported in large breed dogs, especially Siberian huskies and Akitas (Hargis and Myers, 2017e). While Zn deficiency has been linked to thymic atrophy and loss of immune function, the biochemical lesion is

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unknown. Fetal Zn deficiency is associated with severe alterations in normal bone, and, in birds, abnormal feathering. Zn deficiency is teratogenic in all species evaluated (Neldner et al., 1978). Excess In humans, Zn toxicity from food is not encountered under normal dietary conditions. Acute Zn toxicity results in gastric distress, nausea, and disorientation. Emesis occurs with >150 mg. High Zn levels, like Zn deficiency, can cause immunocompromised and decreased food intake. However, even moderately excessive Zn ingestion can disrupt LDL/HDL ratios in an adverse direction (Fosmire, 1990). Zn toxicity has been reported in many species, most often associated with misformulation of mineral supplements or consumption of water stored in galvanized containers. Children, dogs, and birds can also become intoxicated ingesting Zn-containing or Zn-coated objects such as pennies, nuts/bolts/nails/staples, zippers, game board pieces, jewelry, and even lozenges. In ruminants, pancreas, kidney, abomasum, and liver are most commonly affected, with necrotizing pancreatitis followed by a mixture of atrophy and regeneration, renal tubular necrosis with regeneration, abomasal ulcers, and multifocal hepatitis (Allen et al., 1983). Decreased bone mineralization and bone and joint deformities have been reported in several species. Osteochondrosis dissecans of the articularepiphyseal cartilage complex in horses in some cases has been linked to excess Zn consumption (Olson and Carlson, 2017c). These effects, however, are associated with Zn-induced Cu deficiency, rather than Zn itself since persistent Zn overexpose, as noted above, interference with both Cu absorption.

5. DIETARY CONTAMINANTS (Also See Issues In Laboratory Animal Science that Impact Toxicologic Pathology, Vol 1, Chap 29) 5.1. Analyses for Contaminants The issue of pesticides and other contaminants in the diet is a complex and controversial one for both animal and human diets and may not be a biological issue for a given contaminant when one considers the dose consumed and the

increased sensitivity of current detection methods. Much attention has been focused on estimating an acceptable daily intake (ADI) for humans, a judgment based on the NOAEL for a particular compound. The calculation of an NOAEL is based on determining the dose threshold in the most sensitive test or assay in the species most sensitive to the compound in question, with an additional “safety factor” multiplied into the equation. The calculation of an ADI is complicated by a variety of things, one of which from the nutrition aspect is the fact that children consume more food on a body weight basis than adults, and that neonates and females of reproductive age have other considerations when making the calculation. In the context of estimating ADIs for “contaminants” in the diet, it must be remembered that fruits and vegetables contain “naturally” occurring carcinogens, yet overall consumption of such foods consistently has been identified in epidemiologic and experimental studies as “beneficial.” For more in-depth coverage of this topic (see Food and Toxicologic Pathology, Vol 3, Chap 2).

5.2. Pesticides With regard to pesticides much research has been focused on the carbamates and organophosphates, with much debate centered around which rodent assay should take precedence when determining NOAEL and which uncertainty factor should be used for estimating the ADIdthe rodent uncertainty factor of 100 or the human based uncertainty factor of 10. Complicating the debate is the observation that not all carbamates or organophosphates have the same mechanism of action, requiring modification of old or development of new assays. Some scientists have concluded that covalently bound pesticide residues in foods (e.g., edible animal tissues) are either not bioavailable or nonreactive in their covalently bound form and hence of little concern to humans. Much of the evidence to date has been nebulous or inconclusive regarding low levels of pesticides in the diet and the risk of toxicity or cancer in animals or humans. Many of the pesticides (e.g., chlorinated hydrocarbons) that have been shown to be carcinogenic in rodent assay systems have yet to be shown to be associated with cancer in humans.

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Studies using mixtures of common pesticides at levels which might be encountered realistically in the diet have been negative for genotoxic or other DNA damage in in vitro systems or rodents. The problem of pesticide contamination should be considered when purchasing materials for inclusion in dietary formulations when the diets are not tested by the manufacturer for contaminants. For laboratory animals used in toxicological studies only certified diets should be used so that each batch can be assayed for suspected environmental contaminants, and the chemical composition of the diet, including the maximum concentrations of any known or suspected contaminants present be recorded to meet the requirements of regulatory guidelines. Since blanket analysis for all types of pesticides is virtually impossible and prohibitively expensive, a careful and thorough knowledge of the source of the material and any potential pesticides used in growing it or present in it is essential for toxicity studies. For more specific information about the toxicological effects of pesticides (please refer to Agrochemicals, Vol 3, Chap 11).

5.3. Mycotoxins, Heavy Metals, Phytoestrogens, and Other Contaminants Also of relevance to the toxicological pathologist is the potential contamination of experimental diets. Harmful naturally occurring substances as well as manmade contaminants are routinely found at very low concentrations in commercial animal diets. This is a potentially important problem when “natural” ingredients are used, the composition of which may vary considerably from batch to batch. This is particularly true when using plant material that may have widely varying concentrations of phytochemicals, complex organic compounds produced for various reasons such as protection against predators, sequestration of harmful substances absorbed from soil, and storage of unusable metabolic waste. Phytochemicals have no nutritional value for humans or animals consuming them but are often “biologically active,” meaning that, at the very least, they can and will trigger phase I or phase II (or both) detoxication systems to produce the desired biological effect (e.g., consumption of

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herbal supplements containing cannabinoids for alleviation of pain and anxiety associated with chronic disease). In some cases, phytochemicals in a diet are highly toxic; in others, much less so but nevertheless affecting detoxication systems, thus altering baseline metabolism. Phytochemicals vary widely among plant species and even among plants within a plant species, depending on growth conditions, variety of plant grown, and processing for dietary inclusion. Thus, when plants known to contain large quantities of phytochemicals are used in diets, it is often critical to know what the phytochemical composition and concentrations are, especially when different batches of dietary material from different sources or harvest times are used (see Food and Toxicologic Pathology, Vol 3, Chap 2; Poisonous Plants, Vol 3, Chap 7; Mycotoxins, Vol 3, Chap 6). In the human realm, not only can “organic” diets be rich in specific phytochemicals to produce a desired biological effect to enhance overall well-being (e.g., glucosinolates in brassica vegetables to enhance antioxidant status) (Bischoff, 2016), there is the issue of herbal supplements containing specific types of phytochemicals to produce specific biological effects, often neurological, such as the various cannabis preparations containing assorted cannabinoids to alleviate pain, anxiety, depression, and “stress” (see Herbal Remedies, Vol 3, Chap 4). Regulation of composition and quality of these supplements is notoriously lax or even nonexistent and their presence in a daily regimen may substantially alter response to a therapeutic regimen, for example. In addition, certain nutrients can assume exaggerated relevance when some experimental compounds are studied. For example, dietary folate becomes an important covariate when studying contraceptives or anticonvulsants. In rodent cancer studies, low dietary folate increases the risk of colonic cancer and depresses the safety and efficacy of chemotherapeutic agents like cyclophosphamide. In similar fashion, high dietary Fe can exacerbate aminoglycoside ototoxicity due to the generation of free radicals and depletion of glutathione in the ear (see Ear, Vol 4, Chap 10), while high dietary Ca can affect Zn absorption and metabolism, altering appetite and a variety of other metabolic processes. Phytochemicals become potentially

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important when studies involving induction or suppression of phase I or phase II drug metabolizing enzymes are being performed, when the toxicity (as well as safety and/or efficacy) of a drug is dependent upon induction or suppression of a particular detoxification enzyme, or an endocrine disruption study is being performed. Phytoestrogens in lab animal diets have attracted attention regarding their potential deleterious effects in biological systems (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). Phytoestrogens are nonsteroidal compounds (e.g., isoflavones like genistein, daidzein, and glycitein, coumestans, and lignans) produced by many plants, including soybeans, wheat, barley, corn, alfalfa, and oats. Although not steroids, phytoestrogens mimic or antagonize some of the actions of endogenous estrogens, but their potency is much lower than steroidal estrogens. At concentrations found in plants, such as soybeans, phytoestrogens have been shown to have potential health benefits against heart disease, osteoporosis, and certain cancers. However, experimental studies of high doses have shown certain adverse effects and endocrine disruption effects that have promoted some to advocate phytoestrogen-free diets. Removal of these substances from natural foodstuffs would not be advisable; however, knowledge of the types of phytoestrogens and their concentrations might be critical for appropriate interpretation of results and risk assessment. Besides the health benefits of normal levels of phytoestrogens in plants, very low levels may be deleterious to the animal’s health and have the potential to retard normal growth and maturation. Manufacturers can provide phytoestrogen profiles and concentrations present in each batch of certified diets and this approach is recommended for toxicity studies. More attention has been paid to sources of components for dietary formulations in recent years, and the use of certified diets has minimized potential contamination problems. Furthermore, by paying closer attention to component sources, major variations in dietary concentrations of various bioactive compounds that have complicated past studies are now less likely to be major problems in the Good Laboratory Practices (GLP) environment. Past variation has resulted in

appreciable variations in response in certain species to specific drugs or chemicals both within studies and between studies. This is especially relevant in the context of mycotoxin contamination of dietary components, where minuscule concentrations can cause significant alterations in response or even trigger toxicity in a sensitive species (e.g., aflatoxins, fumonisin B1, trichothecenes). Common contaminants of experimental and commercial animal diets that have been identified in the past include mycotoxins such as aflatoxin, heavy metals (lead, mercury, cadmium, and arsenic), nitrates, nitrosamines, chlorinated hydrocarbons, and polychlorinated biphenyls. In addition, microbial contamination remains a potential danger in all foodstuffs, including bacterial contamination from pathogenic Escherichia coli, Salmonella, Listeria, and other microbial pathogens. Even sterilized animal protein sources have been contaminated by heavy metals like organic mercury in fish meal and prion proteins (scrapie, bovine spongiform encephalopathy, and chronic wasting disease) in animal protein products. Therefore, it is of prime importance that all dietary components, natural or formulated, be evaluated, tested, analyzed, and certified before inclusion in any experimental diet intended for a GLP toxicological study. More detailed coverage of specific mycotoxins, phytoestrogens, heavy metals, and other contaminants are offered in other chapters (Food and Toxicologic Pathology, Vol 3, Chap 2, Mycotoxins, Vol 3, Chap 6, Poisonous Plants, Vol 3, Chap 7, Bacterial Toxins, Vol 3, Chap 9, and Metals, Vol 3, Chap 8).

REFERENCES Abdo KM, Rao G, Montgomery CA, et al.: Thirteen-week toxicity study of d-alpha-tocopheryl acetate (Vitamin E) in Fischer 344 rats, Food Chem Toxicol 24:1043–1050, 1986. Abraham A, Kattoor AJ, Saldeen T, et al.: Vitamin E and its anticancer effects, Crit Rev Food Sci Nutr 59(17):2831–2838, 2019. Aburto NJ, Hanson S, Gutierrez H, et al.: Effect of increased potassium intake on cardiovascular risk factors and disease: systematic review and meta-analyses, Br Med J 346: f1378, 2015. Aceves C, Anguiano B, Delgado G: The extrathyronine actions of iodine as antioxidant, apoptotic, and differentiation factor in various tissues, Thyroid 23(8):938–946, 2013.

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4 Herbal Remedies Colin G. Rousseaux University of Ottawa, Ottawa, Ontario, Canada

O U T L I N E 1. Introduction 1.1. Nomenclature

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2. Apothecary to Pharmacy

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8.4. 8.5. 8.6. 8.7.

3. Evidence for Herbal Remedy Efficacy 190 3.1. Empirical Evidence e Traditional Knowledge (Botanical) 190 3.2. Experimental Evidence e Controlled (Single Active Compound) 192 4. The Active Pharmaceutical ingredient(s) 192 4.1. Influencing Factors on the Concentration of the API(s) in the Plant 193 4.2. Dose and Response 193 4.3. Contaminants 194 4.4. Adulterants 194 4.5. A Comparison Between Properties of Herbal Remedies and Conventional Drugs 196 4.6. Acceptability of Herbal Remedies 196 5. Quality, Efficacy and Safety

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7. Efficacy and Effectiveness 7.1. Traditional Knowledge of Efficacy 7.2. Experimental Evidence 7.3. Randomized Clinical Trials Using Herbal Remedies

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8. Safety 8.1. Safety, Side Effects and Toxicity 8.2. Adverse Reactions 8.3. Interactions

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Herb-Herb Interaction Direct Toxicity Indirect Toxicity Hypersensitivity e Idiopathic Allergic Reactions

9. Toxicology of Herbal Remedies 9.1. Lethality 9.2. Genotoxicity and Carcinogenesis 9.3. Herbal Toxicokinetics 9.4. Microbiome 9.5. Herbal Pharmacodynamics 9.6. Organ Toxicity 10. Toxicologic Pathology of Select Herbal Remedies 10.1. Aloe vera e Aloe barbadensis 10.2. Cannabis e Cannabis sativa and Cannabis indica 10.3. Chamomile e Chamomilla recutita 10.4. Coffee e Coffea arabica and C. Canephora 10.5. Cocoa e Theobroma cacao 10.6. Echinacea e Echinacea purpurea 10.7. Ephedra e Ephedra sinica 10.8. Garlic e Allium sativum 10.9. Ginkgo Biloba e Ginkgo biloba 10.10. Ginger e Zingiberis rhizome 10.11. Ginseng e Panax ginseng 10.12. Goldenseal e Hydrastis canadensis 10.13. Green Tea e Camellia sinensis 10.14. Indole-3-Carbinol e Brassica sp. Glucosinolates 10.15. Kava kava e Piper methysticum 10.16. Milk Thistle e Silybum marianum

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10.17. Mint Mentha sp. [Contains Pulegone] 10.18. Rattlepods, Yellow Burrweed, and GroundseldCrotalaria, Amsinckia, and Senecio Containing [Contains Riddelliine a Pyrrolizidine Alkaloid] 10.19. Saw Palmetto e Serenoa repens 10.20. Senna e Senna alexandrina 10.21. St. John’s Wort e Hypericum perforatum 10.22. Tobacco e Nicotiana tabacum

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1. INTRODUCTION Since prehistoric times, humans have used natural products, such as plants, animals, microorganisms, and marine organisms, in medicines to alleviate and treat diseases. These remedies are often prescribed as part of traditional medicine, a discipline that has been built over the ages based on empirical knowledge, which was passed down through the generations (Hopkins et al., 2012). Plants have been particularly important to traditional medicine and still are (Petrovska, 2012). A herbal remedy contains one or more herbs that can contain leaves, stems, flowers, roots, and seeds. As an herbal remedy, these plants can either be sold raw or as extracts, where the plant is macerated with water, alcohol, or other solvents to extract some of the chemicals including the active ingredients. Because herbs contain multiple ingredients, it is difficult to determine the exact concentration of each active ingredient (if known) in combination to maximize a remedy. Plants release secondary metabolites (Table 4.1) as a defense strategy and to adapt to their environment. Under normal conditions, primary metabolism maximizes growth and development of the organism. However, given a biotic or abiotic stress, such as temperature, light intensity, herbivory and microbial attack, plants generate this chemical defense, which can be exploited as herbal remedies. These defence chemicals may also act as poisons (Table 4.1). Plants are not the only biota to use this strategy. Secondary metabolites of fungi have helped us fight infection, e.g., Penicillium chrysogenum expels

10.23. Turmeric Oleoresin e Curcuma longa

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11. International Regulatory Overview 11.1. Select List of Countries and Their Regulatory Requirements

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penicillin into the local environment. Not so with other fungal toxins (mycotoxins), which can be very toxic, carcinogenic, and hazardous to one’s health, e.g., Aspergillus flavus producing aflatoxins that contaminate peanuts and corn (see Mycotoxins, Vol 3, Chap 6). Similarly, plants have maintained their defenses, e.g., Cannabis produces the antifungal THC, others produce alkaloids, tannins, flavonoids, and other molecules such as digitalis from the foxglove, Digitalis purpurea (see Phycotoxins, Vol 3, Chap 5, and Poisonous Plants, Vol 3, Chap 7). The scope of this chapter is limited to herbal remedies. Other supplements will not be part of the discussion except to highlight specific points regarding herbal remedies. In this chapter we explore the use of herbal remedies in medical treatment regimens, differentiate traditional from scientific knowledge and discovery, the issue of quality in herbal remedies, and the difficulties in obtaining precise dose–response data for safety and efficacy.

1.1. Nomenclature Herbal remedies list the Latin names of the plants they contain or may use a common name of which there can be more than one. For this reason, it is best to use the Latin name for nomenclature purposes. Similar issues exist in the field of Toxicologic Pathology – (see Nomenclature and Diagnostic Resources in Anatomic Toxicologic Pathology Vol 1, Chap 25). The following table gives the Latin name of some select herbs and common names to which they are often referred (Table 4.2).

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TABLE 4.1

Biologically Active Secondary Metabolites in Select Herbal Remedies

Class Biologically Active Compounds

Species (Examples)

Indication

Action

Toxicity

ALKALOIDS (>27,000 IN HERBS)

Hyoscyamine, atropine, scopolamine

Datura stramonium (Jimson weed, thorn apple), Atropa belladonna (deadly nightshade), Hyoscyamus niger (henbane)

Mydriasis, preoperative antisecretory, nausea

Anticholinergic, mydriatic spasmolytic, antisecretory, antiemetic

Anticholinergic effects

Pyrrolizidine (only PAs with unsaturated necine bases are hepatotoxic)

Crotalaria, Amsinckia, Symphytum officinale (comfrey), and Senecio

Tea (comfrey)

Wellness

Hepatic (venoocclusive) and pulmonary toxicity e highly toxica

Pyridine

Nicotiana tabacum

Smoking

Nicotinic agonist

Addictive, lung cancer, COPD

Methylxanthines (caffeine)

Camellia sinensis (tea) Stimulant folia, Coffea arabica (coffee) semen, Cola nitida (kola)

CNS stimulant and diuretic

Mildly addictive, insomnia

Methylxanthines (theobromine)

Theobroma cacao

Antioxidant

CNS stimulant

Theobromine: Nausea, vomiting, diarrhea, and increased urination, cardiac arrhythmias, epileptic seizures, internal bleeding

Isoquinoline

Hydrastis canadensis

Antimicrobial, immunostimulant, gastrointestinal tonicdbitter

Berberined antioxidant

Neurotoxic, hepatotoxic, and phototoxic

Terpenes

Cannabis sativa and Cannabis indica

Pain relief, sedation

CB1 and CB2 receptor Paranoia, agonists somnolence, weight gain

Panax sp. (Ginseng)

Energy boost, analgesia (mild)

Humulened modulation of Th1/ Th2 balance, decreased mucus production, inhibition of IL-5, CCL11, and LTB4 levels and P-selectin expression, probably by inhibiting the activation of the transcription factors, NF-kB and AP-1

Hepatotoxicity due to herbedrug interactions

(Continued) II. SELECTED TOXICANT CLASSES

186 TABLE 4.1

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Biologically Active Secondary Metabolites in Select Herbal Remediesdcont’d

Class Biologically Active Compounds

Species (Examples)

Indication

Action

Toxicity

Allium sativum (Garlic)

Wound healing and GIT upset

Nerolidol, alphapinene, and terpinolenedhigh cholesterol, heart disease, and hypertension

Halitosis, digestive upsets

Zingiber officinale (Ginger)

Digestion, joint pains, nonspecific illnesses, alpha-amylase inhibitor, hypoglycemic

Zingiberened antiemetic increases gastric tone and motility, anticholinergic, and antiserotonergic actions

Wheezing and asthma aggravation

Camellia sinensis (Green tea) Ginkgo biloba

Antiinflammatory, antibiotic, ROS scavenger, neurodegenerative diseases

Quercetind antibacterial

Nephropathy at very high doses

Camellia sinensis (Green tea)

Antioxidative, antiinflammatory, antidementia, anticarcinogenic, thermogenic, probiotic, and antimicrobial

Rutin e flavonoid (vitamin P), antimicrobial, and anticancer

Bleeding with a potential for hepatotoxicity

Theobroma cacao (chocolate)

Infectious intestinal diseases and diarrhea, asthma, bronchitis (expectorant), “liver, kidney, and bladder ailments”; diabetes

Epicatechin, apigenindstimulate mitochondrial respiration and biogenesis, inhibits erk signaling (anticancer)

Related to caffeine, bleeding

Curcuma longa (Turmeric)

Antioxidant, antiinflammatory, anticancer improvement of brain function, control of obesity and diabetes

Curcumindinhibits lipoxygenase, inhibits tumor invasion and angiogenesis by irreversibly binding CD13/ aminopeptidase

Antiplatelet e bleeding

Panax ginseng (Ginseng)

Improve psychologic function, immune function, and conditions associated with diabetes

Ginsenosidesd inhibit ROS production, stimulate NO production

Interaction with warfarin, oral hypoglycemic agents, insulin, and phenelzine

PHENOLS

Polyphenols e Flavonoids

(Continued)

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TABLE 4.1 Biologically Active Secondary Metabolites in Select Herbal Remediesdcont’d Class Biologically Active Compounds

Species (Examples)

Indication

Action

Toxicity

Polyketides (anthraquinones)

Aloe barbadensis (Aloe)

Fungicidal, antiviral, antibacterial, antiinflammatory, antimicrobial, laxative, immunomodulating, and anticancer effects

Barbolin, aloeemodin, aloresin (A eE), a growth hormone, interacts with growth factor receptors on the fibroblast, increasing collagen synthesis

Diarrhea, hypokalemia, pseudomelanosis coli, kidney failure, as well as phototoxicity and hypersensitive reactions (group 2 carcinogen)

Cassia senna (Senna)

Laxative

Sennosides (rhein-9- Diarrhea and anthrone) e cathartic stomach cramps laxative

Echinacea purpurea (Echinacea)

Colds, wound healing, immunostimulant respiratory symptoms

Alkamides and caffeic acid derivatives e Immunostimulant, phagocytosis activation, fibroblast stimulation, augmentation of leukocyte mobility

OTHERS

Polysaccharides

Not toxic

a

Veno-occlusive hepatotoxicity is now termed sinusoidal obstruction syndrome (SOS). CB1 and CB2, cannabinoid receptors 1 and 2; NO, nitric oxide; PAs, pyrrolizidine alkaloids; ROS, reactive oxygen species.

TERMINOLOGY (WORLD HEALTH ORGANIZATION, 2019) Complementary Medicines (CM): The terms “complementary medicine” and “alternative medicine” refer to a broad set of healthcare practices that are not part of that country’s own traditional or conventional medicine and are not fully integrated into the dominant healthcare system. They are used interchangeably with traditional medicines in some countries. Conventional pharmaceuticals: Conventional pharmaceuticals are defined as medicinal drugs used in conventional systems of medicine with the intention to treat or prevent disease, or to restore, correct or modify physiological function. Herbal medicines (remedies): Herbal remedies include herbs, herbal materials, herbal preparations and finished herbal products that contain, as active ingredients, parts of plants, other plant materials, or combinations thereof. In some countries, herbal remedies may contain, by tradition, natural organic or inorganic active ingredients that are not of plant origin (e.g., animal, and mineral materials).

Indigenous traditional medicine: Indigenous traditional medicine is defined as the sum of knowledge and practices, whether explicable or not, used in diagnosing, preventing, or eliminating physical, mental, and social diseases. This knowledge or practice may rely exclusively on experience and observation handed down orally, or in writing from generation to generation. These practices are native to the country in which they are practiced. Most of the indigenous traditional medicine has been practiced at the primary healthcare level. Traditional medicine (TM): Traditional medicine has a long history. It is the sum of the knowledge, skills and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement, or treatment of physical and mental illness. Traditional and complementary medicine (TCM): TCM merges the terms TM and CM to encompass products, practices, and practitioners.

188 TABLE 4.2

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Nomenclature for Select Herbal Remedies

Herb e Latin Name

Common Name

Alternative Names

Allium sativum

Garlic

Ail, wild garlic, wild onion

Aloe barbadensis

Aloe vera

First aid plant, Barbados aloe, Curac¸ao aloe, Elephant’s gall, Hsiang dan, Lu hui

Brassica sp.

Broccoli

Depending on the species: cabbage, kale, broccoli, cauliflower, turnip, and mustard

Camellia sinensis

Green tea

White tea, yellow tea, green tea, oolong, dark tea (which includes pu-erh tea), and black tea

Cannabis sativa and indica

Marijuana

Dope, pot, weed, Mary Jane, mojo

Chamomilla recutita

Chamomile

German chamomile, Hungarian chamomile (kamilla), wild chamomile, blue chamomile, or scented mayweed

Coffea arabica

Coffee

Caffeine, cappuccino, espresso, brew, java, mud, perk, and cafe´

Crotalaria juncea

Crotalaria [contains riddelliine, a pyrrolizidine alkaloid]

Devil-bean, rattleweed, shack shack, and wedge-leaf rattlepod

Curcuma longa

Turmeric

Huang jiang, yellow ginger, curcumin, curcuma, curcuma aromatica

Echinacea purpurea

Echinacea

Black Sampson, coneflower, Missouri snakeroot, purple coneflower, Rudbeckia

Ephedra sinica

Ephedra

Joint fir, Ma-huang

Ginkgo biloba

Ginkgo

Eun-haeng, fossil tree, ginkyo, icho, ityo, Japanese silver apricot, kew tree, maidenhair tree, salisburia, and silver apricot

Hydrastis canadensis

Goldenseal

Eyeroot, ground raspberry, Indian dye, Indian turmeric, orange root, yellow Indian paint, yellow puccoon, yellow root

Hypericum perforatum

St. John’s wort

Klammath weed, tipton weed, and goatweed

Mentha piperita, Mentha pulegium

Mint containing pulegone

Pennyroyal, peppermint, mint, mint candy

Nicotiana tabacum

Tobacco

Weed, nicotine, smoke, cancer-stick, chew, smoking, fragrant weed, cigar, cigarette, crop, and leaf

Panax quinquefolium

Ginseng (American)

Nin-sin, Divine herb, Japanese ginseng, Panax schinseng, ren shen, Shen tsao

Piper methysticum

Kava kava

Kawa, waka, lewena, yaqona, grog, sakau, ‘awa, ‘ava, and wati

Senna alexandrina

Senna

Cassia senna (Continued)

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TABLE 4.2

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Nomenclature for Select Herbal Remediesdcont’d

Herb e Latin Name

Common Name

Alternative Names

Serenoa repens

Saw palmetto

Scrub palmetto, American dwarf palm tree, baies du palmier scie, cabbage palm, chou palmiste, ju-zhong, palma enana americana, palmier nain, palmier scie, sabal, serenoa

Silybum marianum

Milk thistle

Lady’s thistle, sow thistle, blessed thistle, holy thistle, sweet sultan

Theobroma cacao

Chocolate

Brunet, nut-brown, cocoa, deep brown, hotchocolate, umber, burnt-umber, drinking chocolate, butterscotch, and nougat

Zingiber officinale

Ginger

Gingerroot, powdered ginger, peppiness, pep

2. APOTHECARY TO PHARMACY An Apothecary sells the ingredients for and prepares herbal remedies, custom medicinal mixtures, and other health products. When Tobacco was still used in medical treatment, it was sold through an apothecary. The mainstay of an apothecary, in addition to offering medical advice and treatments to the public, is the custom mixing of herbs and drugs by a healthcare professional, now referred to as a chemist or pharmacist (Anilkumar, 2019). The role of the pharmacist, or chemist, is like that of the apothecary. Pharmacists dispense prescription medications to patients and offer expertise in the safe use of prescriptions. The advent of synthetic active pharmaceutical ingredients (APIs), e.g., Acetyl Salicylic Acid (ASA) by Bayer in Basil in the 19th century, gave birth to the pharmaceutical industry of today. The ASA synthesized by Bayer was given the name Aspirin, a name derived from the chemical ASAdAcetylspirsa¨ure in German. Spirsa¨ure (salicylic acid) was named after the meadowsweet plant, Spirea ulmaria, from which ASA could be derived). Synthetic pharmaceuticals changed the nature of apothecaries. The refinement of medical practice and the development of synthetic pharmaceutical products, led to the replacement of the classical apothecary with today’s pharmacy (Goggins, 2018). Herbal remedies were first investigated systematically for their pharmacologically active components so that synthetic analogues could be developed. Synthesis resulted in a standardized

concentration of the API, allowing prescription of a medication with known API content and without other plant-based materials. In fact, 60% of the anticancer drugs and 75% of the antiinfectious disease drugs approved from 1981 to 2002, can be traced to natural origins (Lesney, 2004). We have many examples of plant-derived extracts and compounds isolated from plants that have been widely used in the treatment of many diseases. To name just two examples, Poppy (Papaver somniferum) and Marijuana (Cannabis sativa) have been used for as long as 4000 years. The following agents were isolated from the Poppy: the alkaloid Morphine in 1806 by Friedrich Serturner, the antitussive agent Codeine by PierreJean Robiquet, and the antispasmodic alkaloid Papaverine in 1848 by George Merck Fraz. Among the many other important active principles isolated from medicinal plants are Atropine (muscarinic antagonist) from Atropa belladonna by Mein in 1831; Caffeine, obtained by Runge in 1820 from Coffea arabica; Digoxin (digitalis) by Claude-Adolphe Nativelle in 1869 from Digitalis lanata and Curare (muscle relaxant), by Winstersteiner and Dutcher in 1943 from the South American plant Chondrodendron tomentosum. The development of synthetic APIs made controlled experimentation possible, necessary for our understanding of pharmacokinetics and pharmacodynamics – (see Pharmacokinetics and Toxicokinetics and Pharmacodynamics and Toxicodynamics, Vol 1, Chaps 3–5). These potent chemicals helped elucidate the pathogenesis and mechanisms of action of several diseases and, therefore, opening

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the pathway to enable precision treatment of specific defined diseases. In addition, this enabled new molecules not found in nature to be created by chemistry, e.g., Atorvastatin - Lipitor. The use of herbal remedies is increasing. The annual prevalence of dietary supplement use increased from 14.2% in 1998–99 to 18.8% in 2002 (Kelly et al., 2005). In 2000, US$17 billion was spent in the United States on traditional herbal remedies. In 2003, the World Health Organization estimated the annual global market for herbal remedies to be worth US$60 billion and by 2012 the global industry in transitional and complementary medicine (TCM) alone was reported to be worth US$83 billion (Allkin, 2017). More recent data predict that the global herbal remedy market size was estimated to be US$83 billion in 2019 (Jahangir et al., 2020) and is expected to reach US$550 billion by 2030 at a compound annual growth rate (CAGR) 18.9% through 2030. The number of persons in developing countries using herbal remedies is impressive. In Africa, up to 90%, and in India 70%, of the population depend on traditional medicine to help meet their healthcare needs whereas in China, traditional medicine accounts for around 40% of all healthcare delivered; furthermore, greater than 90% of general hospitals in China have units for traditional medicine (WHO, 2005). In fact, public interest in alternative medicine during the COVID-19 pandemic is dramatically escalating (Rokhmah et al., 2020). The use of traditional medicine is not limited to developing countries, and during the past 2 decades public interest in natural therapies has increased greatly in industrialized countries, with expanding use of herbal remedies. In the United States, in 2007, about 38% of adults and 12% of children were using some form of traditional medicine (Barnes et al., 2008; Ernst et al., 2005). According to a survey by the National Center for Complementary and Alternative Medicine: https:// wwwnccihnihgov/ [Accessed April 4, 2021] (Barnes et al., 2008), herbal therapy or the usage of natural products other than vitamins and minerals was the most used alternative medicine (18.9%) when all use of prayer was excluded. A survey conducted in Hong Kong in 2003 reported that 40% of the subjects surveyed showed marked faith in TCM compared with Western medicine (Chan et al.,

2003a). In a survey of 21,923 adults in the United States, 12.8% took at least one herbal supplement (Harrison et al., 2004) and in another more recent survey (Qato et al., 2008), 42% of respondents used dietary or nutritional supplements, with multivitamins and minerals most used, followed by Saw palmetto, Flax, Garlic, and Ginkgo (Table 4.3). The major categories of plant-derived compounds that have medicinal properties are the Terpenoids (e.g., Taxus brevifolia (Pacific yew) – Taxol and various steroids), the glycosides (e.g., Digitalis lanata – Digitalis), Flavonoids (e.g., Ginkgo biloba), Tannins (e.g., Camellia sinensis – black tea), Polyphenols (e.g., Theobroma cacao – Cocoa powder) and the Alkaloids (e.g., Cannabis sativa – Cannabinoids and various opiates).

3. EVIDENCE FOR HERBAL REMEDY EFFICACY Evidence is defined as the “available body of facts or information indicating whether a belief or proposition is true or valid.” Evidence-based is defined as “denoting an approach to medicine, education, and other disciplines that emphasizes the practical application of the findings of the best available current research.” Experience-based knowledge gained from experience is called “empirical knowledge” or “a posteriori knowledge.” The word, “experience” generally refers to know-how rather than propositional knowledge i.e., training on-thejob. Traditional knowledge is usually passed down generation to generation via oral means, often in terms of indigenous stories. Scientific knowledge is objective, is derived from experiments, and is documented; thus alleviating the need for oral transfer (Myerscough, 1998). It has been suggested that a systems biology approach to health and healing, in the sense of predictive, preventive, personalized, and participatory medicine would provide useful data (Lemonniera et al., 2017).

3.1. Empirical Evidence – Traditional Knowledge (Botanical) Traditional, indigenous, and local knowledge generally refer to knowledge systems embedded

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TABLE 4.3

Common Herbal Remedies Used in the United Statesa,b Scientific Evidence for efficacyd

Safety

Insomnia/ gastrointestinal problems

No high-quality data

Rare allergic reactions

Echinacea (Echinacea purpurea)

Upper respiratory tract infection

Inconclusive

Side effects similar to placebo

Garlic (Allium sativum)

Hypercholesterolemia

Likely effective

Mild gastrointestinal side effects and garlic odor; case reports of bleeding

Ginger (Zingiber officinale)

Nausea

Inconclusive

No known side effects

Ginkgo (Ginkgo biloba)

Dementia

Likely effective

Side effects similar to placebo; 16 case reports of bleeding

Claudication

Likely effective

Potential bleeding

Ginseng (Panax quinquefolium)

Physical and cognitive performance

Inconclusive

Limited data; hyperactivity and restlessness in case reports

Kava kava (Piper methysticum)

Anxiety

Likely effective

Case reports of severe hepatotoxicity

Mint (Nepeta cataria, Mentha piperita) contains pulegone

Upset stomach/ irritable bowel syndrome

Inconclusive

Limited data, but side effects appear to be mild

St. John’s wort (Hypericum perforatum)

Depression

Likely effective for mildemoderate depression

Numerous reports of drug interactions

Herb

Common usec

Chamomile (Chamaemelum nobile)

a

Brent RL: Environmental and genetic causes of human congenital malformations: the physician’s role in dealing with these complex clinical problems caused by environmental and genetic factors. In Studd J, Tan SL, Chervenak FA, editors: Progress in obstetrics and gynecology, (vol. 18), Edinburgh, England, Elsevier Ltd., 2008, pp 61–84. Chapter 5. b Percentages are based on estimates from a 2002 National Health Interview Study, age adjusted to the year 2000 US. Standard Population. c Common use was determined from herbal medicine textbooks. d Scientific evidence is based on conclusions from recently published systematic reviews.

in the cultural traditions of regional, indigenous, or local communities, usually based on accumulation of empirical observation and on interaction with the environment. In many cases, traditional knowledge has been passed on for generations from person to person as an oral lesson or igneous stories. Since the dawn of civilization humans have learned to use plants and plant-derived products as remedies for various ailments, perhaps by taking cues from animals or through trial and error, leading to the discovery of various homemade remedies (Hopkins et al. (2012).

Exploration of botanical APIs continues using the science-based approach used in drug development: combining advanced methods of drug discovery (see Overview of the Role of Pathology in Product Discovery and Development, Vol 2, Chap 2), such as combinatorial chemistry, highthroughput screening, and genomics, with conventional approaches using natural products and traditional knowledge (Newman et al., 2003). Thus, traditional knowledge and western science can benefit from each other in our understanding of herbal remedies (Ji et al., 2009).

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Conventional medicine generally demands evidence in the form of randomized controlled trials (RCTs) before accepting the value of a particular herbal remedy. Yet many RCTs have inadequacies (Veal, 2004). To address these inadequacies, the Cochran library (ISSN 1465– 1858) of Systematic Reviews with MetaAnalysis was created. The Cochrane Library, https://www.cochranelibrary.com, contains a collection of databases that contain different types of high-quality, independent evidence to inform healthcare decision-making.

3.2. Experimental Evidence – Controlled (Single Active Compound) Experimental evidence is defined as evidence produced using three fundamental research strategies, which differ in content and context: laboratory experiments, experimental simulations, and field experiments. It is an operation or procedure carried out under controlled conditions to discover an unknown effect or law, to test or establish a hypothesis, or to illustrate a known law. The basis of prospective scientific evidence uses the scientific method: hypothesis, experimental design, control, test methods and materials and then running the experiment. The results may indicate that the hypothesis cannot be disproved, hence true, or may give results that require modification of the hypothesis for an additional experiment. There are many aspects of experimental designs and statistical approaches about a priori experimentation that can be found in Experimental Design and Statistical Analysis for Toxicologic Pathologists, Vol 1, Chap 16. The prospective studies on known plant APIs can be found later in this chapter. The gold standard in determining the safety and efficacy comes from the evaluation of clinical trials, particularly double-blind cross over completely randomized trials. Due to the weak signal regarding effects of herbal remedies and missing data, the Cochrane Collaboration is the best source for science-based evidence as to determining the evidence for the question at hand – the systematic review with or without meta-analysis (see Risk Assessment, Vol 2, Chap 16, Chandler and Hopewell, 2013).

Critically evaluation of published articles and synthesis should be termed a literature review. On the other hand, a systematic review, using a strict protocol, summarizes the results of available carefully designed controlled clinical trials and provides a high level of evidence on the effectiveness of healthcare interventions. In addition, meta-analysis of the combined trials from the systematic review provides pooling of results from the separate studies to give an overall measure of effectiveness (benefits and harms) (Zhang et al., 2015a,b). With respect to herbal remedies, the Cochrane Library https://www.cochranelibrary.com focuses on efficacy and effectiveness from randomized controlled trials that provide evidence for the public and regulators alike rather than addressing safety issues (Lewith et al., 2009). The National Toxicology Program (NTP) of the NIEHS provides information on the potential for toxicity from long-term use of commonly used herbal remedies using well controlled prospective studies in rodents.

4. THE ACTIVE PHARMACEUTICAL INGREDIENT(S) More than one active pharmaceutical ingredient may be present in some herbal remedies. For example, in Marijuana (Cannabis sativa) tetrahydrocannabinol (THC) is the main psychoactive component of cannabis, which is one of the 483 known compounds in the plant, including at least 65 other cannabinoids (Russo, 2016). Prospective research and development of individual APIs led to the development of the conventional single molecule pharmaceuticals: Dronabinol [Marinol] and Nabilone [Cesamet]. To address the issue of multiple APIs, research has focused on therapeutic outcomes from treatment with the plant, rather than attempting to prospectively determine the effect of every compound and their potential interactions. The n ¼ 1 experiments collect positive outcome data for any health condition following exposure to the herbal remedy. As data come in, it is synthesized as a composite record of observations for broad indications responding to an herbal remedy (Lillie et al., 2011). As the database grows, certain conditions become more

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4. THE ACTIVE PHARMACEUTICAL INGREDIENT(S)

prevalent. For example, medicinal Marijuana had little objective data on which to build a medical and treatment program. The n ¼ 1 experiments provided data regarding any cannabinoid responsive condition and those conditions with highest number of positive treatment effects becomes apparent. In other words, n ¼ 1 experiments build traditional knowledge, and this knowledge became sufficient for Canada to allow treatment using medicinal Marijuana for AIDS, multiple sclerosis, cancer, and glaucoma in 1999. Regardless of the number of APIs in Marijuana, there are several factors that interfere with control of the experiments using the whole plant. The following discussion illustrates how defining the API(s) in an herbal remedy can be problematic.

4.1. Influencing Factors on the Concentration of the API(s) in the Plant Active pharmaceutical ingredient concentrations vary because botanical specimens can be regarded as “living factories” producing a variety of chemical compounds, including primary metabolites important for the growth of the plants (amino acids, proteins, carbohydrates) and secondary metabolites (alkaloids, terpenoids, phenylpropanoids, polyketides, flavonoids, saccharides). All these components may work together to deliver a synergistic or antagonistic effect in the finished product or interact with other products and medications. Herbal remedy quality issues can be divided into intrinsic and extrinsic factors (Awang, 2014; Lam et al., 2006; WHO, 2017). Species differences, organ specificity, and diurnal and seasonal variations are examples of intrinsic factors that can affect the qualitative and quantitative accumulation of the biologically or pharmacologically active chemical constituents produced and or accumulated in the herb, such as chemically and naturally variable plants, chemo-varieties and chemo cultivars may be dissimilar. Extrinsic factors such as time of year of harvest, extraordinary weather, the source, storage conditions, and quality of the raw material, methods of harvesting, drying, transportation, and processing (for example, mode of extraction and polarity of the extracting

193

solvent, instability of constituents, etc.) also affect herbal quality (Rousseaux and Schachter, 2003). Standardization of herbal remedies prescribes a set of physical and analytical standards for both intrinsic and extrinsic parameters that give evidence and an assurance of quality, efficacy, safety, and reproducibility. However, selective analytical methods or reference compounds may not be readily available commercially. Determining contaminants in conventional drugs is relatively straight forward in comparison to herbal remedies. Conventional drugs have a known concentration of the API, and excipients, whereas the concentration of the API and other constituents of the plant are variable. In addition, what constitutes the API and what is its concentration? Herbal remedies are labeled as mg of the plant, an extract (in mg or percent), or the active ingredient in mg, e.g., capsules containing 80 mg of the herb [Ginger root 750 mg], or capsules containing 80 mg of the active [Saint John’s Wort 300 mg–0.3% hypericin], or as a straight concentration [Cannabidiol (CBD) oil 2.5%] for the purpose of dosage.

4.2. Dose and Response Without knowledge of the concentration of the API, or whether the plant has more than one API, assessing the dose-response is problematic. Often low dose effects do not follow the classical dose-response curve, an observation that has been known for many years and has only been recently revived under the title of hormesis (Calabrese, 2001; 2015a; 2015b; Calabrese and Blain, 2001). The Arndt-Schulz rule or Schulz’ “law” is a basically a hypothesis concerning the observed effects of many chemicals in low concentrations (Schultz, 1888). According to the Arndt-Schulz rule, highly diluted chemicals enhance life processes, while strong concentrations of the same chemical may inhibit and even terminate these processes (Calabrese and Baldwin, 2001). Herbal remedies show a hormetic low dose-response as do many trace elements. It is proposed that the stimulatory (i.e., low dose) and inhibitory (i.e., high dose) are components of the hormetic dose-response (Wang et al., 2018). Depending on the process affected, this interplay results in either a J-shaped or inverted

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J-shaped dose-response curve, which are sometimes called “bell-shaped,” “U-shaped,” “inverted U-shaped,” “biphasic,” or “b-curve” (Calabrese and Baldwin, 2001). The point at which the hormetic curve crosses the reference level of response (i.e., the threshold) is the zero-equivalent point; in other words, the point at which there is no toxic or stimulatory effect (Agathokleous et al., 2020). In fact, it has been proposed that the application of hormesis to the process of risk assessment for noncarcinogens and carcinogens be explored (Calabrese and Blain, 2005).

4.3. Contaminants Herbal remedies use crude or raw herbs that are collected from the wild or from cultivated fields. Following processing, the herbs become an herbal remedy, somewhat similar to the development of a conventional drug product. The herbal products may contain the dry herb, a herbal mixture or herbal nutrient-supplement mixture. Toxic contaminants may come from environments and conditions that the medicinal plants are grown in or collected from, the conditions under which they are dried and processed, the storage conditions and conditions during transport, and during the manufacturing processes of finished medicinal products, e.g., toxic metals (Locatelli et al., 2014), pesticides residues and microbes. As such, tests for acceptable levels of pesticide and herbicide contamination of herbal ingredients are needed. In addition, herbal supplements may contain ingredients made of different parts of a plant, or herbal extracts (Slifman et al., 1998), the concentration of which had decreased in the past few years (Steinhoff, 2021). Herbal preparations may be contaminated with a wide spectrum of microorganisms (Kosalec et al., 2009) that are enhanced by improper storage conditions (Martins et al., 2020). The main microbial contaminants of the herbal remedy are aerobic, mesophilic microorganisms: intestinal bacteria, yeasts, and molds. These microorganisms may be classified as primary (forming the plant’s microbiota), and secondary contaminations, i.e., such contaminations which have penetrated the product during its manufacturing. Plant-associated microbiota are complex; aerobic bacteria and fungi are normally present in plant material and may increase due to faulty growing,

harvesting, storage or processing. Herbal ingredients, particularly those with high starch content, may be prone to increased microbial growth (de Sousa Lima et al., 2020). Furthermore, pathogenic organisms including Enterobacter, Enterococcus, Clostridium, Pseudomonas, Shigella, and Streptococcus are known herbal remedy contaminants (Barnes et al., 2008). Numerous natural occurrences of mycotoxins in medicinal plants and traditional herbal remedies (Altyn and Twaruzek, 2020) have been reported from various countries including Spain, China, Germany, India, Turkey, and from Middle East; hence, not only the possibility of infection exists but intoxication with mycotoxins can have serious consequences (Ashiq et al., 2014) (see Mycotoxins, Vol 3, Chap 6). Contamination of end products may also result from their improper handling during manufacturing, packaging, and transportation (Barnes, 2003). However, contaminants can be controlled by implementing standard operating procedures (SOPs) leading to Good Agricultural Practice (GAP), Good Laboratory Practice (GLP), Good Supply Practice (GSP), and Good Manufacturing Practice (GMP) for producing these medicinal products from herbal or natural sources (Chan, 2003a).

4.4. Adulterants Perhaps the largest impediment to the integration of herbal remedy into conventional medicine is the intentional adulteration of herbal remedy products with synthetic pharmaceutical drugs (Calahan et al., 2016) (Table 4.4). Intentional adulteration of herbal remedies with pharmaceuticals to substantiate medicinal claims has resulted in several serious adverse effects, including some fatal cases (Xu et al., 2019). Multicomponent Chinese or Ayurvedic herbal remedies have long been documented to be adulterated with synthetic antiinflammatory drugs such as phenylbutazone, indomethacin, or corticoid steroids in arthritis remedies. In fact, about 24% of 2609 herbal remedy samples collected by eight major hospitals in Taiwan were found to contain one or more synthetic therapeutic agents (Huang et al., 1997). Cases of metal intoxication have been reported from their use as active ingredients or their presence as contaminants. Substituting a more toxic herb

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4. THE ACTIVE PHARMACEUTICAL INGREDIENT(S)

TABLE 4.4 The Top Adulterants Identified in Herbal Preparations Adulterants

Frequency Purpose

Abietic acid

20

Antioxidant agent, cardiovascular agent

Glibenclamide

16

To mimic the function of treating diabetes

Auramine O

16

Dye used in herbals to increase quantity

Rumex madaio

8

Substitution to increase quantity

808 scarlet

8

Dye used in herbals to increase quantity

Phenformin

7

To potentiate antidiabetes effects

Diazepam

6

To potentiate sedative effects

Sildenafil

6

To potentiate effects on sexual dysfunction

Prednisone

6

To potentiate antitussive and antiasthmatic effects

Total Ash

6

To increase weight

Orange II

6

Dye used to increase quantity

Free quercetin

6

To reflect the change of manufacturing process

Kaempferide

6

To mimic the change of manufacturing process

Carmine

5

To dye herbals to increase quantity

Foreign organic substances

5

To increase weight

Acetaminophen/ Paracetamol

4

To potentiate antiasthmatic effects

Sibutramine

4

To potentiate weight loss effects

Sunset yellow

4

Dye used to increase quantity of the therapeutic product

195

for a benign one, either by misidentification or for economic gain, can also result in adverse effects (Fraser and Wen, 1998). A study of 1234 adulterants identified approved drugs, banned drugs, drug analogues and animal thyroid tissue. The six most common categories of adulterants detected were nonsteroidal antiinflammatory drugs (17.7%), anorectics (15.3%), corticosteroids (13.8%), diuretics and laxatives (11.4%), oral antidiabetic agents (10.0%) and erectile dysfunction drugs (6.0%). Sibutramine was the most common adulterant (n ¼ 155), which is a serotonin and norepinephrine reuptake inhibitor which has been used for correcting obesity but was withdrawn from use in the United States because of the increased risk of cardiovascular events (Table 4.5). The reported sources of these illicit products included over-the-counter drug stores, the internet and Chinese medicine practitioners. A significant proportion of patients (65.1%) had adverse effects attributable to these illicit products, including 14 severe and two fatal cases. Psychosis, iatrogenic Cushing syndrome and hypoglycemia were the three most frequently encountered adverse effects (Ching et al., 2018). Ninety representative samples of herbal remedies were randomly purchased from New York City’s Chinatown in the form of pills, tablets, creams, and teas then analyzed for adulterants. Drugs identified included promethazine, chlormethiazole, chlorpheniramine, diclofenac, chlordiazepoxide, hydrochlorothiazide, triamterene, diphenhydramine, and sildenafil citrate (Viagra) in five of the samples (Miller and Stripp, 2007). Twenty-six systematic reviews concerning adulteration and contamination of herbal remedies were conducted (Posadzki et al., 2013a). Most commonly, herbal remedies were adulterated or contaminated with dust, pollens, insects, rodents, parasites, microbes, fungi, mold, toxins, pesticides, toxic heavy metals, and prescription drugs. The most severe adverse effects caused by these adulterations were agranulocytosis, meningitis, multiorgan failure, perinatal stroke, arsenic, lead or mercury poisoning, malignancies or carcinomas, hepatic encephalopathy, hepatorenal syndrome, nephrotoxicity, rhabdomyolysis, metabolic acidosis, renal or liver failure, cerebral edema, coma, intracerebral hemorrhage, and death. Adulteration and contamination of herbal remedies

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196 TABLE 4.5

4. HERBAL REMEDIES

Common Pharmaceutical Adulterants in Herbal Remedies

Adulterant

Number Samples

Number Positive

Claim

Amoxicillin

22

2

Tracheitis

Captopril

19

8

Hypertension

Dextromethorphan

22

4

Alleviation of cough and expectorant effect

Diazepam

11

3

Dysphoria and insomnia

Famotidine

47

18

Ibuprofen

14

3

Arthritis

Nifedipine

16

6

Hypertension, angina pectoris, and cardiovascular health

Promethazine

19

2

Tranquilizer effect or improving “health status” of old people

Sildenafil

81

28

Enhancing sexual performance

Digestive disorders

Ernst E: Adulteration of Chinese herbal medicines with synthetic drugs: a systematic review, J Intern Med 252:107–113, 2002; Xu M, Huang B, Gao F, Zhai C, et al.: Assessment of adulterated traditional Chinese medicines in China: 2003-2017, Front Pharmacol 10:1446, 2019.

were most noted for traditional Indian and Chinese remedies, respectively (Posadzki et al., 2013b). Adulteration of Chinese materia medica is not new, appearing in ancient China; therefore, it is somewhat understandable as to why intentional contamination of herbal remedies with prescription medicines is mainly a Chinese phenomenon – history repeats itself. Methods used to adulterate and produce fraudulent medicines were developed in the Northern-Southern Dynasties, and counterfeit medicines began to appear in the Tang Dynasty. These herbal remedies went unchecked since the Ming and Qing Dynasty (Xie and Wang, 2013).

4.5. A Comparison Between Properties of Herbal Remedies and Conventional Drugs Poisoning from medicinal plants, when reported, is usually due to misidentification of the plants in the form in which they are sold, or incorrect preparation and administration by inadequately trained personnel. Some herbal remedies have similar pharmacologic activities to those of small molecule pharmaceuticals; however, the mechanisms by which the herbs exert their pharmacodynamic activity are often unknown (Karimi et al., 2015) (Table 4.6). There

is some reassurance that most herbal remedies are less toxic, in terms of dose [mg/kg], than synthetic APIs. Indeed, Traditional Chinese medicine, Ayurveda, Kampo, traditional Korean medicine, and Unani have been practiced in some areas of the world for centuries without negative reports and patients indicate satisfaction with such treatments (Ameade et al., 2018), which may be in part due to poor accessibility to prescription pharmaceuticals.

4.6. Acceptability of Herbal Remedies Two distinct opinions and beliefs regarding the use of herbal remedies exist: those that don’t believe they work and are a waste of money, others who believe they are effective and do not contain harmful chemicals, and that the government regulates safety, efficacy, and quality. The regulatory authorities also determine whether certain herbs, such as Marijuana and raw opiates should be permitted for use. Some of the outcomes surrounding decisions of access can be politicized. A good example is hemp and Marijuana when the petrochemical industry developed synthetic fiber and the obvious competitor was hemp fiber - sales of hemp threatened DuPont’s attempt to create

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5. QUALITY, EFFICACY AND SAFETY

TABLE 4.6 A Comparison of Properties of Herbal Products and Conventional Drugs Properties

Herbal Remedy

Conventional Drug

Active ingredients

Often unknown

Known

Availability of raw material

Limited

Yes

Quality of raw material

Variable

Good

Stability of preparation

Uncertain

Good

Mechanism of action

Often unknown

Usually known

Toxicological tests

Often not available in animals

Mandatory

Empirical data

Very important

Often meaningless

Specific adverse effects

Rare (may be more than reported)

Infrequent

Tolerance of therapy

Usually, good

Limited

Suitability for chronic use

Often not well tested

Rigorous testing

Placebo controls

Difficult to achieve

Achievable

Controlled clinical trial

Usually not available

Mandatory

PHYSICOCHEMICAL PROPERTIES

BIOMEDICAL PROPERTIES

a market for synthetic fibers. To illustrate the change in acceptability the contentious herb, an abridged summary of some milestones in medicinal use of Marijuana can be seen in Table 4.7.

5. QUALITY, EFFICACY AND SAFETY In order to integrate herbal remedies into modern medical practices, including cancer treatments, the interrelated issues of quality, safety, and efficacy must be considered for the pharmaceutical, the herb and potentially harmful contaminant, intentional or not. Due to the difficulty in quantifying the efficacy and safety of an herbal remedy, the quality of the health product is one aspect that can be controlled and is essential for the consumer. If accepted as

a traditional medicine, the herbal remedy can be assumed qualitatively to be safe and effective even if this is not always the case. Although the consumer may be warned caveat emptor, it is essential that they know the ingredients present in the medication, particularly the concentration of the active ingredients. Not only do plants contain multiple pharmacologically active components, but they also usually contain fiber and nutrients (e.g., fatty acids and vitamins), which may, or may not, alter the pharmacokinetics, pharmacodynamics, or both, of the remedy (see ADME: principles in small molecule drug discovery and development – an industrial perspective and Principles of pharmacodynamics and toxicodynamics, Vol 1, Chaps 3 and 5, respectively). Plants are not sterile; hence plant diseases, opportunistic pathogens or

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198 TABLE 4.7

4. HERBAL REMEDIES

An Abridged Synopsis of the History of the Medical and Recreational Use of Marijuana

Date

Event

2900 BCE

Chinese emperor Fu Hsi references Marijuana as a popular medicine

2700 BCE

First written record of cannabis use, in the pharmacopoeia of Shen Nung

1500 BCE

The earliest written reference is found in the 15th century BC Chinese pharmacopeia, the Rh-Ya

1450 BCE

Book of Exodus references Holy anointing oil made from cannabis (Exodus (30:22e23))

1213 BCE

Egyptians use cannabis for glaucoma, inflammation, and enemas

1000 BCE

Bhang, a drink of cannabis and milk, is used in India as an anesthetic

700 BCE

Persian prophet Zoroaster gives hemp first place in the sacred text, the Zend-Avesta

600 BCE

Indian medicine treatise cites cannabis as a cure for leprosy

450 BCE

Greek historian Herodotus describes throwing hemp onto heated stones under canvas

1 CE

Ancient Chinese text recommends cannabis for more than 100 ailments

45 CE

St Mark establishes the ethiopian Coptic church. The Copts claim that Marijuana as a sacrament has a lineage descending from the Jewish sect, the essenes, who are considered to be responsible for the dead sea Scrolls

70 CE

Roman emperor Nero’s surgeon, Dioscorides, praises cannabis for its medical properties

200 CE

Chinese surgeon Hua T’o uses cannabis resin and wine as anesthetic

800 CE

Mohammed allows cannabis but forbids alcohol use

1500 CE

Muslim doctors use Marijuana to reduce sexuality

1538 CE

During the Middle ages, hemp was central to any herbalist’s medicine cabinet

1621 CE

Popular English mental health book recommends cannabis to treat depression

1632 CE

Pilgrims bring cannabis to New england

1799 CE

Napoleon’s forces bring Marijuana from Egypt to France

1812 CE

Blockade of Napoleon in the war of 1812 by Britain prevented provision of cannabis hemp from Russia for refitting sails and rigging

1839 CE

Homeopathy journal publishes first of many reports on the effects of cannabis

1841 CE

Dr. W.B. O’Shaughnessy of Scotland works in India then introduces cannabis to western medicine and for treatment of the Queen’s menstrual cramps

1845 CE

Psychologist and ‘inventor’ of modern psychopharmacology and psychotomimetic drug treatment; Jacques-Joseph Moreau de Tours documents physical and mental benefits of cannabis

1850 CE

Marijuana added to US pharmacopeia

1857 CE

Smith Brothers of Edinburgh market a highly active extract of Cannabis indica used as a basis for innumerable tinctures

1870 CE

Cannabis is listed in the US pharmacopoeia as a medicine for various ailments

1906 CE

Pure Food and Drugs Act requires labeling of medicine, including cannabis

1925 CE

League of nations sign multilateral treaty restricting cannabis use to scientific and medical only

1930 CE

American pharmaceutical firms sell extracts of Marijuana as medicines

1936 CE

Reefer Madness film cautions against Marijuana (Continued) II. SELECTED TOXICANT CLASSES

199

6. QUALITY

TABLE 4.7

An Abridged Synopsis of the History of the Medical and Recreational Use of Marijuanadcont’d

Date

Event

1942 CE

Marijuana removed from US pharmacopeia

1956 CE

Inclusion of Marijuana in Narcotics Control Act leads to stricter penalties for Marijuana possession

1961 CE

1961dUN convention provides basis for future international prohibition of Marijuana

1976 CE

Marijuana decriminalized in The Netherlands

1980 CE

Marinol, a synthetic version of THC, and smoked Marijuana tested on cancer patients

1986 CE

Anti-Drug Abuse Act increases penalties for Marijuana possession and dealing

1990 CE

Scientists discover Cannabinoid receptors

1992 CE

Scientists discover first endocannabinoid

1998 CE

UK house of Lords Committee recommends legalizing Medical Marijuana

1999 CE

Canada commences the Medical Marijuana research plan

2008 CE

Two pounds of cannabis found buried in 2700-year-old Chinese tomb

2011 CE

Study finds legal medical Marijuana reduces fatal car accidents

2018 CE

US FDA approves its first Marijuana-based drug “herbal remedy” [CBD]

2018 CE

Canada legalizes marijuana for recreational use

2020 CE

US house passes Marijuana Decriminalization Bill

mycotoxins may pose a potential health risk to individuals.” Numerous examples of plant pathogens exist that may act as opportunistic pathogens in individuals taking the herbal remedy and the plant material may act as a fomite carrying virulent pathogens, such as enteropathogenic Escherichia coli or Salmonella sp. Examples of plant pathogens that may cause disease by either infection, allergy or mycotoxin production include but are not limited to Pseudomonas sp. (Bacterial), Alternaria sp; Fusarium oxysporum; Fusarium sulphureum; Phomopsis sp.; and Botrys sp. (Fungal). In addition, without regulatory control of quality, adulterants may be included in the formulation (Slifman et al., 1998). Many regulatory authorities are addressing the needs for marketing authorization (see section on Regulatory issues) of herbal remedies to ensure that marketed products meet standards for quality and safety (Barnes, 2003). Because most complementary medicines are not licensed as medicines in many countries, evidence of quality, efficacy and safety is not required before marketing (Heinrich, 2015). There is a concerted

effort to address these regulatory problems at present.

6. QUALITY Quality issues concerning herbal remedies can be classified as external and internal factors (Zhang et al., 2012). External factors include environmental factors; field collection methods such as cultivation, harvest, postharvest transport and storage; manufacturing practices; inadvertent contamination and substitution; and intentional adulteration. Source plant materials that are contaminated with microbes, microbial toxins (see Bacterial Toxins, Vol 3, Chap 9), environmental pollutants (see Environmental Toxicologic Pathology, Vol 3, Chap 1), or heavy metals (see Heavy Metals, Vol 3, Chap 10); or finished products that are adulterated with foreign toxic plants or synthetic pharmaceutical agents can lead to adverse events (Gosh, 2018). Internal factors can be limited by the rigorous implementation of Good Agricultural and Collection

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200

4. HERBAL REMEDIES

Practices (GACP) while Good Manufacturing Practices (GMP) would undoubtedly reduce the risk of external issues (Pan et al., 2013).

6.1. The Active Pharmaceutical Ingredient Herbal remedies may have one or more APIs, all of which should be accounted for via analytical methods. Single synthetic molecular conventional drugs are well characterized regarding the API, excipients, impurities, stability, etc. In contrast, herbal remedies, be they single herbs or polyherbal products, are not uniform in the API due to growth conditions storage, etc., as previously mentioned. Complicating the issue further, Ayurvedic medicine employs complex mixtures of plant, animal, and minerals such as lead, mercury, cadmium, arsenic, and gold in certain formulations (Ernst and Thompson Coon, 2001), making the quality of the herbal remedies difficult to standardize.

6.2. Quality Control Quality of herbal remedies is affected by several factors not pertinent to synthetic small molecular APIs including mixtures of many constituents, the API(s) may not be known, analytical methods or reference compounds may not be available commercially, plant materials are chemically and naturally variable and the source and quality of the raw material are inconsistent. Potency, and hence toxicity, depends on the part of the plant useddfor example root, stem, leaves, or fruitsdthe time of the year it is picked, and the actual species of plant used. Potency refers to the concentration of drug required to evoke a response, if the potency of the formulation of the herbal remedy is unknown it is difficult to know what dose to prescribe to get the desired effect without causing problems of lack of efficacy and possible toxicity. An example of this problem is provided by Mistletoe, the popular name for species of evergreen, including the European variety Viscum album. Mistletoe is used as an antispasmodic, diuretic, and hypotensive, and some claim it has anticancer properties. But Mistletoe extract contains at least three types of potentially toxic compounds: alkaloids, viscotoxins, and lectins

(Teschke et al., 2013). The question arises as how to regulate this type of situation? Standardization and quality control of herbal remedies require a process to define the physicochemical evaluation of crude drug covering aspects, such as selection and handling of crude material, safety, efficacy and stability assessment of finished product, documentation of safety and risk based on experience, and provision of product information to consumer and product promotion. Assessment involves quality indices such as macro and microscopic examination to identify the herb and search of adulterants and foreign organic matter, ash values to determine the identity and purity of crude drug, and moisture content to determine the weight of the API. In fact, drier plant material may indicate better stability and extractable chemical constituents of herb and fiber content (Gosh 2018).

6.3. Manufacturing Processes and Controls Product quality improvement may be achieved by implementing control measures from the point of medicinal plant procurement under good agricultural practices (GAPs) and the manufacture of the finished botanical products under good manufacturing practices (GMPs), plus postmarketing quality assurance surveillance. A robust quality assurance system for the collection, harvest, storage, and primary processing of the plant material is essential to ensure consistent composition of the active compound. The traceability of herbal raw materials for use in herbal remedies is essential to avoid the risk of adulteration and to deliver consistent quality in products to the consumer (Fong, 2002). Items with required quality specifications include GAPs, GMPs, definition of plant family, subfamily, species, subspecies and variety, plant part, solvents and solubilizers, impurities, adulterants and misidentifications, and minimum batch-to-batch, product-toproduct, and variety-to-variety variability (Teschke et al., 2013).

7. EFFICACY AND EFFECTIVENESS Natural products are rarely evaluated in the well-controlled clinical trials that biopharmaceuticals are, which may be in part due

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7. EFFICACY AND EFFECTIVENESS

to the high cost of research and development for many herbal remedy producing companies (See ADME Principles in Small Molecule Drug Discovery and Development – An Industrial Perspective, Vol 1, Chap 3) and biotherapeutics (see Biotherapeutics ADME and PK/PD principles, Vol 1 Chap 4) to receive approval by regulatory bodies (see Overview of Drug Development, Vol 2, Chap 1); therefore, they tend to have less scientific evidence to support their efficacy. The gold standard for determining efficacy comes from randomized clinical trials (RCTs), to which few herbal remedies have been subjected (Boozari and Hosseinzadeh, 2021).

7.1. Traditional Knowledge of Efficacy Efficacy, as described by those with traditional knowledge uses cultural norms and practices to promote health maintenance, and diagnose and treat disease. For example, using interviews and surveys, respondents cite the medicinal plants that they have used. These data can be quantitatively analyzed using various indices such as Informant Consensus Factor (F(ic)), Fidelity Level (FL), Informant Agreement on Remedies (IAR), and Cultural Importance Index (CII). Quantitative analysis of such data has shown a consensus regarding the knowledge they impart (Mutheeswaran et al., 2011).

7.2. Experimental Evidence Four of the top 10 herbs (Ginkgo, Garlic, St. John’s wort, and Kava kava) have substantial scientific evidence suggesting efficacy for specific indications. However, even for these commonly used herbs, the scientific evidence often suffers from poor methodology, inconsistent outcome measures, selection of dose, different preparations of the herb, and conflicting results. It has been estimated that there are 20,000 herbal products. There is limited evidence to support the efficacy of even the top 10 herbs, and there is far less evidence for the remaining 19,990. This lack of evidence does not indicate a lack of benefit, but primarily indicates a lack of conclusive studies, positive or negative, for the efficacy of most

201

herbal products (Brent, 2008). Unfortunately, there is a misperception that herbal remedies are natural and, therefore, are safe.

7.3. Randomized Clinical Trials Using Herbal Remedies In their review of published systematic reviews, the Cochrane Collaboration, concluded that there is inadequate good quality clinical trial evidence to make any conclusion about the efficacy of a majority of evaluated herbal treatments, while the others indicated a suggestion of benefit, which was qualified by a caveat about the poor quality and quantity of studies (Table 4.8). Most reviews included many distinct interventions, controls, outcomes, and populations, providing a wealth of comparisons to be critiqued (Brent, 2008). Although some herbal remedies show efficacy in the systematic review and meta-analysis, the majority have not (Manheimer et al., 2009). Botanical drugs such as Panax ginseng (Ginseng) herbs used as a tonic, Tanacetum parthenium (Feverfew) used to treat migraine headache, Allium sativum (Garlic) used to lower low-density protein cholesterol and some cardiovascular disturbances, Matricaria chamomilla (Chamomile) recommended as a carminative, antiinflammatory and antispasmodic, Silybum marianum (Milk thistle) used for repairing liver function including cirrhosis, Valeriana officinalis (Valerian), used as a sedative and sleeping aid, Piper methysticum (Kava kava) used as an anxiolytic, Aesculus hippocastanum (Horse chestnut) used for the treatment of chronic venous insufficiency, Cassia acutifolia (Senna) and Rhamnus purshiana (Cascara sagrada) which are used as laxatives, Echinacea purpura (Echinacea) used as an antiinflammatory and immunostimulant, Arnica montana (Arnica) used to treat posttraumatic and postoperative conditions, and Serenoa repens (Saw palmetto) used for the treatment of benign prostatic hyperplasia are all herbal remedies with the largest worldwide foot print. Some have clinical trials to support efficacy. A summary of herbal remedies assessed for efficacy using systematic review and meta-analysis methodology can be found in Table 4.8.

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202

4. HERBAL REMEDIES

TABLE 4.8 A Summary of Select Systematic Reviews From the Cochrane Librarya Result Versus Placebo

Adverse Events

1

1¼

Allergy

Antirheumatic (low back pain)

1

1¼

None reported

Atopic dermatitis

5

2? \, 3 ¼

None reported

Rheumatoid arthritis

3

3? \

Psoriatic arthritis

1

1¼

Premenstrual syndrome

2

2¼

Menopausal flushing

1

1¼

Obesity

1

1¼

Ulcerative colitis

1

1¼

Hyperactivity

2

2¼

Raynaud’s syndrome

1

1¼

Sjo¨gren’s syndrome

1

1¼

Psoriasis

2

2¼

Feverfew (Tanacetum parthenium)

Migraine

2

2\

Rheumatoid arthritis

1

1¼

Garlic (Allium sativum)

BP. Lowering

7

7? \

17

17? \

Herbal Remedy

Traditional Use

Chamomile, German (Chamomilla recutita)

Mouthwash, oral mucositis

Devils claw (Harpagophytum procumbens) Evening Primrose (Oenothera biennis)

Cholesterol lowering

Number of RCTs

Allergy

Halitosis

Seasickness

1

1¼

Hyperemesis gravidarum

1

1¼

Post-op N/V

3

2 \, 1 ¼

Dementia progression

5

5\

Tinnitus

2

1 \, 1 ¼

American ginseng (Panax quinquefolium)

Exercise performance

1

1¼

None reported

Korean ginseng (Panax ginseng)

Exercise performance

2

2¼

None reported

Psychomotor performance

1

1¼

None reported

Flu Vaccine immun. Resp.

1

1\

None reported

Exercise performance

1

1¼

None reported

Ginger (Zingiber officinale)

Ginkgo (Ginkgo biloba)

Siberian ginseng (Eleutherococcus senticosus)

None reported

Bleeding

(Continued) II. SELECTED TOXICANT CLASSES

203

8. SAFETY

TABLE 4.8 A Summary of Select Systematic Reviews From the Cochrane Libraryadcont’d Herbal Remedy

Traditional Use

Saint John’s wort (Hypericum perforatum)

Antidepressant

Saw palmetto (Serenoa repens)

Prostatic hyperplasia

Number of RCTs

Result Versus Placebo

Adverse Events

2

2\

Photosensitization

2

2\

None reported

a

https://www.cochranelibrary.com [Accessed March 25, 2021]. ¼, same as compared to placebo; ? \, reported benefit unlikely (due to design or analytical flaw); \, benefit as compared to placebo; RCT, randomized controlled trials.

8. SAFETY 8.1. Safety, Side Effects and Toxicity The safety of using most herbs with drugs is not well established, as most of this information comes from case reports rather than systematic investigations (Ernst, 2002). Because herbs are plants, they are often perceived as “natural” and therefore safe. However, many different side effects of herbs have been reported including effects from biologically active constituents from herbs, side effects caused by contaminants, and herb–drug interactions. Although case reports of nephropathy caused using certain Chinese herbs are common (Nortier and Vanherweghem, 2002), hepatic damage is more common. Unintentional addition of another toxic plant can impact safety, e.g., pyrrolizidine alkaloids are found in many species of plants that may be growing with the herb, some which may be intentionally used (comfrey) or inadvertently added to herbal remedies (see Poisonous plants, Vol 3, Chap 7). These alkaloids produce hepatotoxicity resulting in characteristic veno-occlusive disease that may be rapidly progressive and fatal. Herbs that may harm the fetus are of particular interest. Notwithstanding the potential interactions, there are some herbs that are known teratogens that should not be taken under any circumstances during pregnancy (Rousseaux and Schachter 2003). Some herbs known to cause problems during pregnancy include, but are not limited to, Semen Crotonis (Ba Dou), Semen Pharbitidis (Qian Niu Zi), Semen Persicae (Tao Ren), Radix Euphorbiae (Da Ji), Mylabris (Ban Mao), Radix Phytolaccae (Shang Lu), Moschus (She Xiang), Rhizoma Sparganii (San Leng), Rhizoma Zedoariae (EZhu), Hirudo seu Whitmania (Shui

Zhi) and Tabanus (Meng Chong) Flos Carthami (Hong Hua), Radix and Rhizoma Rhei (Da Huang) Fructus Aurantii (Zi Shi) Radix Aconiti (Fu Zhi), Rhizoma Zingiberis (Gan Jiang), and Cortex Cin namomi (Rou Gui) (Liu et al., 2015). The potential for toxicity from certain herbs is compounded by the frequent use of misleading marketing information. Illegal and erroneous marketing claims for herbal products are common. For example, a systematic review of citrus aurantium for weight loss identified only one methodologically flawed study examining the effect of the herb, which incorrectly reported a statistically significant benefit for weight loss (the herb was no more effective than placebo). This misleading article is often cited as “published scientific evidence” of the efficacy with no mention of possible side effects – fake news. In one study of internet marketing, more than half of the marketing illegally claimed the herbal products as effective in treatment, prevention, diagnosis, or curative of specific diseases (Ekor, 2014).

8.2. Adverse Reactions Mild adverse reactions, or side effects, can occur as an herb-related effect of a type-A (drug actions) which are predictable and dose-dependent, and type-B (patient reactions), which are rare, unpredictable, and dose-independent. Type-A reactions represent about 80% of all adverse drug reactions and can be predicted based on the drug’s pharmacology. Instead, type-B reactions are independent of the drug’s pharmacology and, in most cases, occur at any dose (Ahmed et al., 2020.) Serious adverse effects were shown in four systematic reviews for Herbae pulvis standardisatus, Larrea tridentata, Piper methysticum, and

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4. HERBAL REMEDIES

Cassia senna. The most severe adverse effects were liver or kidney damage, colon perforation, carcinoma, coma, and death. Moderately severe adverse effects were noted for 15 herbal remedies containing Pelargonium sidoides, Perna canaliculus, Aloe vera, Mentha piperita, Medicago sativa, Cimicifuga racemosa, Caulophyllum thalictroides, Serenoa repens, Taraxacum officinale, Camellia sinensis, Commiphora mukul, Hoodia gordonii, Viscum album, Trifolium pratense, and/or Stevia rebaudiana. Minor adverse effects were noted for 31 herbal remedies: Thymus vulgaris, Lavandula angustifolia Miller, Boswellia serrata, Calendula officinalis, Harpagophytum procumbens, Panax ginseng, Vitex agnus-castus, Crataegus spp., Cinnamomum spp., Petasites hybridus, Agave americana, Hypericum perforatum, Echinacea spp., Silybum marianum, Capsicum spp., Genus phyllanthus, Ginkgo biloba, Valeriana officinalis, Hippocastanaceae, Melissa officinalis, Trigonella foenum-graecum, Lagerstroemia speciosa, Cnicus benedictus, Salvia hispanica, Vaccinium myrtillus, Mentha spicata, Rosmarinus officinalis, Crocus sativus, Gymnema sylvestre, Morinda citrifolia, and/or Curcuma longa. Most of the HMs evaluated in SRs were associated with only moderately severe or minor adverse effects (de L Moreira et al., 2014; Izzo et al., 2016).

8.3. Interactions Herb–Drug Interaction Reports on herb–drug interactions are mainly from inadequately described case reports or based on in vitro studies. When herbs are administered in combination with prescription and over the counter drugs, and an adverse drug reaction occurs, the reporting of herb-drug interactions is rare and often overlooked (Posadzki et al., 2013c). Since herbal remedies are often used in conjunction with conventional drugs, kinetic and clinical interactions are a cause for concern. A summary of class of drug and their interaction with herbal remedies is portrayed in Table 4.9. Both pharmacokinetic and pharmacodynamic actions are probably involved in herb–drug interactions, although metabolic induction or inhibition is a common underlying mechanism for many herb–drug interactions (de L. Moreira et al., 2014). Drugs that have a high potential

to interact with herbal remedies usually have a narrow therapeutic index, including warfarin, digoxin, cyclosporine, tacrolimus, amitriptyline, midazolam, indinavir, and irinotecan (Table 4.10). Many of them are substrates of cytochrome P450s (CYPs) or P-glycoprotein (P-gp). Herbal remedies that are reported to interact with drugs include Garlic (Allium sativum), Ginger (Zingiber officinale), Ginkgo (Ginkgo biloba), Ginseng (Panax ginseng), and St. John’s wort (Hypericum perforatum). Similarly, case reports have suggested that Ginkgo may potentiate bleeding when combined with warfarin or aspirin, increases blood pressure when combined with thiazide diuretics, and has even led to a coma when combined with trazodone, a serotonin antagonist and reuptake inhibitor used to treat depression. Furthermore, Ginseng reduced the blood levels of warfarin and alcohol as well as induced mania if taken concomitantly with phenelzine, a nonselective and irreversible monoamine oxidase inhibitor used as an antidepressant and anxiolytic agent. Lastly, multiple herb-drug interactions have been identified with St. John’s wort that result in a significantly reduced area under the curve (AUC) and blood concentrations of warfarin, digoxin, indinavir, theophylline, cyclosporine, tacrolimus, amitriptyline, midazolam, and phenprocoumon. The clinical consequence of herb-drug interactions varies, from being well-tolerated to moderate or serious adverse reactions, or possibly life-threatening events (Chen et al., 2011; Parvez et al., 2019). It has been suggested that a demonstration of the safety of herbal remedies for registration purposes should include at least in vitro and in vivo genotoxicity assays, long-term rodent carcinogenicity tests (for drugs intended to be continuously used for >3 months or intermittently for >6 months), reproductive and developmental toxicity studies (for drugs used by women of childbearing age), and investigation of the effects on drug-metabolizing enzymes (de L. Moreira et al., 2014).

8.4. Herb-Herb Interaction Herb–herb interactions are documented in ancient textbooks on Traditional Chinese Medicine medicinal formulae (i.e., a mixture of herbs) and practitioners prescribe herbal formulae

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8. SAFETY

TABLE 4.9 Herb–Drug Interactions for Each Class of Medicationa Type of Interaction Class of Medication

Herb

Synergism

Alzheimer’s agents

Saffron (Crocus sativus)

x

Anesthetics

Ginkgo (Ginkgo biloba)

x

Analgesics

Ginkgo (Ginkgo biloba)

x

Green tea (Camellia sinensis)

x

Antiarrhythmias

Ginseng (Panax ginseng)

x

Antibiotics

Rosemary (Rosmarinus officinalis)

x

Saw palmetto (Serenoa repens)

x

Anticoagulants and antiplatelet agents

Antihypertensives

Antiinflammatory agents

x

St John’s wort (Hypericum perforatum)

x

Ginkgo (Ginkgo biloba)

x

Ginseng (Panax ginseng)

x

Guggul (Commifora mukul)

x

Lavender (Lavandula angustifolia Miller)

x

Noni (Morinda citrifolia)

Antidiabetics

Antagonism

x

Reishi mushroom (Ganoderma lucidum)

x

Saw palmetto (Serenoa repens)

x

Senna (Cassia senna)

x

Turmeric (Curcuma longa)

x

Aloe (Aloe vera)

x

Cinnamon (Cinnamomum spp.)

x

Fenugreek (Trigonella foenum-graecum)

x

Ginseng (Panax ginseng)

x

Gymnema (Gymnema sylvestre)

x

Turmeric (Curcuma longa)

x

Black cohosh (Cimicifuga racemosa)

x

Chia (Salvia hispanica)

x

Mistletoe (Viscum album)

x

Saffron (Crocus sativus)

x

Stevia (Stevia rebaudiana)

x

Aloe (Aloe vera)

x

Saw palmetto (Serenoa repens)

x

Reishi mushroom (Ganoderma lucidum)

x (Continued)

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4. HERBAL REMEDIES

TABLE 4.9 HerbeDrug Interactions for Each Class of Medicationadcont’d Type of Interaction Class of Medication

Herb

Synergism

Antilipemic

Alfalfa (Medicago sativa)

x

Fenugreek (Trigonella foenum-graecum)

x

Ginseng (Panax ginseng)

x

Green tea (Camellia sinensis)

x

Gymnema (Gymnema sylvestre)

x

Black cohosh (Cimicifuga racemosa)

x

Antineoplastics

St John’s wort (Hypericum perforatum)

x

Thyme (Thymus vulgaris)

x

Antioxidants

Chia (Salvia hispanica)

x

Antiseizures

Green tea (Camellia sinensis)

x

Lavender (Lavandula angustifolia Miller)

x

Aloe (Aloe vera)

x

Green tea (Camellia sinensis)

x

Maitake mushroom (Grifola frondosa)

x

Antivirals

St John’s wort (Hypericum perforatum)

x

Anxiolytics

Rosemary (Rosmarinus officinalis)

x

b-Adrenoceptor blockers

Green tea (Camellia sinensis)

x

Cholinergic agents

CNS depressants

Cytochrome P450e metabolized agents

Antagonism

Guggul (Commifora mukul)

x

Turmeric (Curcuma longa)

x

Butterbur (Petasites hybridus)

x

Mistletoe (Viscum album)

x

Kava (Piper methysticum)

x

Mistletoe (Viscum album)

x

Echinacea spp.

x

x

Green tea (Camellia sinensis)

x

St John’s wort (Hypericum perforatum)

x

Dopamine agonists and antagonists

Kava (Piper methysticum)

x

Diuretics

Ginseng (Panax ginseng)

x

Gastroprotective agents

Mistletoe (Viscum album)

x

Stevia (Stevia rebaudiana)

x

Belladonna (Herbae pulvis standardisatus)

x (Continued)

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TABLE 4.9 HerbeDrug Interactions for Each Class of Medicationadcont’d Type of Interaction Class of Medication

Herb

Synergism

Hepatotoxic agents

Echinacea spp.

x

Green tea (Camellia sinensis)

x

Noni (Morinda citrifolia)

x

Umckaloabo (Pelargonium sidoides)

x

Chasteberry (Vitex agnus-castus)

x

Ginseng (Panax ginseng)

x

Hormonal agents

Green tea (Camellia sinensis)

Hypoglycemics

Immunomodulators

x

Red clover (Trifolium pratense)

x

Saffron (Crocus sativus)

x

Saw palmetto (Serenoa repens)

x

Alfalfa (Medicago sativa)

x

Bitter melon (Momordica charantia)

x

Saw palmetto (Serenoa repens)

x

St John’s wort (Hypericum perforatum)

x

x

x

Laxatives

Umckaloabo (Pelargonium sidoides)

Leukotriene inhibitors

Boswellia (Boswellia serrata)

Monoamine oxidase inhibitors

Saffron (Crocus sativus)

x

Sedatives

Calendula (Calendula officinalis)

x

x x

Green tea (Camellia sinensis)

Selective serotonin reuptake inhibitors

Antagonism

x

Lavender (Lavandula angustifolia Miller)

x

Lemon balm (Melissa officinalis)

x

Saffron (Crocus sativus)

x

a

Izzo AA, Ernst E: Interactions between herbal medicines and prescribed drugs: an updated systematic review, Drugs 69(13):1777–1798, 2009. Posadzki P, Watson L, Ernst E: Herb-drug interactions: an overview of systematic reviews, Br J Clin Pharmacol 75(3):603–618, 2013c.

based on disease manifestation and characteristics of the herbs using traditional knowledge. The most well-documented herb–herb interactions were 18-incompatible herbs and 19counteracting herbs. For example, Aconitum rhizome (Wu Tou) cannot be used with Pinellia ternata rhizome (Ban Xia), and Radix aconiti (Fu Zi) is incompatible with Bulbus fritillariae (Bei Mu). It should be noted that evidence of the

adverse reactions and/or toxicity of the combined use of these herbs was mainly derived from clinical observations in ancient times. Regardless, experienced Traditional Chinese Medical practitioners may choose to use some combinations for various conditions (Amadi and Mgbahurike, 2018). Interest is shifting from the one-drug-onetarget paradigm to combination therapy or

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208 TABLE 4.10

4. HERBAL REMEDIES

Interaction of Herbal Remedies and Prescription Medicinesa

Herbal Remedy

Indication

Drug Interaction

Aloe vera (Aloe barbadensis)

Constipation and wound healing

Anticoagulants and antiplatelet drugs

Red sage (Salvia miltiorrhiza)

Coronary heart diseases, cerebrovascular disorders. Angina pectoris, antihyperlipidemic, ischemic stroke

Bleeding may be induced in combination with warfarin

Black Cohosh (Actaea racemosa)

Vaginitis, menopausal disorders (“hot flashes”), uterine spasms, and painful menstruation

Could reduce the effectiveness of such drugs as amiodarone, fexofenadine (Allegra), glyburide, and many statin medications, acetaminophen, and alcohol

Coenzyme Q10

Natural immune booster, antioxidant

Use with warfarin may cause blood clotting

Cranberry (Vaccinium sp.)

Supplement of vitamin C

Cranberry may increase effect on anticoagulants

Echinacea (Echinacea purpurea)

Improve immune system against colds

Potential interaction with antipsychotics and antidepressant medications

Ephedra (Ephedra sinica)

Asthma, bronchitis, hay fever, cold and flu, nasal congestion, cough

Combined with caffeine it may cause lifethreatening conditions

Garlic (Allium sativum)

Atherosclerosis high blood pressure (hypertension)

Plasma concentration may be increased when combined with saquinavir, hypoglycemia interactions with colchicine, digoxin, doxorubicin [Adriamycin], quinidine, rosuvastatin [Crestor], tacrolimus [Prograf], verapamil

Ginger (Zingiber officinale)

Treat colds, nausea, migraines, and high blood pressure

Ginger induces interaction with anticoagulants like warfarin, aspirin, or other anticoagulants

Ginkgo (Ginkgo biloba)

Memory problems, Alzheimer’s disease, asthma, bronchitis, fatigue, and tinnitus

Exacerbate bleeding in combination with warfarin and aspirin. Gastric irritability, spontaneous bleeding

Ginseng e American (Panax quinquefolium)

Reduce inflammation and boost immunity, brain function, and energy levels

Modestly reduce blood glucose level e insulin potential interaction with warfarin. Central nervous system stimulation, hypertension, skin eruptions

Goldenseal (Hydrastis canadensis)

Diarrhea and eye and skin irritations. It is also used as an antiseptic

Most over the counter and prescription medications

Green tea (Camellia sinensis)

Promote weight loss, blood sugar regulation, disease prevention, and exercise recovery

It increases simvastatin (Zocor) concentrations, fluoroquinolones, some beta blockers, imatinib (Gleevec), and antiretrovirals

Hawthorn (Crataegus oxycantha) flowers, roots, berries

Mild to moderate congestive heart failure

Cardiac arrythmias, lowered blood pressure

(Continued)

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8. SAFETY

TABLE 4.10

Interaction of Herbal Remedies and Prescription Medicinesadcont’d

Herbal Remedy

Indication

Drug Interaction

Kava kava (Piper methysticum)

Sedative and anxiolytic

Increased risk of drowsiness and motor reflex depression. Hepatotoxic, cytochrome P450 enzyme inhibitor

Licorice (Glycyrrhiza glabra)

Flavoring

Many interactionsdhypokalemia

Milk thistle (Silybum marianum)

Treat liver conditions and high cholesterol, and to reduce the growth of cancer cells

May decrease concentrations of medications metabolized by CYP2C9, such as warfarin, phenytoin (Dilantin), and diazepam (Valium)

Pulegone (Nepeta cataria, Mentha piperita)

Peppermint oil and mint oil are mainly related to common cold and gastrointestinal disturbances

Drugs that induce CYP450 enzymes (e.g., phenobarbitone) will increase pulegone’s hepatotoxicity

Saw palmetto (Serenoa repens)

Urine symptoms from benign prostatic hypertrophy (BPH)

None

Senna (Senna alexandrina)

Laxative

Diureticsdhypokalemia

St. John’s wort (Hypericum perforatum)

Hemorrhoids, stomach upset anxiety, insomnia, mild to moderate depression, fluid retention, aid wound healing and alleviate insomnia, depression, and various kidney and lung diseases

Can reduce effectiveness of cyclosporine (Sandimmune), tacrolimus (Prograf), warfarin (Coumadin), protease inhibitors, irinotecan (Camptosar), theophylline, digoxin, venlafaxine, and oral contraceptives; avoid combining with over the counter and prescription medications

Turmeric (Curcuma longa)

Chronic inflammation, pain, metabolic syndrome, and anxiety

May cause decreased levels of many antidepressant and antipsychotic medications

a

Parvez MK, Rishi V: Herb-drug interactions and hepatotoxicity, Curr Drug Metabol 20(4):275–282, 2019.

polypharmacy to achieve therapeutic benefits. There is momentum to explore new knowledge by tapping the past empirical experiences of herb-herb combinations (Chen et al., 2011). Traditional knowledge will be essential in addressing the problem.

8.5. Direct Toxicity Herbal remedies can be toxic when taken in excessive amounts. Many herbs contain pharmacologically active compounds, which can augment the pharmacodynamics of prescription medications used to treat an illness, to the level of toxicity. For example, Ephedra, which contains ephedrine, is more likely to lead to an adverse drug reaction probably because of its additive effect with other stimulants, such as caffeine. A systematic review

found that Ephedra led to a 2- to 3-fold increased risk of nausea, vomiting, psychiatric symptoms, and palpitations when compared with placebo (Shekelle et al., 2003) indicating that it has potent effect and should be of concern when used in energy drinks. Unfortunately, the true frequency of toxic effects for most herbs is not known because surveillance systems are much less extensive than those in place for pharmaceutical products. The following table (Table 4.11) highlights a few of the herbal remedies that have been associated with direct toxicity.

8.6. Indirect Toxicity Indirect toxicity includes all other factors that can contribute to toxicity, e.g., lack of quality control of the herbal remedy may lead to the

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210 TABLE 4.11

4. HERBAL REMEDIES

Toxicity of Selected Herbal Remedies (Severe)a

Herbal Remedy

Use

Toxicity

Aloe vera (Aloe barbadensis)

Mainly used as laxative, treating superficial wounds

Hypokalemia, hypoglycemia, diarrhea, nephrotoxicity, hematuria, muscle weakness, weight loss, and cardiotoxicity (long-term use at high doses) and hepatotoxicity (rare)

Anthranoid laxatives (Senna sp.)

Laxative

Associated with colorectal cancer in epidemiological studies. Nausea, emesis, diarrhea, muscle spasm and weakness, extrasystole, dizziness, hypouresis, mental and mood changes (such as confusion)

Comfrey (Symphytum tuberosum)

It is used for treatments of wounds. Comfrey herbal tea is for wellness

Pyrrolizidine alkaloids are hepatotoxic and hepatocarcinogenic

Ephedra (Ephedra sinica)

Used for obesity treatment and for mood elevation

Anxiety, hypertension, hepatotoxicity, and cardiotoxicity. Long-term uses of Ephedra may cause hepatotoxicity and cardiotoxicity

Ginkgo (Ginkgo biloba)

Mainly used for improvement of mental alertness and memory power

Long-term use of hemorrhage and reduction platelet activating factor

Ginseng (Panax quinquefolium)

Used for improvement of general immunity, an antihypertensive agent

Long-term use may cause coagulopathy

Goldenseal (Hydrastis canadensis)

Applied to the skin for rashes, ulcers, wound infections, itching, eczema, acne, dandruff, ringworm, herpes blisters, and cold sores. It is used as a mouthwash for sore gums and mouth

Cardiac necrosis and depression, hypotension, nausea and vomiting, nervousness

Green tea (Camellia sinensis)

Improves blood flow and lowers cholesterol. Prevents a range of heartrelated issues, from high blood pressure to congestive heart failure

Hepatotoxicity and potential carcinogenicity

Kava kava (Piper methysticum)

Anxiety disorders, insomnia, benzodiazepine withdrawal, common cold, upper respiratory tract infections, depression, epilepsy, headaches, migraines, insomnia, myalgia, psychosis, and stress

Hepatotoxicity, urticaria, dizziness, drowsiness, mydriasis, and headache

Pulegone (Nepeta cataria, Mentha piperita)

Used in flavoring agents, in perfumery, and in aromatherapy; Memory enhancer, antianxiety, and psychostimulant properties

Hepatotoxicity, pulmonary edema, and hemorrhage

St John’s wort (Hypericum perforatum)

Antidepressant. Mild and moderate depression, and sometimes seasonal affective disorder (SAD), mild anxiety and sleep problems

Photosensitivity, dry mouth, excitation, “serotonin syndrome” dizziness, and confusion

a

Liu R, Li X, Huang N, Fan M, et al.: Toxicity of traditional Chinese medicine herbal and mineral products, Adv Pharmacol 87:301–346, 2020. II. SELECTED TOXICANT CLASSES

9. TOXICOLOGY OF HERBAL REMEDIES

supply of poor quality, poorly processed, incorrect or substitute herbs. Use of the wrong herb can lead to unexpected side effects. For example, in Hong Kong Tupistra chinensis (root) was supplied instead of Panax notoginseng (root) and Datura metel (flower) instead of Campsis grandiflora (flower) resulting in cardiac toxicity and atropine poisoning, respectively (Wong et al., 2005). Unexpected toxicity may result from changing formulations; even safe herbs may cause adverse effects if used inappropriately. Indirect toxicity can occur when plants produce a host of secondary metabolites (see Poisonous Plants, Vol 3, Chap 7), or house fungi that produce secondary metabolites (see Mycotoxins, Vol 3, Chap 6) or bacteria (see Bacterial Toxins, Vol 3, Chap 8) that can be toxic to eukaryotic cells with subsequent systemic toxicity (Shitan, 2016).

8.7. Hypersensitivity – Idiopathic Allergic Reactions Like conventional drugs, herbal preparations can result in hypersensitivity reactions, which can range from dermatitis to anaphylactic shock. For example, Chamomile can cause allergic reactions (Pokladnikova et al., 2016). The ethnobotanicity index shows that 7.24% of the local flora can cause adverse reactions. Six plants (Murraya koenigii L., Datura stramonium L., Atropa belladonna L., Rubus alceifolius Poir., Piper betle L., Phoenix dactylifera L.) have the highest index of severity of adverse reactions (ISARs) In addition, eight possible herb-herb and herb-drug interactions can occur concurrently (Mahomoodally et al., 2018; Urumarudappa et al., 2019).

9. TOXICOLOGY OF HERBAL REMEDIES The adage, “The dose makes the poison,” is perhaps the most famous quote in the history of toxicology. It was coined by the Swiss physician, natural philosopher, and radical church reformer Theophrastus Bombast of Hohenheim, called Paracelsus (1493/94–1541). In the case of conventional drugs developed from plant, the active ingredient is isolated from the plant, chemically standardized, subjected to

211

critical clinical assessment and then often replaced with a synthetic analogue. In contrast, the herbalist uses mixtures of diverse herbal ingredients of varying potency (Efferth and Kaina, 2011).

9.1. Lethality The intraperitoneal LD50 values for some herbal remedies are within the range of 178 mg/kg to greater than 5000 mg/kg body weight and the oral LD50 values are greater than 5000 mg/kg body weight (Table 4.12).

9.2. Genotoxicity and Carcinogenesis Many herbal remedies are genotoxic (Table 4.13), and some are carcinogenic (Tewari et al., 2019), e.g., anthranoid laxatives such as Aloe, Cascara, Rhubarb and Senna have genotoxic potential and have been associated with colorectal cancer in epidemiological studies in humans (Ning et al., 2018). The Ames assay, using Escherichia coli (0157:H7) has shown positivity for Morinda lucida [Oruwo (Root)], Azadirachta indica [Dongoyaro (Leaf)], Terapleura tetraptera [Aridan (Fruit)], Xylopia aethiopica [Erunje (Fruit)], Newbouldia laevis [Akoko (Leaf)], Alstonia boonei [Ahun (Bark)], and Enantia chlorantha [Awopa (Bark)] and not Rauvolfia vomitoria [Asofeyeje (Root)] and Plumbago zeylanica [Inabiri (Root)] (Akintonwa et al., 2009). Details of genetic toxicology for each herbal remedy can be found under the section “Toxicology and toxicologic pathology of select herbal remedies.” In the investigation of genotoxicity, mutagenicity and carcinogenicity associated with herbal remedies, the National Toxicology Program used individual herbs or herb extracts as the test substance (Table 4.14). In these studies, the individual herb or herbal product was administered to F344/N rats and B6C3F1 mice by oral administration for up to 2 years. The spectrum of carcinogenic responses ranged from no or equivocal evidence for carcinogenic activity (Ginseng, Milk thistle, and Turmeric oleoresin) to a liver tumor response (Ginkgo, Goldenseal, Kava kava), thyroid tumor response (Ginkgo), or an intestinal tumor response (Aloe vera whole leaf nondecolorized extract). Different mechanisms may be involved in the occurrence of liver (Ginkgo, Goldenseal, and

II. SELECTED TOXICANT CLASSES

212 TABLE 4.12

4. HERBAL REMEDIES

Acute Lethality for Select Herbal Remediesa

Plant Species

Common Name

Parts Tested

LD50 (g/kg)

Alchemilla vulgaris

Common lady’s mantle

Leaves

17.3

Atriplex halimus

Mediterranean saltbush, sea orache, shrubby orache, silvery orache

Leaves

21.5

Cichorium pumilum

Dwarf chicory

Leaves

23.6

Crataegus azarolus

Azarole, azerole, and Mediterranean medlar

Leaves

23.4

Eruca sativa

Arugula, garden rocket, rocket salad, roka, roquette, rucola or rugula

Leaves

21.6

Eryngium creticum

Eryngo and sea holly

Leaves

20.7

Ferula hermonis

Zallouh and Lebanese viagra

Roots

8.8

Hypericum triquetrifolium

Saint John’s wort

Leaves

14.7

Inula viscosa

False yellowhead, woody fleabane, sticky fleabane, and yellow fleabane

Leaves

11.9

Juglans regia

English walnut or Persian walnut

Leaves

16.9

Mentha longofolia

Horse mint

Leaves

14.8

Nigella sativa

Black cumin, black seed, black caraway, Roman coriander, kalonji, or fennel flower

Seeds

19.8

Olea europaea

Olive

Leaves

19.3

Portulaca oleracea

Garden purslane, little hogweed, pusley, and wild portulaca

Above ground parts

23.8

Saponaria officinalis

Bouncing bet, soapwort, saponaire officinale, savonie`re, herbe a` savon

Leaves

Silene aegyptiaca

Rose campion

Above ground parts

25.2

Uritica dioica

Stinging nettle, common nettle

Leaves

22.1

Ziziphus spina-christi

Christ’s thorn jujube

Leaves

22.2

a

5.1

Plant extracts prepared from dried plant material were used. Values are the means of 30–50 rats (Saad et al., 2006).

Kava kava) and gastrointestinal toxicity (Turmeric oleoresin and Aloe vera whole leaf nondecolorized extract), while the toxic lesion is the same (Dunnick and Nyska, 2013). The following table provides a summary of the carcinogenic response seen following exposure to select herbal remedies.

9.3. Herbal Toxicokinetics Efficacy of herbal remedies depends on the multiple constituents absorbed in the body and their pharmacokinetics. Thus, many factors will

influence the clinical practice of herbal remedies, i.e., their absorption, distribution, metabolism, and excretion (ADME). Among these factors, herb–drug interaction has been widely discussed, as these compounds may share the same drug-metabolizing enzymes and drug transporters. There are many other potential factors that can also change the ADME of herbal remedies, including herb pretreatment, herb– herb interactions, disease status, gender, age of patient, and chemical and physical modification of certain ingredients (Sun et al., 2019).

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9. TOXICOLOGY OF HERBAL REMEDIES

TABLE 4.13 Genotoxicity of Select Herbal Remedies Herbal Remedy

Genotoxicity

Model

Exposure

Aloe vera (Aloe barbadensis)

Negative

Salmonella typhimurium strains TA98 or TA100

Not available

Positive

Bacillus subtilis

Not available

Negative

Salmonella typhimurium 3 mg/plate strains TA97, TA98, TA100, TA102, TA1535, TA1537, and TA1538 (activationdgut flora enzymes)

Positive TA1537 only

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 without and with S9 mix

5 mg/plate Negative 1000 mg/plate

Negative

V79 cells

1000 mg/mL without S9 mix and up to 5000 mg/mL with S9 mix

Positive clastogenicity No clastogenicity was observed with S9 mix at concentrations up to 4750 mg/mL

Chinese hamster ovary

3000 mg/mL (30-hour harvest) and 4000 mg/mL (24-hour harvest)

Negative

Mouse micronucleus assay

1.5 mg/kg (orally)

Chamomile Doseeresponse decrease in (Chamaemelum nobile) sister chromatid exchange

Sister chromatid exchange

5 mg/kg (47.5% inhibition), 50 mg/kg (61.9% inhibition), and 500 mg/kg (93.5%)

Echinacea (Echinacea Negative purpurea) Negative

Ames test

8 to 5000 mg/plate

Chinese hamster ovary

500 mg/mL

Ephedrine

Negative

Salmonella typhimurium strains TA1535, TA1537, TA97, TA98, and TA100 without and with S9 mix

10 mg/mL

Negative

Chinese hamster ovary

3 mg/kg

Negative

Mouse micronucleus

0, 2.5, 5.0, 7.5 g/kg 2 malesþ2 females/dose oral administration

Negative

Maximal dose tested: 160 mL/ Salmonella typhimurium strains TA98 and TA100 with plate and without metabolic þ/ S9 activation

Dose-dependent increase of micronucleated cells and polychromatocytes on the bone marrow cells

Chinese hamster ovary

Garlic (Allium sativum)

Not available

(Continued)

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4. HERBAL REMEDIES

TABLE 4.13 Genotoxicity of Select Herbal Remediesdcont’d Herbal Remedy

Genotoxicity

Model

Exposure

Ginger (Zingiber officinale)

Positive mutagenic activity

Salmonella typhimurium strains TA98, TA100, and TA1535 with and without metabolic activation

Ethanol extract 25e50 mg/ plate

Positive mutagenic activity

Salmonella typhimurium strains TA98, TA100, and TA1535 with and without metabolic activation

Essential oil 5e10 mg/plate

Negative

Salmonella typhimurium strains TA100 and TA1838 with and without metabolic activation

Ethanol extract 10 and 200 mg/plate

Negative

Comet and mouse micronucleus assays

Doses up to 2000 mg/kg/day

Negative

Human cells of hepatic origin 0.0005e1.2 mg/mL (HepG2 and THLE-2)

Negative

Escherichia coli strain WP2 3 months to 1000 to 5000 mg/ uvrA/pKM101 with and kg ginseng via gavage without metabolic activation

Negative

Bacterial strains tested included S. typhimurium strains TA97, TA98, TA100, TA102, TA104, and TA1535 with and without metabolic activation

3 months to 1000 to 5000 mg/ kg ginseng via gavage

Negative

Male or female B6C3F1 micronucleus assay

3 months to 1000 to 5000 mg/ kg ginseng via gavage

Negative

S. typhimurium tester strains TA97, TA98, TA100, and TA1535 with and without metabolic activation

Up to 10 mg/plate

Negative

E. coli strain (WP2 uvrA/ pKM101) with and without metabolic activation

Up to 10 mg/plate

Negative

Mouse micronucleus test

B6C3F1 mice administered kava kava extract by gavage for 3 months

Negative

L5178Y mouse lymphoma cells

0.3 mg/mL

Negative

Mouse micronucleus test

Up to 2 g/kg/day

Ginkgo (ginkgo biloba)

Ginseng (Panax quinquefolium)

Kava kava (Piper methysticum)

(Continued)

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TABLE 4.13 Genotoxicity of Select Herbal Remediesdcont’d Herbal Remedy

Genotoxicity

Model

Milk thistle (Silybum Negative in three of the five Ames assay and E. colid Five studies marianum) studies, with and without exogenous metabolic activation. Mutagenic in S. typhimurium strain TA98 in the presence of exogenous metabolic activation enzymes e two studies

Pulegone (Nepeta cataria, Mentha piperita) [peppermint]

Exposure Bacterial mutagenicity studies using a variety of S. typhimurium tester strains and one strain. Silymarin, a major constituent of milk thistle extract, silybin, another component of milk thistle extract, was negative in a Salmonella typhimurium gene mutation assay, with and without liver S9 activation enzymes

Negative

Mouse micronucleus test

male or female B6C3F1 mice 3 months

Positive with presence of exogenous metabolic activation enzymes

Salmonella typhimurium strains TA98 and TA100

Not available

Negative

Salmonella typhimurium Concentrations of up to strains TA1537, TA1535, 800 mg/plate TA100, TA98 and TA97 with and without metabolic activation

Negative

Concentrations of up to Salmonella typhimurium 800 mg/plate strains TA1537, TA1535, TA100, TA98, and TA97 with and without metabolic activation

Negative

In vivo micronucleus test in mice

9.375e150 mg/kg

Saw palmetto (Serenoa repens)

Tests on genotoxicity, carcinogenicity, and reproductive toxicity have not been performed

Senna (Senna alexandrina)

Negative

trp þ revertants in Escherichia Not available coli

Positive: Dose-related increases in mutations

S. typhimurium strains TA97a, Mutagenicity was observed in TA98, TA100, and TA102 in TA97a and TA102 the presence of S9

Positive: Induced his þ revertants in

S. typhimurium strains TA98, with and without liver microsomes (S9), and in TA1537 in the absence of S9

Induced structural chromosomal changes

Chinese hamster ovary (CHO) Not available cells in the presence and absence of S9

Negative

Mouse micronucleus

Negative

Salmonella typhimurium TA98 None reported

Not available

2000 mg/kg

(Continued) II. SELECTED TOXICANT CLASSES

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TABLE 4.13 Genotoxicity of Select Herbal Remediesdcont’d Herbal Remedy

Genotoxicity

Model

St. John’s wort (Hypericum perforatum)

Weak positive

None reported Salmonella typhimurium strains TA100 and TA 98 with and without metabolic activation

Genotoxic e 120 mg/mL

Chinese hamster ovary (V79 cells)

30, 60, 120, and 240 mg/mL

Cytotoxic e 156.2 mg/mL

Mouse micronucleus assay

30, 60, 120, and 240 mg/mL

Turmeric (Curcuma longa)

Exposure

Moderately cytotoxic LDHRat hepatocytes leakage from rat hepatocytes. This increase was accompanied by an increase in GSH-depletion

Concentration of 5  103 M curcumin susceptibility to cytotoxicity

Negative

Sister chromatid exchange

Mice fed turmeric or (0.5%) or curcumin (0.015%)

Negative

Salmonella typhimurium strains TA1535, TA100, and TA98 with or without metabolic activation

None reported

Negative

Ames assay

Purity up to 85%

Negative

Ames assay

Not mutagenic

Chromosomal aberrations

Sister chromatid exchange

79%e85% purity

Some herbal components may be converted to toxic, or even mutagenetic and carcinogenic metabolites by cytochrome P450s (CYPs) and less frequently by Phase II conjugating enzymes. This is exemplified by aristolochic acids (AAs) in Aristolochia sp., which undergo reduction of the nitro group by hepatic CYP1A1, CYP1A2, or peroxidases in extrahepatic tissues to generate highly reactive cyclic nitrenium ions. The latter can react with macromolecules (DNA and protein), resulting in activation of H-ras oncogene and gene mutation in renal cells and finally carcinogenesis in the kidneys. Some naturally occurring flavonoids (e.g., quercetin) and alkenylbenzenes (e.g., safrole, methyleugenol, and estragole) can undergo metabolic activation by sequential 1-hydroxylation and sulfation, resulting in reactive intermediates capable of forming DNA adducts and finally genotoxicity. Additional examples are Pulegone present in essential oils from many mint species; and teucrin A, a diterpenoid found in germander (Teucrium

chamaedrys) used as an adjuvant to slimming dietary supplements but cause severe hepatotoxicity. Some herbal constituents (e.g., capsaicin from Chili peppers, glabridin from Licorice root, oleuropein in Olive oil, diallyl sulfone in Garlic, and resveratrol found in Red wine) behave as mechanism-based inhibitors of various CYP450s. This may provide an explanation for some reported herb–drug interactions where the concentration of the conventional drug is elevated, sometimes to toxic levels due to inhibition of CYPs. In addition, the inhibition of CYPs by herbal constituents may decrease the formation of toxic metabolites and thus inhibit carcinogenesis, as CYPs play an important role in procarcinogen activation (Zhou et al., 2003a; Zhou et al., 2013a).

9.4. Microbiome Herbal remedies have been successfully used in the clinical management of type 2 diabetes

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TABLE 4.14 Herbal Remedies With Carcinogenic Activitiesa Herbal Remedy

Use

Carcinogenesis

Aloe vera (Aloe barbadensis) Wound treatments and laxative

Gastrointestinal tumors in animals

Anthranoid such as aloe, cascara, rhubarb, and senna

Laxative

Colorectal carcinoma

Aristolochia (Aristolochia fanghi)

Used as the antibacterial, antiviral, antifungal, and antitumor agent

Renal carcinoma [A. clementis e Balkan nephropathy]

Chaparral (Larrea tridentata)

Used to relieve pain and inflammation, Cystic renal cell carcinoma colds, diabetes, digestive problems, gallbladder and kidney stones, and cancer

Comfrey (Symphytum officinale)

Tea

Hepatocarcinogenesis

Ginkgo (Ginkgo biloba)

Treat pulmonary disorders (asthma, cough, and enuresis), alcohol abuse, and bladder inflammation, heart and lung “dysfunctions,” and skin infections

Hepatocellular adenomas and carcinomas; thyroid follicular cell tumors

Goldenseal (Hydrastis canadensis)

Treat digestive disorders, urinary tract infection, and upper respiratory inflammation

Hepatocellular adenoma and hepatoblastoma

Kava kava (Piper methysticum)

Sleep aid

Hepatocellular carcinoma

Vinca leaves (Catharanthus roseus)

Chemotherapy

Vincristine (VCR), Vinblastine (VBL), and Vinorelbine (VRL) are antcancer agents which may induce genotoxicity

a

Dunnick JK, Nyska A: The toxicity and pathology of selected dietary herbal medicines, Toxicol Pathol 41(2):374–386, 2013; Tewari I, Shukla P, Sehgal VK: Carcinogenic herbs: a review, Int J Res Med Sci 7(2):649–655, 2019.

mellitus for centuries. However, the related underlying mechanisms are still unclear. It has been found out that the microbiota are implicated in the pathogenesis and treatment of diabetes mellitus. An interplay between gut microbiota and host occurs mainly at the gastrointestinal mucosal barrier where the host bowel movements influence the composition and abundance of gut microbiota, whereas gut microbiota in turn modulate the metabolic and immunological activities of the host. Intestinal dysbiosis, endotoxin-induced metabolic inflammation, immune response disorder, bacterial components and metabolites, and decreased production of short-chain fatty acids are considered significant pathogenic mechanisms underlying diabetes mellitus 2. Herbal remedies alter the composition of beneficial and harmful bacteria and decrease the

inflammation caused by gut microbiota. Furthermore, the metabolism of gut microbiota modulates the herbal remedy’s biotransformation (Zheng et al., 2020). So, interactions between the gut microbiota and herbal remedies occur primarily through two pathways: the gut microbiota “digests” the herbal remedies into absorbable active small molecules, which induce physiological changes, and regulation of the composition of the gut microbiota and its secretions, thereby changing gut microbiota and its secretions inducing physiological changes (An et al., 2019; Xu et al., 2017). Garlic (Allium sativum L.) contains prebiotic components, fructans, antibacterial compounds, and organosulfur compounds. The complex ingredients of Garlic seem to impart a paradoxical result on the gut microbiome. Gut microbiota characterization by high throughput 16S

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4. HERBAL REMEDIES

rRNA gene sequencing revealed that whole Garlic supplementation increased the a-diversity of the gut microbiome, especially increasing the relative abundance of Lachnospiraceae and reducing the relative abundance of Prevotella. Thus, whole Garlic supplementation could meliorate the high fat diet-induced dyslipidemia and disturbance of gut microbiome (Chen et al., 2019).

Muhammad et al., 2019; Stedman, 2002; Tesche et al., 2014, 2016). It has been proposed that in certain situations herbal products may be just as harmful to the liver as conventional drugs (Liu et al., 2016; Stedman 2002; Zhang et al., 2016), as hepatic injury from herbal remedies is like that from conventional medicines (Roytman et al., 2018). Herbal remedies may be important in polypharmacy induced hepatotoxicity (Jing and Teschke, 2018; Liu et al., 2019).

9.5. Herbal Pharmacodynamics

Renal Toxicity Some of the nephrotoxic components from herbs are aristolochic acids, alkaloids, anthraquinones, flavonoids, and glycosides (Table 4.16). Renal manifestations of nephrotoxicity associated with an herbal remedy include acute tubular necrosis, chronic interstitial nephritis and fibrosis, chronic progressive nephropathy, nephrolithiasis, rhabdomyolysis with myoglobinuric nephropathy, Fanconi syndrome, and urothelial carcinoma. The exact incidence of kidney injury due to nephrotoxic herbal remedy is not known for the reasons stated before (Brown, 2017; Yang et al., 2018). Nephrotoxicity following herbal remedy exposure may not be limited to its pharmacokinetic and pharmacodynamic properties alone. Other factors such as unintentional inclusion of toxic plant material, such as the ubiquitous pyrrolizidine alkaloids toxins, during careless preparation; and the addition of adulterants, heavy metals, and some pharmaceutical products intentionally to reduce cost or increase efficacy can result in nephrotoxicity (Xu et al., 2016). The nephrotoxic mechanism of action has been established for a few herbal remedies. Herbs such as Tripterygium wilfordii Hook [Thunder god vine] contain diterpenoid epoxide, which causes renal necrosis. Averrhoa carambola L. [Star fruit] contains oxalates that crystalize in the kidney causing acute tubular necrosis. Guaiacum officinale L. [Rough bark] and Arctostaphylos uva-ursi [Cranberry] increase renal calculi formation. Aristolochia fangchi causes chronic interstitial nephritis, renal interstitial fibrosis, Fanconi syndrome, and urothelial carcinoma. Callilepis laureola DC [Impila] inhibits mitochondrial ATP synthesis. Uncaria tomentosa wild DC [Peruvian’s cat claw] causes acute allergic interstitial nephritis. Studies are being conducted on Salix alba L. [Willow bark] analgesic nephropathy

The characteristic of multiple components of herbal remedies acting on different biological targets poses a dilemma for the evaluation of therapeutic efficacy of herbal remedies. Advances in metabolomics enable efficient identification of the various changes in biological systems exposed to different treatments or conditions and the use of serum pharmacochemistry of a herbal remedy has significant implications for tackling the major issue in herbal remedies developmentdpharmacodynamic effects. Chinmedomics integrates metabolomics and serum pharmacochemistry of herbal remedies to investigate the pharmacodynamic activity and mechanisms of action of herbal remedies (Han et al., 2020). Indeed, ancient knowledge of herbal species and concoctions may serve as a possible treasure rather than Pandora’s box (Eigenschink et al., 2020).

9.6. Organ Toxicity Although adverse events are seldom reported, still there are many reported cases (Liu et al., 2020). The organ systems most affected include liver, kidney, heart, and finally gastrointestinal tract. Hepatotoxicity Severe hepatic injury, including acute and chronic abnormalities and even cirrhosis and liver failure, have been described after the ingestion of a wide range of herbal remedies (Navarro and Seef, 2013) (Table 4.15). The range of liver injury includes minor transaminase elevations, acute and chronic hepatitis, steatosis, cholestasis, zonal or diffuse hepatic necrosis, hepatic fibrosis, and cirrhosis (Thoolen et al., 2010), veno-occlusive disease, and acute liver failure requiring transplantation (Frenzel, 2016;

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TABLE 4.15 Reported Human Cases of Liver Toxicity Related to Herb Consumption Common Name

Scientific Name

Suggested Active Compounds

Celandine

Chelidonium majus

Chaparral

Uses

Side effects (Liver)

Isoquinoline alkaloids

Externally for skin conditions (warts, eczema); liver and gallstones

Hepatitis

Larrea divaricata

Nordihydroguaiaretic acid (NDGA)

Cancer (melanoma), bronchitis, colds, rheumatic pain, stomach pain, and chicken pox

Hepatitis, liver toxicity, and liver failure

Comfrey

Symphytum officinale Symphytum asperum

Pyrrolizidine alkaloids

Bruises, sprains, and broken bones, digestive tract problems, rheumatism, and pleuritis. Gum disease, pharyngitis, and strep throat

Veno-occlusive diseasea, liver toxicity and failure, and liver cancer

Dai-Saiko-to

Combination Combination of seven different plants: Bupleuri radix, Pinelliae tuber, Scuterlarie radix, Zipiphy fructus, Ginseng radix, Glycyrrhizae radix, and Zingeberis rhizoma

Fever, flu, bronchitis, lung infections, TB, malaria, jaundice, and hepatitis

Hepatitis

Germander

Teucrium chamaedrys

Diterpenes

Weight loss, gout, digestive aid, fever

Liver toxicity, fatal hepatitis

Groundsel

Senecio vulgaris

Pyrrolizidine alkaloids

Colic, epilepsy, and worms

Carcinogenic pyrrolizidine alkaloid toxicity

Hathisunda

Heliotropium eichwaldii

Pyrrolizidine alkaloids e heliotrine

Epilepsy, fever, vitiligo

Hepatic failure and cirrhosis

Impila

Callilepsis laureola

Atractylside

Multipurpose remedy

Hepatotoxicity

Levotetrahydropalmatine; pyrrolizidine alkaloids

Sedative, analgesic, and for indigestion

Acute hepatitis

Kava lactones (kava pyrones)

Sedative, analgesic, and psychotropic properties, nervousness, and insomnia

Hepatitis

Jin Bu Huan (JBH) Lycopodium serratum

Kava

Piper methysticum

(Continued)

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TABLE 4.15 Reported Human Cases of Liver Toxicity Related to Herb Consumptiondcont’d Suggested Active Compounds

Uses

Side effects (Liver)

Viscum album

Toxic proteinsdphoratoxins and viscotoxins

Anxiolytic, hypertension, and antispasmodic

Hepatitis

Pennyroyal

Mentha pulegium

Pulegone

Liver toxicity, death Used in hispanic cultures to treat colic, stimulate menses, and induce abortion

Senna

Cassia angustifolia

Menthofuran

Laxative

Hepatitis

Skullcap

Scutellaria lateriflora

Cytotoxic flavonoids

Nervousness, insomnia

Hepatitis, liver failure, and death (Pau d’arco also taken)

Common Name

Scientific Name

Mistletoe

a

Veno-occlusive disease is now termed sinusoidal obstruction syndrome (SOS).

induction. Ephedra sinica Stapf [Chinese ephedra] affects the renin-angiotensin-aldosterone system. Glycyrrhiza glabra L. [Licorice] and Harpagophytum procumbens DC [Devil’s claw] inhibit renal transport processes (Fatima and Nayeem, 2016). Cardiotoxicity Herbal remedies prepared from plants including Digitalis purpurea [Digitalis], Catharanthus roseus [Vinca], Aconitum napellus [Monk’s hood], Atropa belladonna [Deadly nightshade], Ephedra distachya [Sea grape], Mandragora officinarum [Mandrake), Glycyrrhiza glabra [Licorice] Areca catechu [Betel nut], Thevetia peruviana [Yellow oleander], and Cleistanthus collinus (Oduvan) may show cardiotoxicity (Table 4.17). Herbs mixed with lead, copper and/or mercury are known to be highly cardiotoxic and show typical myocardial degeneration and necrosis (Anaeigoudari et al., 2020; Dwivedi et al., 2011; Fatima and Nayeem, 2016; Li et al., 2016). Neurotoxicity Some common plants used as herbal remedies have demonstrated neurotoxic effects including Papaver somniferum [Opium], Catharanthus roseus [Vinca], Datura stramonium Thorn apple], Atropa belladonna [Deadly nightshade], Hyoscyamus niger [Henbane], Cannabis indica [Marijuana], Conium maculatum [Hemlock], Coscinium fenestratum [Yellow vine], and Brugmansia species [Angel’s trumpet] (Fatima and Nayeem, 2016).

Dermal Toxicity Dermal toxicity reactions of herbal remedies include primary irritant dermatitis, allergic dermatitis, and photosensitization. PRIMARY IRRITANT DERMATITIS

This is a direct irritation of skin by the herbal remedy which results in clinical signs of redness, itching, pain, blusters, peeling, or open wounds. Primary irritant dermatitis may be caused by plants such as Cannabis sativa [Marijuana], Dieffenbachia amoena [Dumb canes] Asclepias syriaca [Milk weed], Narcissus pseudonarcissus [Daffodils], Digitalis purpurea [Foxglove] Ricinus communis [Castor bean] Tulipa gesneriana [Tulip bulb], Primula veris [Cowslip], Hevea brasiliensis [Rubber tree], Ficus carica [Fig tree sap] Ranunculus acris [Butter cup], etc. ALLERGIC CONTACT DERMATITIS

This immunological event can occur following dermal exposure plants such as Toxicodendron vernix [Sumac], Cedrus deodara [Cedar], Dendranthema grandiflorum [Chrysanthemum], Pinus sabiniana [Pine], Apium graveolens [Celery], Allium sativum [Garlic], and Allium cepa [Onions]. PHOTOSENSITIZATION DERMATITIS

The classic herbal remedy that causes photosensitization is Hypericum perforatum [St. John’s

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TABLE 4.16 Reported Human Cases of Kidney Toxicity Related to Herb Consumptiona Possible Toxic Compound

Latin Name

Aristolochic acid

Aristolochia spp.

Aristolochia, Guan To induce weight Mu tong, Han Fang Ji loss, liver disease, arthritis, headache, edema

Chronic interstitial nephritis, renal interstitial fibrosis, Fanconi syndrome, urothelial carcinoma

Flavonoid (sciadopitysin)

Taxus celebica

Chinese yew

Diabetes, vascular diseases

Acute tubular necrosis, acute interstitial nephritis

Flavonoid

Cupressus funebris Endl Mourning cypress

Vascular diseases, instead of “yew”

Acute tubular necrosis, acute interstitial nephritis

Flavonoid oligomeric Crataegus orientalis procyanthins

English Name/ Chinese Name

Indications

Kidney Manifestations

Hawthorn

Congestive heart Acute kidney injury failure, hypertension, (AKI) hyperlipidemia

Ephedrine, norephedrine, pseudoephedrine

Ephedra sinica

Ma huang

Cough, to induce AKI, nephrolithiasis weight loss, to cause sexual arousal

Glycyrrhetinic acid, glycyrrhizic acid

Glycyrrhiza glabra

Licorice Gancao

Cough, sore throat, arthritis, to induce weight loss

Acute tubular necrosis, hypokalemic nephropathy, Fanconi syndrome

Anthraquinones, oxalic acid

Rhizoma rhei

Rhubarb

Laxative, antiinflammatory

Interstitial fibrosis, tubular atrophy

Active moiety triptolide

Tripterygium wifordii

Lei Gong Teng

Acute tubular Arthritis, necrosis antiinflammatory, immunosuppressant

Cape aloe

Constipation, insect bites

Acute tubular necrosis, parenchymatous nephritis

Oxidative Aloe capensis degradation products

Colchicine

Colchicum autumnale

Autumn crocus

Gout

AKI

Dioscorine, dioscine

Dioscorea quartiniana

Yam

Taken as food, to cause poisoning or induce suicide

Acute tubular necrosis

Spurge

Edema, to induce abortion

Acute tubular necrosis

Irritant chemicals in Euphorbia the latex of the plant matabelensis, Euphorbia paralias Andrographolide

a

Andrographis paniculata Chuan-Xin-Lian Infectious disease, (heart piercing lotus) such as upper and lower respiratory tract infection, acute enteritis, bacillary dysentery

AKI, acute tubular necrosis

Xu XL, Yang LJ, Jiang JG: Renal toxic ingredients and their toxicology from traditional Chinese medicine, Expet Opin Drug Metabol Toxicol 12:149–159, 2016; Yang B, Xie Y, Guo M, et al.: Nephrotoxicity and Chinese herbal medicine, Clin J Am Soc Nephrol 13(10): 1605–1611, 2018.

222 TABLE 4.17

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Reported Human Cases of Heart Toxicity Related to Herb Consumption

Common Name

Scientific Name

Chuanwu Caowu Aconitum carmichaeli, Aconitum kusnezoffii

Suggested Active Compounds

Uses

Side Effects

Alkaloids of aconite (mesaconite, hyperconitine)

Analgesics, antiinflammatory

Nausea, vomiting, tachycardia, fibrillation, cardiac arrest

Foxglove

Digitalis lanata

Cardiac glycosides

Congestive heart failure

Tachycardia; ventricular fibrillation and death

Henbane

Hyoscyamus niger

Tropane alkaloids e Hyoscyamine

Stomach complaints, toothaches, ulcers, and tumors

Impaired vision, constipation, flushed skin, irregular heartbeat

Jin Bu Huan

Lycopodium serratum

Levotetrahydropalmatine; pyrrolizidine alkaloids

Used as a sedative, analgesic, and indigestion aid

Life-threatening bradycardia, respiratory distress, liver damage, acute hepatitis

Licorice

Glycyrrhiza glabra

Triterpene saponins, Hydroxycoumarins

Cough, bronchitis, ulcers, inflammation, and epilepsy

Hypertension, hypokalemia, hypernatremia, edema, heart failure, death

Lily of the Valley

Convallaria majalis

Cardiac glycosides similar to those in foxglove plant e convallarin (concallamarin)

Arrhythmia, cardiac insufficiency, and nervous heart complaints

Nausea, vomiting, arrhythmia, cardiac shock

Ma huang

Ephedra sinica

Ephedrine alkaloids

Bronchitis, asthma, edema, and weight loss

Extrasystole, increased blood pressure, chest pain, anxiety, nervousness, tremor, hyperactivity, insomnia, heart attack, stroke, psychoses, hallucinations, seizure, and death

Squill

Urginea maritima

Cardiac glycosides

Cardiac insufficiency, arrhythmia, nervous heart complaints, bronchitis, asthma, whooping cough, and wounds

Nausea, vomiting, hyperkalemia, arrhythmias, and atrioventricular block

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10. TOXICOLOGIC PATHOLOGY OF SELECT HERBAL REMEDIES

wort] (Fatima and Nayeem, 2016). However, the appearance of a sunburn-like may be a result of a Type IV hypersensitivity, such as Toxicodendron diversilobum [Poison oak] and Toxicodendron rydbergii [Poison ivy].

223

extremely toxic, 5–50 mg/kg highly toxic, 50– 500 mg Moderately toxic, 500–5000 mg/kg slightly toxic, 5000–15,000 practically nontoxic, and >15,000 relatively harmless (Erhirhie at al, 2018).

10.1. Aloe vera – Aloe barbadensis 10. TOXICOLOGIC PATHOLOGY OF SELECT HERBAL REMEDIES Most rodent studies that evaluate the toxicologic pathology of individual herbs or herb extracts as the test substance have been done under the auspices of the NIEHS National Toxicology Program. The purpose of these studies was mainly aimed at determining genotoxicity, mutagenicity and carcinogenicity, although nonneoplastic changes were also described. In these studies, the individual herb or herbal product was administered usually to F344/N rats and B6C3F1 mice by oral administration for up to 2 years. The spectrum of carcinogenic responses ranges from no or equivocal evidence for carcinogenic activity (Ginseng, Milk thistle, and Turmeric oleoresin) to a liver tumor response (Ginkgo, Goldenseal, Kava kava), thyroid tumor response (Ginkgo), or an intestinal tumor response (Aloe vera whole leaf nondecolorized extract). Different mechanisms may be involved in the occurrence of liver (Ginkgo, Goldenseal, and Kava kava) and gastrointestinal toxicity (Turmeric oleoresin and Aloe vera whole leaf nondecolorized extract), while the toxic lesion is the same (Dunnick and Nyska, 2013). However, these effects were seen at doses orders of magnitude greater than used in herbal remedies. The following sections summarize studies undertaken to elucidate the toxicology, gross and microscopic events that occur following exposure to the herbal remedies listed above and others. For each herbal remedy experimental study, other animal studies and human exposure studies will be provided. A summary of experimental studies summarizing LD50, acute, subacute, and chronic life-time exposure, carcinogenic–mutagenic response, reproduction toxicity, NOAEL/LOAEL, and human adverse events can be seen in Table 4.18. For the purpose of discussion, the following toxicity dose scale will be used: < 5 mg/kg

The Aloe barbadensis Miller, referred to as Aloe vera, has green fleshy leaves of three identifiable layers: the rough and thick cuticle, rind tissue underneath, and the water parenchyma beneath the rind tissue. The aloe gel is the mucilaginous clear component extracted from the inner pulp. Decolorized aloe vera is also known as “whole leaf aloe vera” since it undergoes activated carbon adsorption to remove the phenolic components of aloe latex (EMA, 1999; Guo and Mei, 2016) (Figure 4.1). Aloe’s name is derived from the Arabic word alloeh meaning “bitter and shiny substance,” and vera from the Latin word for “truth.” The “plant of immortality” used in early Egypt, has been used as a traditional medicine in Arab, Chinese, Egyptian, Greek, Indian, Japanese, Korean, and Roman cultures (Reynolds and Dweck, 1999). It has enjoyed a long history of providing a myriad of health benefits, skin care and as a laxative (Snow and Fantus, 1923) for more than 2000 years and is one of the most frequently used herbal remedies employed throughout the world (Vogler and Ernst, 1999). There are more than 400 species of Aloe, but the most popular and widely used species is Aloe barbadensis Miller. Other species used in health and medicine include but are not limited to Aloe arborescens Miller (a member of the Asphodelaceae family), Aloe perryi Baker, Aloe andongensis, and Aloe ferox (Cosmetic Ingredient Review Expert Panel, 2007). Aloe has fungicidal, antiviral, antibacterial, antiinflammatory, antimicrobial (Boudreau et al., 2017), laxative, immunomodulating, and anticancer properties. Although Aloe vera has long been considered as a safe functional food material that can be used orally and topically, on many occasions it has not been as safe as commonly thought (Steenkamp and Stewart, 2007). It is not recognized as generally safe (GRAS), partly because Aloe vera reduces the synthesis of prostaglandin, thus inhibiting secondary aggregation of platelets interfering with hemostasis (Lee et al.,

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TABLE 4.18 Herb Common Name

Summary of Toxicity Findings for Select Herbal Remedies Acute Toxicity Studies

Subacute Toxicity Studies

Chronic Toxicity Studies

Genotoxicity Lifetime and Exposure Mutagenicity Studies

Taxonomy

LD50

Aloe vera

Aloe barbadensis

Rat: >50 mg/kg im; >15 mg/kg ip. Mouse: >200 mg/kg im; >80 mg/lg ip. Dog: >50 mg/kg im; >10 mg/kg ip [moderate toxicity]

Chamomile

Chamomilla Rat: 8.6 g/kg po recutita Rabbits: > 5 g/ kg [practically nontoxic]

Mouse: 14-day no finding at 1.4 g/ kg Rat: 14-day no findings at 4 g/ kg Rabbits: No dermal toxicity

4-week rat: NSF No studies (2% w/v tea) found 4-week mouse: NSF (2% w/v tea)

Not genotoxic No studies but found antimutagenic

Cocoa

Theobroma cacao

6-day mouse: 2% cocoa powder NOAEL 2% 10-day mouse: 1.0 mg/kg/day epicatechin NOAEL 1.0 mg/kg 1-day mouse: 1.0 g/kg cocoa NOAEL 1.0 g/ kg 10-day rat: 5.0, 10.0% quercetin NOAEL 10% 2-week rat: 1.0 mg/kg quercetin NOAEL 1.0 mg/kg quercetin

Oral sensitization rat: 10% cocoa NOAEL 10% 19-day rat: 10% cocoa diet NOAEL 10% 4-week rat: Cocoa diet (0.2% polyphenols), NOAEL (0.2% polyphenols) 4-week Rat: 400 mg/kg/day cocoa extract, NOAEL 400 mg/kg extract 3-week rat: 0, 5/0% and 10% cocoa NOAEL 10%

Mouse: Comet assay. Not genotoxic Human: randomized placebocontrolled double-blind study 2 g/day for 6 months e prevents DNA damage

Theobromine human: 1000 mg/kg oral Rat: 1265 mg/kg oral Mouse: 837 mg/ kg oral Dog: 250e500 mg/kg oral

14-day rat and None found mouse study e no significant findings (NSF)

13-week rat and Generally, not mouse e genotoxic or intestinal mutagenic goblet cell (group 2B hyperplasia carcinogen)a

20-week rat: Diet 10% cocoa for 20 weeks, NOAEL 10% 12-week apoE knockout mouse: 0%, 0.2%, and 2% cocoa powder, NOAEL 2% 28-week mouse: 8% cocoa powder, NOAEL 8% 18-week mouse: 8% cocoa powder, NOAEL 8% 10-week rat: 14 and 140 mg/kg/ day. NOAEL 140 mg/kg

Rat: Large intestinal adenomas and carcinomas Clear evidence of carcinogenicity Mouse: no evidence of carcinogenicity (group 2B carcinogen)

104-week rat: 0%, 1.5%, 3.5%, and 5.0% cocoa powder, NOAEL 3.5% - no evidence of carcinogenicity 104-week rat: 0, 500, 2000, and 10,000 ppm cocoa, NOAEL 10,000 ppm. No evidence of carcinogenicity

Reproduction Toxicity Studies

Others

Long-term exposure affects general reproduction; not teratogenic, embryolethal, or fetotoxicity

1-year photocarcinogenesis study e phototoxic to both UVA and visible light. It is photocarcinogenic

Allergy e topical; phototoxicity; acute hepatitis; laxative effects; drug interactions; contact dermatitis; cardiac arrhythmias; hypoglycemia; abdominal cramping; diarrhea; muscle weakness; hypokalemia

No studies found

No studies found

Case reports serious allergic reaction; skin reactions

7-day male reproductive study rat: cocoa containing 500 mg/kg theobromine e no testicular toxicity 31-day male reproduction study rat: 2.14 g/kg (117 mg) theobromine/g extract e vacuolation of Sertoli cells 3 generation rat: 0.0%, 1.5%, 3.5%, and 5.0% renal tubular mineralization, NOAEL 3.5%. No evidence of carcinogenicity Teratogenicity study rat: 0, 2.5, 5.0, or 7.5% cocoa in diet GD 0 e21. NOAEL 7.5% cocoa

31-500 ug/mL inhibits 3T3-L1 adipocytes lipid accumulation and induces browning during differen tiation Microbiome rat: 0, 10% cocoa e decreased E. coli count Filamentous fungi may contaminate many stages in cocoa processing, and poor practices may have a strong influence on the quality Filamentous fungi may also produce aflatoxins and ochratoxin A

12-week human: 2000 mg/day cocoa flavanol, NOAEL 2000 mg/day Human: randomized double-blind placebocontrolled trial using 900 mg/ day, NAOEL 900 mg/day

Human Adverse Events

Coffee

Coffea arabica Coffea canephora

(Caffeine) rat: 127 Rat: No effect at 28-day rat: > e367 mg/kg oral 2000 mg/k 75 mg/kg daily Mouse: 127 mg/ single dose NSF kg oral Rabbit: 224 mg/ kg oral [Moderate toxicity]

90-day rat: 8.8 g/kg/day female and 4.0 g/kg male NOAEL

Not genotoxic, have some protective effect on other carcinogens Not mutagenic

Echinacea

Echinacea purpurea

Rats: >30 mg/kg po; >15 g/kg; iv Mice: 2.5 g/kg ip; >10 g/kg po; >5 g/kg iv [practically nontoxic]

No studies found

Negative in No studies found mouse and rat (Ames and hamster embryo cells)

Ephedra (ma huang)

Ephedra sinica

Mouse: 610 mg/ 14-day rat oral: None found kg [slightly toxic] up to 2.0 g/kg (ephedrine/ caffeine) heart e coagulative necrosis particularly the IV septa 14-day mouse oral: up to 2.0 g/ kg NSF

13-week rat: 0, 1,250, 2,500, and 5000 ppm in food lesions in salivary glands and renal tubular injury 13-week 0, 1,250, 2,500, and 5000 ppm in food. NSF

Not genotoxic or mutagenic in Ames TA100, TA1535, TA97, or TA98, sister chromatid exchange No evidence of carcinogenicity

Garlic

Allium sativum

Rat e 635 mg/ Rat: NOAEL kg. 30 mL/kg ip Rabbit oral: 3.3 g/kg Rat oral: >2 g/kg [slightly toxic]

4-week rat: > 2.4 g/kg decreased prothrombin time in male

4-week rat: NSF 4-week mouse: NSF

21-day rat: hepatic Rat: 250 mg/ necrosis 100 mg/ kg/day cardiac, kg hepatic, and renal lesions

Anticarcinogenic (group 3 carcinogen) GRASb 2-year rat: Effects seen at > 25% coffeedtime to tumor but not tumors per se 2-year rat: decrease in benign, malignant, or both 2-year mouse: No increase in tumors at 5%

Possible sperm damage; pregnancy loss possible in humans Rats: NOAEL 80e120 mg/ kg/day Rat: LOAEL 330 mg/kg/ day; Mouse: LOAEL 50 e75 mg/kg Potential behavioral changes Rat: Sperm number and quality decrease at 25 mg/ kg Rat: Maternal toxicity at 25% and 100% coffee

Antioxidant; chemoprotective; antiinflammatory; protects against a number of cancers; neuroprotective; improves live and cardiac health liver disease; increase in fractures women Caffeinated coffee > Decaf in its effects

Reduces the risk of a number of cancers; occupational hazard; reduced cardiovascular disease; risk increase of metabolic syndrome and type II diabetes; risk of stroke

No studies found

No studies found

Not teratogenic in humans

Cardiotoxicity in rats, mice, and humans

Cardiovascular effects e stroke, myocardial infarction, arrhythmias, hypertension, tremor, headaches

GRAS

Halitosis; lowers cholesterol; potential risk of bleeding

Rats: 0, 125, 250, No studies found 500, 1,000, or 2000 mg/kg/day ephedrine Mouse: 0, 310, 630, 1,250, 2,500, or 5000 mg/kg/day ephedrine decreased hepatocellular adenomas and carcinomas. Cardiac lesions

Negative No studies found Ames but positive mouse micronuclei test and hamster embryos

No studies found

(Continued)

TABLE 4.18 Herb Common Name

Summary of Toxicity Findings for Select Herbal Remediesdcont’d

Taxonomy

LD50

Ginkgo

Ginkgo biloba Mouse oral: 7.7 g/kg, 1.1 g/ kg ip Rat: 1.1 g/kg ip [practically nontoxic]

Ginger

Zingiberis rhizoma

Acute Toxicity Studies

Subacute Toxicity Studies

No study found No study found

Rat: 500 mg/kg No study found mild pulmonary changes Mouse: 1551 mg/ kg ip [slightly toxic]

Chronic Toxicity Studies

Genotoxicity Lifetime and Exposure Mutagenicity Studies

Reproduction Toxicity Studies

3-month rat: 1.0 mg/kg hepatocellular hypertrophy, thyroid follicular hypertrophy Mouse: 3-month 2.0 g/kg hepatocellular hypertrophy NOAEL 2.0 mg/ kg/day

Mutagenic in Ames test, positive (weak) mouse micronucleus assay, positive comet assay (DNA strand breaks in vitro) (Group 2B carcinogen)

Mouse Segment I: No studies found Preimplantation loss at GRAS 100 mg/kg/day oral Mouse segment II: conflicting results at similar doses in the same species 30 mg/kg/day. NOAEL Rabbit: NOAEL 1.0 mg/ kg/day

Bleeding risk especially with warfarin e antagonism with plateletactivating factor

No studies found Mouse Segment II: significantly reduced the number of live fetuses and increased fetal death, resorption, and a significant decrease in implantation sites, fertility and fetotoxicity study, maternal toxicity, and alteration of estrus cycle at 2.0 g/kg/day enhances testosterone production, increase in testicular weight and blood flow Mouse Segment I: 0, 100, and 200 mg/kg methanolic extract and 150 and 300 mg/kg water extract; increased fertility index, sexual organs weight, serum testosterone level, and sperm motility and count

Uncommon adverse effects of GIT disturbance; Affects fibrinolytic activity and platelet aggregation, hence interaction with Warfarin; does not have an effect on the vestibular system; has an antiemetic effect

2-Year rat: dose eresponse increase in hepatic adenomas and hepatocellular carcinoma 100 e1000 mg/kg/day 2-Year mouse: dose eresponse increase in hepatic adenomas and hepatocellular carcinoma 100 e1000 mg/kg/day

35-day rat oral: 13-week rat oral: No studies No studies found NOAEL ¼ 1.0 g/ NOAEL found for mg based on body 500 mg/kg genotoxicity weight decrease at Mutagenic in 2.0 g/kg Salmonella 30-day rat: typhimurium. significant Strains TA100 inhibition of and T1838 cholangio with S9 carcinoma 2.0 g/ metabolic kg/day activation

Others

Human Adverse Events

Ginseng

Panax ginseng

Rat: up to 1.0 mL NSF Rat: oral dose >1.6 g/kg Mouse: oral dose >0.8 g/kg [slightly toxic]

16-day rat: Dose up to 2.0 g/kg/ day. No findings except that weight gain of 2.0 mg/kg group was greater than the rest

Rat: oral 5.0 g/kg NSF 28-day rat: NOAEL of 2.0 g/ kg/day NSF

13-week rat oral e No effects in all groups, NOAEL 1.0 g/ kg 13-week rat oral e NOAEL 2.0 g/ kg/day 14-week rat: NOAEL 5.0 g/ kg 26-week rat oral: NOAEL 375 mg/kg/day 14-week mouse oral: NOAEL of the top treatment group of 5.0 g/kg

3-month mouse: Not mutagenic in Ames and mouse micronucleus and not genotoxic

2-year rat oral: up to 5.0 g/kg no findings, NOAEL 5.0 g/kg. Significant decrease in the incidence of mammary gland fibroadenoma at 5.0 g/kg/day

Goldenseal

Hydrastis canadensis

Rat: 500 mg/kg ip; oral >1.0 g/ kg; 14.5 mg/kg im. Mouse: 329 mg/ kg oral Mouse: 18 mg/ kg sc Mouse: 24.3 mg/ kg ip [highly toxic]

15-day mouse: up to 50,000 ppm e hepatocellular hypertrophy

No studies found 3-month rat: up to 50,000 ppm e hepatocellular hypertrophy 3-month mouse: up to 50,000 ppm e hepatocellular hypertrophy

Not genotoxic or mutagenic (group 2B carcinogen)

2-Year rat: up to Rat Segment II: no effect at No studies found 25,000 ppm diet. 65 times the human dose Increased hepatocellular adenoma and carcinomas, and hepatoblastoma 2-Year mouse: up to 25,000 ppm diet. Increased hepatocellular adenoma and carcinomas, and hepatoblastoma

Overdose causes cardiac damage, depression, hypotension, spasms, and seizures; traditionally contraindicated in pregnancy

Green tea

Camellia sinensis

Mouse oral 3.3 g/ 14-day rat: NSF No studies found 13-week rat oral: kg [slightly toxic] at a single dose up to 4.0 g/kg. up to 12.0 g/kg No effects 14-week mouse oral: up to 1.0 g/ kg e hepatic necrosis and thymic atrophy 14-week mouse oral: up to 1.0 g/ kg e hepatic degeneration and olfactory epithelial atrophy

No genotoxic activity. Has an antimutagenic effect and can be considered genoprotective

2-year rat oral: up Rat 5% extract: Increase in to 1.0 g/mg/day. sperm concentration; not Hepatic necrosis, a teratogen gastric mucosal necrosis, olfactory atrophy and necrosis, marrow hyperplasia, and bronchop neumonia 2-Year mouse oral: up to 300 mg/kg/ day increased hepatocellular carcinoma, bone marrow hyperplasia, islet cell hypertrophy

Use has an increased lung cancer risk, hepatotoxic, and teratogenic

Rat Segment I/II: oral up to 2.0 g/kg did not affect fertility, early embryonic development, the dam or fertility parameters, NOAEL 2.0 g/kg/day

Interaction with herbal products and conventional drugs to some extent GRAS

Interaction. With blood thinners because of vitamin K levels GRAS

A number of systematic reviews have failed to show changes medicines and foods on physical, psychomotor performance, cognitive function, diabetes mellitus, and immune modulation

(Continued)

TABLE 4.18 Herb Common Name

Summary of Toxicity Findings for Select Herbal Remediesdcont’d Acute Toxicity Studies

Subacute Toxicity Studies

Chronic Toxicity Studies

Genotoxicity Lifetime and Exposure Mutagenicity Studies

Reproduction Toxicity Studies Rat: single-dose GD 15, 1.0, or 100 mg/kg/day no biologically significant effects Rat Segment II: up to 1.5 g/kg: day 62 postnatally sperm count decreased and epididymal transit time Rat Segment II: 0, 200, or 300 mg/kg on GD 9e11. NSF

Taxonomy

LD50

Indole-3Carbanol

Brassica glucos inolate gluco brassicin

Rat: 500 mg/kg sc [Moderately toxic]

4-week mouse: 10 umole was the LOAEL but an NOAEL was not established

14-week rat: Lymphatic ectasia and dilation of lymphatics 14-week mouse: 300 mg/kg/day NOAEL

Nonmutagenic up to 250 mg/ kg/day, but was toxic to bone marrow

2-Year rat: up to 300 mg/kg/day: Uterine metaplasia and fibroma/ sarcoma, thyroid follicular hyperplasia 2-year mouse oral: up to 250 mg/kg/ day: Increased hepatocellular adenomas and carcinomas, olfactory nerve atrophy, respiratory epithelia necrosis, and hyperplasia

Kava kava

Piper Mice: 750 mg/kg 2-week rat: up to No studies found methysticum [slightly toxic] 2.0 g/mg hepatocellular hypertrophy at 2.0 g/mg 2-week mouse: up to 2.0 g/mg hepatocellular hypertrophy

3-month rat: oral 0, 0.125, 0.25, 0.5, 1.0, and 2.0 g/ kg/day, dose eresponse hepatocellular hypertrophy 3-month mouse: 0, 0.125, 0.25, 0.5, 1.0, and 2.0 g/ kg/day, dose eresponse hepatocellular hypertrophy

None of the kavalactones was found to be positive in the experimental concentration ranges tested. Not genotoxic or mutagenic (group 2B carcinogen)

2-Year rat oral: up No studies found to 1.0 g/kg/day. Doseeresponse testicular adenomas, hepatocellular hypertrophy, pancreatic acinar metaplasia, nephropathy, pituitary adenomas 2-Year mouse oral: up to 1.0 g/kg. Doseeresponse increase in hepatoblastomas, hepatic adenomas, and carcinomas

Others

Human Adverse Events

Induces P450 (CYP) Decreased risk 1A1; protection by of cancer CYP-mediated estrogen metabolism; intestine is the target organ

No studies found

Reported cases of severe hepatotoxicity, cirrhosis, and liver failure

Marijuana

Cannabis sativa and Cannabis indica (studies usually done with delat-9tetrahydro cannabinol e THC)

Rat: 800 e1900 mg/kg (THC) Rat: 5000 mg/kg Dog: 130 mg/kg iv Dog: > 9.0 g/kg oral Monkey: 130 mg/day iv Monkey: >9 g/ kg [Moderate toxicity]

Milk thistle

Silybum marianum

Mouse: iv Rat e protects 400 mg/kg against CCL4 Rat: 385 mg/kg intoxication Rabbits: 140 mg/ kg Dogs: 140 mg/kg [Moderately toxic]

Rat and mouse: Ataxia in minutes gait alterations, gait width increased, and rotarod impairments. Pathology NSF 14-day rat: 0, 1.0, 2.0, and 4.0 g/ kg/day. Reduced food consumption, piloerection centrilobular hepatocellular hypertrophy 24-hour mouse: 0, 246, 738, or 2460 mg/kg of CBD (acute toxicity, 24 h) or with daily doses of 0, 61.5, 184.5, or 615 mg/kg for 10 days (subacute toxicity). Clear signs of hepatotoxicity

A 14-day rat: 1000, 2000, and 4000 mg/kg/day resulted in effects where an NOAEL could not be concluded

Persistently alters the structure and function of the rat hippocampus, a paleocortical brain region involved with learning and memory processes Conflicting reports as to whether it is neurotoxic or neuroprotective 90-day rat: 0, 200, 400, or 800 mg/kg/day. Periacinar hepatomegaly with increased liver weight which reversed in 28 days. NOAEL is 800 for females and 400 mg/kg for males 90-day rats: 0, 100, 360, and 720 mg/kg/day, followed by a 28-day recovery period. Significant decreases in body weight. NOAEL 100 for males and 360 mg/kg/ day for female

No evidence of genotoxicity was found in a bacterial reverse mutation test (Ames), in an in vitro mammalian chromosomal aberration test, or in an in vivo mouse micronucleus study no evidence of genotoxicity

No studies found 3-month rat: Not cytotoxic NOAEL or genotoxic 50,000 ppm 3-month mouse: NOAEL 50,000 ppm

Cancer: Immuno suppression does stop the anticancer properties of THC. 2-year rat: 50 mg/ kg/day oral. Animals increased survival. Growth arrest and death of tumor cells. Cannabinoid as selective antitumor compounds that kill tumor cells without damage to surrounding normal cells

Cannabis and THC act on the hypothalamic-pituitary adrenal axis. Rat and mouse studies show effects on adrenocorticotropic hormone, thyroidstimulating hormone, and growth hormone levels (by 90% within 60 min of treatment in rats) and stimulate the release of adrenocorticotropic hormone and glucocorticoids

2-Year mouse: up to 50,000 ppm in feed: no increase in tumor, no other significant findings 2-Year mouse: up to 50,000 ppm in feed: no increase in tumors but decrease in hepatic tumors

Rat Segment I: doses up to Antioxidant and 3600 ppm (NOAEL) and antifibrotic activity 12,000 ppm LOAEL. Pup GRAS loss and lower growth rates. Not a strong signal and may not have any reproductive implications

In vitro THC produces a biphasic effect on heart rate with an initial increase followed by a decrease. THC also decreases coronary blood flow, cardiac contractile force, and gut motility Guinea pigs and mouse: THC doses in the range of 0.2 mg/kg to 100 mg/kg. THC has the potential to compromise host resistance to both viruses and bacteria by decreased cellular and humoral immunity

Sedation dysphoria, euphoria, munchies, cardiac effects, memory loss, and short-term spatial learning tasks Can cause liver injury Drug interactions Male reproductive toxicity Cumulative exposure long t1/2 Special populations exaggerated effects (elderly, children, adolescents, pregnant and lactating women) May have utility in Alzheimer’s disease

GI irritant effects: Nausea, etc. Not shown to be hazardous during pregnancy

(Continued)

TABLE 4.18 Herb Common Name

Summary of Toxicity Findings for Select Herbal Remediesdcont’d

Taxonomy

LD50

Pulegone American penny-royal and mints

Mentha pulegium

Rats: 150 mg/kg ip Mouse: 1709 ip. [Moderately toxic]

Riddelliine

Pyrroli See Table 4.19 zidine [highly toxic] alkaloids from, e.g., Crotalaria, Amsinckia, and Senecio

Saw palmetto Serenoa repens

Rat: >50 g/kg ip Mouse: > 1080 mg/kg ip Dog: > 10 g/kg ip [relatively harmless]

Acute Toxicity Studies Hepatotoxicity is potentiated by enzyme induction (CYP450) Considered nontoxic by some 16-day rat: dose up to 300 mg/ kg/day. Liver toxicity 16-day mouse: dose up to 300 mg/kg/day. Liver toxicity

Subacute Toxicity Studies

Chronic Toxicity Studies

Genotoxicity Lifetime and Exposure Mutagenicity Studies

28-day rat up to 160 mg/kg gave biologically insignificant findings: NOAEL 20 mg/kg

3-month rat oral: up to 150 mg/ kg/day e glomerulopathy and bile duct hyperplasia with periportal fibrosis 3-month mouse oral: up to 150 mg/kg/day e no effects

Not genotoxic or mutagenic (group 2B carcinogen)

2-year rat: up to Rat Segment II: up to 50 150 mg/kg oral: ug/kg/day NSF treatment-related nephropathy hepatocellular alterations, ovarian atrophy, nasal lesions 2-year mouse: up to 150 mg/kg oral: treatment-related hepatocellular adenomas, hepatoblastomas, nasal lesion

13-week rat: up to 10 mg/kg oral: focal hepato cytomegaly 13-week mouse oral: up to 25 mg/kg NSF

Genotoxic and mutagenic in many systems (group 2B carcinogen)

2-Year rat oral: up to 1.0 mg/kg e hemangiosarcoma hepatocellular necrosis, hepatic necrosis, renal tubular necrosis, mononuclear cell leukemia 2-Year mouse oral: up to 3.0 mg/kg e hemangiosarcoma, hepatic necrosis, nephropathy

No studies found, but No studies found teratogenic, embryotoxic, and fetotoxic in livestock e possibly carcinogenic to humans

Following contamination of crops (or herbs) causes venoocclusive disease with liver failure

No studies found

No studies found

No studies found

Dizziness, headache, nausea, and vomiting. Rare report (2) of unspecified live damage and (1) pancreatic damage

2-week rat oral: No studies were up to 25 mg/kg: found Pulmonary hemorrhage 2-week mouse oral: up to 25 mg/kg: Hepatocellular cytomegaly

Rat: given 50 g/ kg once ip showed clinical signs of depression, dyspnea, and greasy fir. No mortality

No studies 30-day rat: 150 and 300 mg/kg/ found day. Creatinine and Kþ elevated in both groups so NOAEL could not be determined

Reproduction Toxicity Studies

Others

Human Adverse Events

P450 catalyzed reactive metabolites may be responsible for hepatotoxicity

Beneficial for irritable bowel syndrome, gas, and diarrhea

No studies found

Senna alexandrina

Rat: 5 g/kg oral No studies Mouse: 1.0 g/kg found ip Mouse: 5.0 g/kg oral [practically nontoxic]

30-day rat: oral up to 5.0 g/kg/day NSF 5-week mouse: up to 10,000 ppm e increase epithelial hyperplasia of the cecum and colon at 5000 ppm 30-day rabbit: up to 4% senna in the feed. Those in the 4% group showed myocardial necrosis and centrilobular degeneration with a reduction in CYP450 activity in the glycogenolytic fibers, together with muscle atrophy, confirmed by morphometrics

St. John’s wort Hypericum perforatum

Rat: 5628 mg/kg No studies oral found Mouse: 9.8 g/kg sc Rabbit: 15.8 g kg dermal Rabbit: 1826 mg/ kg ip [practically nontoxic]

No studies found 45-day rat: (quercetin) 100 mg/kg and rotenoned reduction of neuronal damage

Senna

4-week rat: 12 or 58 mg/kg/day. NSF 13-week rat: up to 1500 mg/kg/ daydreduced body weight at 750 mg/kg and above. Pathology NSF 5-week mouse: up to 10,000 ppm in feed NSF

Not cytotoxic or mutagenic (Ames) Double-strand breaks in plasmid DNA; potentially antioxidant and antimutagenic activity Not carcinogenic

2-Year rat: up to 300 mg/kg/day orally. Reduced body weights in 300 mg/kg/day. Pathology, no biologically significant findings considered not carcinogenetic 2-year rat: up to 25 mg/kg/day. NSF

No evidence of any embryolethal, teratogenic, or fetotoxic action in rats and rabbits

90-day rat: up to 2% seeds in feed e M/E ratio decrease and increased reticulocytes 40-week mouse: carcinogenesis in p53() mice e epithelial hyperplasia colon and rectum but no neoplasia

Diarrhea and GIT hypermotility; liver and kidney toxicity; allergic reactions; potential link to colon cancer Considered safe for pregnancies

Not cytotoxic or mutagenic and some protection against the clastogenic effect of cyclophos phamide

No studies found

21-day rat Segment I: 100 and 1000 mg/kg necrosis and degeneration in liver and kidneys at 100 mg/kg. An NOAEL could not be determined 60-day rat: Male reproduction studydMaternal exposure NSF at 100 mg/kg Rat Segment II: study at 36 mg/kg during embryonic and fetal development did not show toxicity to mother or interfere with the progression of gestation during organogenesis 5-week mouse postnatal development study: 182 mg/kg/day e hypericin did not have major impact on cognitive tasks

In vitro eye phototoxicity human lens epithelial cells e necrosis and apoptosis (hypericin) e positive phototoxicity In vitro cytokine activation e protection in rat and human pancreatic islet cells Rat: Angiogenesis inhibitor Inhibits neoplastic cell proliferation via apoptosis induction 50e300 mg/kg inhibits edematous response and antinociceptive activity

hypericin (a naphthodia nthrone) and hyperforin (a lipophilic phloroglucinol) dhave the greatest medical activity Adverse effects are gastrointestinal symptoms, allergic reactions, dizziness, confusion, restlessness, lethargy, and dryness of the mouth, effects that are generally mild, moderate, or transient Antidementia potential Serious drug interactions (Continued)

TABLE 4.18 Herb Common Name Tobacco

Summary of Toxicity Findings for Select Herbal Remediesdcont’d

Taxonomy

LD50

Acute Toxicity Studies

Only available No studies for nicotine found Rats: 50 mg/kg oral Mouse: 3 mg/kg oral Human: 0.5 e1.0 mg/kg oral [extremely toxic]

Subacute Toxicity Studies

Chronic Toxicity Studies

No studies found 96-week rat: Squamous metaplasia of the laryngeal and tracheal epithelium 1-Year hamster: Laryngeal carcinomas and leukoplakia 70-week hamster: Laryngeal carcinomas but longer survival in exposed group 66-month rabbit: NSF 60-month dog: NSF 4-month A/J mice mouse: Significantly more lung tumors than other mice

Genotoxicity Lifetime and Exposure Mutagenicity Studies

Reproduction Toxicity Studies

Produces DNA adducts and extensive DNA damage in bladder cancer cells Is mutagenic e positive Ames, sister chromatid exchange, DNA strand breaks, and micronuclei suggestion that it might be a germ cell mutagen Genotoxic amniotic fluid (group 1 carcinogen)

Reproductive and developmental toxicity Rat fertility study postnatal decreased proliferation of bronchiolar epithelial cells and altered surfactant Most other studies use nicotine and not tobacco Decreased fetal weights in most studies e intrauterine growth retardation

2-year rat tobacco extract at 0, 0.2, 2.0, or 5.0 mg nicotine/ kg/day: Significant trends for mammary gland adenoma (female), basal cell carcinoma, and thyroid follicular cell adenoma but were within historical control values 2-year rat: Respiratory neoplasia e nasal squamous cell carcinoma, nasal adenocarcinoma, pulmonary adenoma, and alveolar carcinomas 126-week rat: Increased pulmonary adenocarcinomas in females only 2-Year mouse: Doseeresponse increase in pulmonary adenomas but not among other groups 100-week hamster: nasopharyngeal and laryngeal squamous carcinomas 125-week dog: Bronchioalveolar carcinomas but no metastases. Control dogs also had similar lesions but to a lesser extent

Others Condensates from different cigarette brands, tar categories and styles vary in their concentrations of the carcinogenic compounds Rabbits and mouse: dermal exposure to cigarette smoke condensate causes skin tumors Mouse: Increase in mutations in the p53 tumors suppressor gene and the K-ras proto-oncogene Mouse: Skin application dose eresponse skin tumors Rat: CO-carcinogen with beta irradiation Hamster: NSF Rabbit: Large papilloma and skin cancer

Human Adverse Events Cigarette smoking and lung cancer Chronic obstructive pulmonary disease Lung, bladder, renal, pancreatic, oral, and colorectal cancer Cardiovascular disease Several factors determine the biologically effective dose of carcinogens, including the number of cigarettes smoked per day, type of cigarette, smoking topography, carcinogen metabolism, and DNA repair Nicotine does not cause of modulate cancer

Turmeric oleoresin

a b

Curcuma longa

Rats: >5.0 g/kg No studies oral found Mouse: >2.0 mg/ kg oral [practically nontoxic]

No studies found 109-day pig: 0, 60, 296, and 1551 mg/kg. statistically significant doserelated increases in the weight of the liver and the thyroid were recorded at all dose levels. Pericholangitis, hyperplasia of the thyroid, and epithelial changes in the kidney and urinary bladder were observed in the two higher dose groups 90-day mouse and rat: turmeric up to 5% in feed. Hepatic necrosis (focal) with or without regeneration. mouse more sensitive 13week rat: 0, 1,000, 5,000, 25,000, or 50,000 ppm in feed. Mucosal hyperplasia cecum and colon 13-week mouse: 0, 1,000, 5,000, 25,000, or 50,000 ppm in feed NSF 13week rat: oral 0, 0.1, 0.25, 0.5 g/ kg. NSF 90-day rat: 0, 100, 200, and 400 mg/kg/ day oral. NSF 90-day rat: 0, 180, 360, and 720 mg/kg/day. NSF 90-day rat: 1000 mg/kg oral. NSF

90-day rat: 1000 mg/kg oral. Not mutagenic Amesd inhibits microsomal activation edependent mutagenicity of 2-acetamido fluorene Ames, bone marrow chromosome aberration test, erythrocyte micro nucleusdall tests negative

103-week rat: 0, 2,000, 10,000, and 50,000 ppm in the feed. increased incidence of ulcers, hyperplasia, and hyperkeratosis of the forestomach. Ulcers, chronic active inflammation, and hyperplasia of the cecum. Increased clitoral adenomas but not carcinomas 103-week mouse: 0, 2,000, 10,000, and 50,000 ppm in the feed. Increased hepatocellular neoplasms and thyroid follicular cell hyperplasia

90-day rat Segment III: 0, 100, 200, and 400 mg/kg/ day oral. NSF Rat Segment II: 0, 100, or 200 mg/kg oral e a concentration-dependent toxicity ex vivo in the embryos on gestation day 9.5. Placental calcification and inhibited vascular endothelial growth factor probably blocked by sesquiterpenoids Rat Segment II: 0, 1,500, 3,000, and 10,000 ppm in the feed. NSF

International Agency for Research on Cancer: Some drugs and herbal products, IARC Monogr Eval Carcinog Risks Hum 108:7–419, 2016. Generally regarded as safe.

In vitro reduces inflammatory cytokine and chemokine expression in synovial fibroblasts GRAS

None observed following 2 daily dose for 30 days Phase I study NSF

234

4. HERBAL REMEDIES

FIGURE 4.1

Aloe vera (Aloe barbadensis).

2004). In addition, long term use of anthranoid laxatives appears to correlate with the risk of developing colon cancer (van Gorkom et al., 1999). Like most other drugs, idiopathic reactions do occur, e.g., Aloe-induced HenochSchonlein purpura, an idiopathic vasculitis of the small vessels (Cholongitas et al., 2005). Generally, allergy, including urticaria, contact dermatitis, and widespread dermatitis, cardiac arrhythmias, hypoglycemia, abdominal cramping and diarrhea, muscle weakness, and hypokalemia may occur following Aloe vera exposure (Ulbricht et al., 2007). Toxicity studies can be stratified with respect to the whole leaf extract, latex, and gel. The LD50 in mice is > 200 mg/kg, rats >50 mg/kg, and dogs >50 mg/kg for unspecified Aloe vera parts and in intravenous studies the LD50 in mice was >80 mg/kg, rats >15 mg/kg, and dogs >10 mg/kg (Cosmetic Ingredient Review Expert Panel, 2007). Using the toxicity scale outlined in Erhirhie et al. (2018), the lowest dose would classify the Aloe acute experiment as moderately toxic based on the LD50 results. Whole Leaf Extract Studies of acute lethality have shown that exposure to Aloe vera dried leaf extract has an LC50 in brine shrimp of 3.59 mg/mL and the LD50 in Swiss albino mice was 120.65 mg/kg (Cosmetic Ingredient Review Expert Panel, 2007). An MTD of 100 mg/kg ip in mice was established (Dhar et al., 1968).

In vitro, Aloe vera whole-leaf material caused a dose-dependent decrease in the viability of HeLa and HepG2 cells with half-maximal cytotoxic concentration (CC50) values of 413.9 and 439.0 mg/mL, respectively, following a 4-h treatment (du Plessis and Hamman, 2014) It also caused a dose-dependent increase of apoptosis in HeLa cells at concentrations up to 1000 mg/mL. Subchronic studies showed renal degeneration in rats exposed to 8.0 g/kg for 90 days (Zhou et al., 2003b) and B6C3F1 mice (Boudreau et al., 2013a; National Toxicology Program 2013a), characterized by renal cystic degeneration, with hemorrhage, lymphoid hyperplasia, pigmentation, and plasma cell infiltrate. In contrast, no significant pathological findings were observed after subchronic exposure for 13 weeks (Sehgal et al., 2013; Shao et al., 2013). No genotoxic effects were observed in the Ames test and DNA repair assays using Aloe vera decolorized extract beverage (Sehgal et al., 2013). In the mouse lymphoma assay (MLA) a concentration-dependent cytotoxicity and mutagenicity in the mouse lymphoma cells followed exposure to the whole extract, showing a positive response at lower concentrations than the decolorized extract after a 24-h treatment (Boudreau et al., 2013b). Significant sperm damage, hematological changes, inflammation, and mortality as compared to control animals was observed after chronic oral ingestion of 100 mg/kg Aloe vera extract per day for 3 months (Cosmetic Ingredient Review Expert Panel, 2007). Goblet cell hyperplasia was observed in the cecum and large intestine of rats and mice exposed to Aloe vera whole leaf extract (National Toxicology Program 2013a). There is clear evidence of carcinogenic activity in F344/N rats following oral administration of Aloe vera whole leaf extract in drinking water for 2 years (National Toxicology Program, 2013a). A significant dose-related increase in the number of adenomas and/or carcinomas of the ileocecal and cecal-colic junction, cecum, and the ascending and transverse colon in male and female rats in the high-dose groups was seen with a dose-response increase in goblet cell hyperplasia, associated with cellular infiltration of the mesenteric lymph nodes (Figure 4.2). (National Toxicology Program, 2013a) In the

II. SELECTED TOXICANT CLASSES

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FIGURE 4.2 Tumor sections from the large intestine of F344 rats exposed to Aloe vera nondecolorized whole leaf extract in drinking water, ad libitum, for 2 years. (A) Adenoma (X50). The exophytic mass with dysplastic hyperchromatic cells (arrows and inset box) was limited to the mucosa without evidence of invasion into the muscularis mucosa. (B) Carcinoma (X60). Note the neoplastic cells invading past the muscularis mucosa (arrows) and the fibroplasia surrounding the neoplastic cells (inset box). Reproduced with permission from Pandiri et al., Toxicol Pathol 39(7):1065–1074, 2011 [Page 1067].

subsequent 2-year study, adenomas or adenocarcinomas in the cecum, colon, and rectum were prominent in males, whereas adenomas only were observed in the females. The irritation of the intestinal tract may contribute to the equivocal carcinogenic potential in the colon (Yokohira et al., 2009). A weak enhancing effect on the photocarcinogenic activity of simulated solar light was seen when Aloe whole leaf or decolorized whole leaf creams were applied topically to male and female SKH-1 hairless mice for 1 year (National Toxicology Program, 2010a). Latex Prolonged use of the laxative is associated with electrolyte imbalance due to diarrhea, abdominal pain, vomiting, hypokalemia, pseudomelanosis coli, and the development of a cathartic colondthe colon becomes atonic and dilated. The long-term use of anthranoid laxatives might be correlated with the risk of developing colon cancer (van Gorkom et al., 1999) or stimulate uterine contractions thereby increasing the risk for premature labor or miscarriage (Guo and Mei, 2016). Aloe-emodin induces apoptosis through various mechanisms, including a p53-dependent pathway

in T24 human bladder cancer cells (Lin et al., 2006) and G2/M cell cycle arrest in human promyelocytic leukemia HL-60 cells (Chen et al., 2004). The apoptotic process caused by aloeemodin and emodin in human lung squamous carcinoma cells (CH27) and human lung non– small cell carcinoma cells (H460), increases cytosolic cytochrome c, caspase-3 activation, and changes of protein kinase c (PKC) isozymes (Lee, 2001). This study also demonstrated that PKC stimulation occurred at a site downstream of caspase-3 in the emodin-mediated apoptotic pathway. In addition, aloe-emodine significantly inhibits proliferation and induces apoptosis in adult human keratinocytes (Popadic et al., 2012). The impairment of keratinocyte proliferation could be observed at concentrations far below the industry standards for commercial products containing Aloe extract. The toxicity of aloin has also been investigated in human Jurkat T lymphocytes using flow cytometry and microscopy (Buenz, 2008). Aloin treatment resulted in a dose-dependent decreased cell size, increased granularity, a block at the G2/M phase of the cell cycle, and loss of both membrane integrity and mitochondrial membrane potential,

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suggesting a mitochondrial-dependent pathway for aloin-induced apoptosis. Aloe vera crude leaf extract does not show cytotoxicity to mouse bone marrow cells. No significant increase in structural abnormalities in chromosomes was observed, but a marked increase in cells with chromosome-number anomalies was found. The extract, however, significantly increased the mitotic index of both cell types (Verma et al., 2012). The genotoxicity of some anthraquinones has been confirmed in vitro and in vivo. Many anthraquinones are mutagenic in several strains of Salmonella typhimurium, particularly sensitive to frameshift mutagens, e.g., TA1537, TA1538, TA102, and TA98 (Brown et al., 1977; Nesslany et al., 2009; Westendorf et al., 1990). Aloeemodin also induces increased revertant colonies in strains TA1537, TA1538, and TA98 (Heidemann et al., 1996). Emodin, danthron, and aloeemodin causes a dose-response increase in micronuclei and moderate increases in mutant frequency in the mouse lymphoma assay and micronucleus test, as well as increased DNA breaks in the Comet assay with renal organ specific genotoxicity (Nesslany et al., 2009) due to the inhibition of the catalytic activity of topomerase II (Topo II) (Muller et al., 1996; Muller and Stopper, 1999). Aloe-emodin also causes DNA damage in human lung carcinoma cells through the production of reactive oxygen species (ROS), which induces micronuclei in TK6 human lymphoblastoid cells, and chromosomal aberration in Chinese hamster ovary cells (Heidmann et al., 1996). Finally, it causes DNA damage and caspase cascade-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways (Chiang et al., 2011). Tumor promotion activities using nine hydroxyanthraquinones (HAs) including danthron, aloe-emodin, and emodin, show an increase in DNA synthesis in primary rat hepatocytes, malignant transformation of C3H/M2 mouse fibroblasts pretreated with N-methyl-N0 -nitro-Nnitrosoguanidine or 3-methylcholanthrene suggesting that HA with hydroxy groups in the 1,8-positions may have tumor-promoting activity (Wolfe et al., 1990). Since rats developed adenomas or adenocarcinomas in the cecum or upper portion of the colon, as well as liver hepatocellular adenomas and carcinomas (Boudreau et al., 2013b), there is strong evidence that HA is carcinogenic in rodents. Carcinogenicity was initially considered

questionable by the finding that feeding emodin to rats and mice gave only equivocal evidence of carcinogenic activity due to a marginal increase in Zymbal’s gland carcinoma numbers and a low incidence of renal tubule neoplasms (Pandiri et al., 2011). A combined treatment of ultraviolet radiation and aloe-emodin in ethanol vehicle resulted in melanoma, which provides evidence for aloe-emodin dermal photocarcinogenicity (Strickland et al., 2000). Gel Aloe vera gel is a transparent mucilaginous jelly-like substance contained in the parenchymatous cells of fresh Aloe vera leaf pulp, with a highwater content (99%–99.5%) and a (pH) of 4.4–4.7 (Guo and Mei, 2016). There is a dearth of conflicting studies elucidating the toxic effects of Aloe vera gel probably due to the differences in the gel contents that can be influenced by a variety of factors including season, location, irrigation, harvest time, and, most importantly, the lack of standardization of the gel preparations. The LD50 for the gel is > 21.5 g/kg in rats and >31.6 g/kg in dogs (European Medicines Agency, 1999) The cytotoxicity of Aloe vera gel has been confirmed in monolayers of chicken fibroblasts based on the observations of disrupted intercellular junctions and the formation of cell-free gaps in the monolayers after treatment (Avila et al., 1997). Aloe barbadensis leaves, native gel, purified gel, and the low molecular weight fraction caused this type of cell injury. Supporting the cytotoxic effects of the gel, an Aloe vera dehydrated gel material induced dosedependent cytotoxicity in HeLa cells, with a CC50 value of 269.3 mg/mL (du Plessis and Hamman, 2014). Dose-related increases in urine glucose levels in female rats and dose-related decreasing trends in serum levels of triglycerides, cholesterol, and albumin occurred at concentrations of 1.5% or greater in female rats and of 3.0% have been seen (Dunnick and Nyska, 2013). In addition, serum testosterone, sperm count, and sperm fertility were significantly decreased compared to the control rats (Asgharzade et al., 2015). Chronic studies revealed no changes in feed consumption, body weight gain, and serum chemistry results following a 13-week subchronic exposure in B6C3F1 mice following a 5.5-month ingestion of crude skinned Aloe filet (Sehgal et al., 2013). An NOAEL in CD-1 mice with oral administration of up to 2 g/kg/d for

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14 days or up to 1 g/kg/d for 90 days (Yimam et al., 2014). Similarly, no adverse effects were observed on body weight gain, food intake, gastrointestinal transit time, or gross pathology at dietary concentrations of 1% or 10% (corresponding to doses of Aloe vera gel of w0.33 and 3.3 g/kg/day). Life-long ingestion of low dose (1%) Aloe vera filet caused no obvious harmful effects or deleterious changes in the rat (Ikeno et al., 2002), indicating that the gel is unlikely to be the major toxic component of the herb. Agarose gel electrophoresis assay showed that Aloe vera pulp extract produced dosedependent single-strand breaks in the plasmid of Escherichia coli–deficient repair mutant, revealing the genotoxic properties of the gel, but not cytotoxicity (Paes-Leme et al., 2005). Mutagenicity could not be supported in in vitro and in vivo safety studies in the Ames test, the chromosomal aberration test, and the in vivo bone marrow mouse micronucleus test, following oral administration at doses up to 5.0 g/kg/day to rats for 90 days (Williams et al., 2010). Similarly, Aloe vera gel did not induce a significant increase in SOS DNA repair in Escherichia coli or mutagenesis in Salmonella TA100 (Sehgal et al., 2013). Carcinogenicity data are not available for Aloe gel and no severe adverse effects or carcinogenicity have been reported in humans using Aloe vera gel (Guo and Mei, 2016). There is clear evidence that the Aloe vera whole leaf has carcinogenic activity in rats and has been classified by the International Agency for Research on Cancer (IARC) as a possible human carcinogen (Group 2B) (Boudreau and Beland 2006; Dunnick and Niska 2013; Guo and Mei 2016; IARC, 2002; 2016).

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preparations of Marijuana vary in strength and genetic manipulation is a hobby for some. However, the THC concentration in cannabis varies with climate, soil, and cultivation techniques to name a few extrinsic factors. Finally, the amount absorbed by the body varies with the route of administration (Joy et al., 1999) – smoking or edibles (Figure 4.3). Many aspects of the legalization of the herb are controversial due to its political nature. With the advent of synthetic fiber from the petrochemical industry, an effort to remove the competing fiber was based on the psychotropic nature of the smoked plant. “Refer madness” and creeping legislation put Marijuana on the narcotics list. “Clearing the smoke” has been difficult due to the controversies surrounding the scientific enquiry undertaken at the time. Even now much discussion continues on the adequacy of clinical design to demonstrate effect without confounding variables (Russo, 2016).

10.2. Cannabis – Cannabis sativa and Cannabis indica The term “Marijuana” typically refers to the tobacco-like preparations of the leaves and flowers of the plant Cannabis sativa and Cannabis indica that contain many psychoactive compounds: cannabinoids. The primary psychoactive ingredient responsible for most of the intoxicating effects is delta-9tetrahydrocannabinol (THC). Different

FIGURE 4.3 Cannabis [Marijuana] (Cannabis sativa and Cannabis indica).

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Not all effects of cannabis intoxication are welcomed by users as some experience unpleasant psychological reactions such as panic, fear, or depression. Acute intoxication also affects the heart and vascular system, resulting in cannabis-induced tachycardia and postural hypotension. CNS and respiratory depression have been noted in animal models given very high doses of THC. Studies show that inhaled doses of 2–3 mg of THC and ingested doses of 5–20 mg THC can cause impairment of attention, executive functioning, and short-term memory. Doses >7.5 mg/m2 inhaled in adults and oral doses from 5 to 300 mg in pediatrics can produce more severe symptoms such as hypotension, panic, anxiety, myoclonic jerking/hyperkinesis, delirium, respiratory depression, and ataxia (Saad et al., 2006). Chronic use may lead to long-term effects on cognitive performance. Cannabis intoxication can lead to acute psychosis in many individuals and can produce short-term exacerbations of preexisting psychotic diseases such as schizophrenia (Joy et al., 1999). The endocannabinoid system is complex. Research continues to elucidate an understanding of its function. Cannabinoids, whether endogenous or exogenous, act on specific cannabinoid binding receptors (CBs), cannabinoid binding receptor 1 (CB1) and cannabinoid binding receptor 2 (CB2). CB1 is primarily central in the CNS but is also present in the periphery, e.g., viscera, while the opposite is true for CB2, which is primarily peripherally located but also found in the CNS. CB1 receptors are mainly involved in the pharmacodynamic effects of cannabinoids, which include consequences on learning, memory, cognition, emotion, movement, sensory perception, and nausea, as well as the psychoactive properties associated with cannabinoids (Kelly and Nappe, 2021). Medical Marijuana is aimed more at the CB2 receptors; hence, the CB2 agonist cannabidiol (CBD) is used with minimal amounts of THC. CB2 receptors are thought to affect inflammation and immune system regulation. Cannabinoid receptors are G-protein linked receptors that inhibit adenylyl cyclase and thereby cyclic AMP, which affects calcium channels and potassium channels, leading to overall decreased intracellular calcium and extracellular potassium concentrations. This subsequently leads

to decreased neurotransmission. However, depending on the specific location of the CB and specific G-protein involved, stimulation of CB1 may result in the inhibition or stimulation of various neurotransmitters, including L-glutamate, g-aminobutyric acetylcholine, acid, dopamine, norepinephrine, and 5hydroxytryptamine. This neurotransmitter modulation may contribute to the central and peripheral effects observed in cannabinoid toxicity (Rousseaux, 2008a; 2008b). The absorption kinetics of cannabinoids depends on the exposure route, with inhalation reaching peak serum concentrations in less than 30 minutes (Tmax), and ingestion peaking in concentration (Cmax) at around 2–4 h (or longer) after consumption. Duration of toxicity secondary to inhalation and ingestion lasts approximately 2–6 h and 8–12 h, respectively. THC’s volume of distribution (Vd) is approximately 3 L/kg and collects in fat due to its high lipid solubility. Chronic exposures lead to increased accumulation in fat (Kelly and Nappe, 2021) and ongoing intoxication. Toxic effects can be separated into acute and chronic effects. Acute toxicity studies show that Marijuana virtually nontoxic; however, chronic toxicity causes lesions in the respiratory, hepatic, immune and endocrine systems. Unfortunately, many of these studies are difficult to interpret due to confounding variables and knowledge of the “dose” given, i.e., THC, extract, CBD, etc. As previously mentioned, any adverse effect occurs at very high concentrations of the cannabinoids (Beaulieu, 2005).

10.3. Chamomile – Chamomilla recutita The dried flowers of Chamomile (Chamomilla recutita) and Roman Chamomile (Chamaemelum nobile) contain many terpenoids and flavonoids contributing to its medicinal properties. Chamomile preparations are commonly used for many human ailments such as hay fever, inflammation, muscle spasms, menstrual disorders, insomnia, ulcers, wounds, gastrointestinal disorders, rheumatic pain, and hemorrhoids. Essential oils of Chamomile are used extensively in cosmetics and aromatherapy. Many different preparations of Chamomile have been developed, the most popular of which is in the form of herbal tea consumed at more than one million

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cups per day (Singh et al., 2011; Srivastava et al., 2010) (Figure 4.4). Chamomile oils are generally nonirritant to human skin and animals, except for rabbits that show moderate irritation. The irritation caused by inhalation of Chamomile dust has been an occupational health matter in the tea factory workplace (Dutkiewicz et al., 2001). Severe allergic reactions have been seen in a few individuals following consumption of Chamomile tea. Skin reactions have occurred after contact with the Chamomile plants or Chamomile-containing ointment, and conjunctivitis developed in hay fever in patients following eye washing with Chamomile tea (Wang et al., 2005). Median lethal oral dose lethality (LD50) in rabbits exceeded 5.0 g/kg (Opdyke 1974). Acute oral LD50 values of 8,560 mg/kg and 10,000 mg/ kg in rats (Pauli 2008) were reported. The lowest dose would classify the Chamomile acute experiment as practically non-toxic based on the LD50 results.

FIGURE 4.4

Chamomile (Chamomilla recutita).

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Toxicity testing revealed no significant findings when a lyophilized infusion of Chamomilla recutita (maricaria) flowers was given to SwissNOS mice as a single oral dose of 720 and 1440 mg/kg (Srivastava et al., 2010), and rats fed doses of flower extract up to 4.0 g/kg were similarly unaffected (Maliakil and Wanwimolruk 2001; Shivananda et al., 2007). Chamomile is not genotoxic, but rather it has a strong antimutagenic effect (HernandezCeruelos et al., 2002). No developmental or reproductive studies were found.

10.4. Coffee – Coffea arabica and C. Canephora Coffee originated in the Ethiopian highlands, where the legend is that 9th century goat herder, Kaldi, noticed how “spirited” his goats became after eating berries from a certain tree, and so informed the local monastery as to his problem with the goats. After hearing this news, experimentation by a monk created a brew from the berries that the goats had eaten. Coffee became popular at the monastery as the monks were able stay up later praying. Eventually news of this new brew spread into Egypt and into the Arabian Peninsula, where Coffee traveled east and west, finally landing in Southeast Asia and the Americas (Fredholm, 2011) (Figure 4.5). The use of Coffee became more widespread in the 15th and 16th centuries, and in Europe in the 18th and 19th centuries. Coffee was mostly an upper-class drink in Arabia and remained a relative luxury in Europe until quite recently. The use of other methylxanthine-containing beverages, such as Mate´, is less well known. Coffee dust in the occupational setting may cause severe lower airway disease (Thomas et al., 1991), ranging from exacerbation of preexisting asthma (Osterman et al., 1982) to bronchiolitis obliterans (Bailey et al., 2015; Centers for Disease Control, 2013). Initially, Coffee was used as an herbal remedy indicating that its pharmacological actions have long been noted (Fredholm, 2011). Now it is used as a drink that has become the leading worldwide beverage after water (Butt and Sultan, 2011). For adults consuming moderate amounts of Coffee (three to four cups/day providing 300–400 mg/d of caffeine) (Higdon

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FIGURE 4.5 Coffee (Coffea arabica).

and Frei, 2006), there is little evidence of health risks and some evidence of health benefits (Nieber, 2017). Generally, three to five cups a day maximizes the health benefits (Rodrı´guez-Artalejo and Lo´pez-Garcı´a, 2018). Pharmacokinetics Caffeine is rapidly and nearly completely (up to 90%) absorbed by the stomach with peak Tmax of 20–40 min. Its metabolism, clearance, and pharmacokinetics are affected by many factors such as age, sex and hormones, liver disease, obesity, smoking, and diet (Nehlig, 2018; Romualdo et al., 2019). As a result, toxic levels can be reached quickly and last for prolonged periods of time secondary to caffeine’s 3- to 10-hour half-life. The liver metabolizes caffeine via N-demethylation, acetylation, and oxidation. The half-life of caffeine can be prolonged (up to 72%) by other substances that use these same pathways, such as alcohol or medications (Murray and Traylor, 2021). The pharmacological agents in Coffee include caffeine, hydroxyhydroquinone, acrylamide chlorogenic acids, phenolic compounds, polyphenols, acrylamide, quinic acids and quercetin,

and diterpenes (Inoue and Tsugane, 2019); polycyclic aromatic hydrocarbons have been found in some ground Coffee brands (Grover et al., 2013). The diterpene, at multiple times the human dose, will cause hyperlipidemia in rats (Terpstra et al., 2000). Quercetin is sold as an herbal remedy to promote heath. The pharmacokinetics of Coffee show that Coffee is consumption significantly affects the absorption, distribution, metabolism, and excretion of many drugs (Belayneh and Molla, 2020). As the polycyclic aromatic hydrocarboninducible cytochrome P450 (CYP) 1A2 participates in the metabolism of caffeine, an herb– drug interaction can be seen with a number of drugs, including certain selective serotonin reuptake inhibitors (particularly fluvoxamine), antiarrhythmics (mexiletine), antipsychotics (clozapine), psoralens, idrocilamide and phenylpropanolamine, bronchodilators (furafylline and theophylline), and quinolones (enoxacin). Thus, pharmacokinetic interactions with Coffee at the CYP1A2 enzyme may cause toxic drug concentrations due to impaired Phase I metabolism (Carrillo and Benitez, 2000). Most work done on the pharmacokinetics of Coffee is limited to caffeine (Nawrot et al., 2003). The pharmacokinetics of caffeine are highly variable among individuals due to a polymorphism at the level of the CYP1A2 isoform of cytochrome P450, which metabolizes 95% of the caffeine ingested, and a polymorphism of the enzyme, N-acetyltransferase 2 (Nehlig, 2018). Six loci are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR, and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine (Cornelis, 2019). The three major components, Coffee diterpenes cafestol and kahweol, caffeine, and chlorogenic acid contribute to the beneficial effects of Coffee by reducing reactive oxygen species (ROS) (Wierzejska, 2015). A number of pharmacological activities are attributed to these components that include antioxidant, antiinflammatory, immunomodulatory, antimicrobial, anticancer, cardioprotective, and neuroprotective effects (Islam et al., 2018). Coffee phytochemicals regulate DNA repair, phase II enzymatic activity, apoptosis, and have antiproliferative, antiangiogenetic and antimetastatic effects (Bøhn et al., 2014).

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These components induce phase II detoxifying and antioxidant enzymes and inhibit the expression or decrease the activity of phase I activating enzymes thus preventing elevated concentrations of carcinogens that require bioactivation. They target different stages of a common pathway, Kelch-like ECH-associated protein 1 (Keap1)dNF-E2-related factor-2 (Nrf2)d antioxidant-responsive-element (ARE) signal pathway and alter the ARE-dependent expression of genes needed in the antitumorigenic effects (Tao et al., 2008). The microbiome affects the bioavailability of polyphenols (Nishitsuki et al., 2018). Polyphenols are biotransformed into their metabolites by gut microbiota which modulates the gut microbiota composition. Although there are studies on the in vivo bioavailability of polyphenols, the mutual relationship between polyphenols and gut microbiota is not fully understood (Ozdal et al., 2016). Human Health Coffee and its impact on health continue to be the topic of much heated debate. Until recently, Coffee consumption has been believed to be associated with adverse effects, mainly cardiovascular problems. However, the vast majority of contemporary sources not only emphasize a lack of detrimental effect, but also suggest a beneficial effect of Coffee intake (Wierzejska 2015). Regardless, certain atopic individuals can develop allergies to Coffee bean allergens (Lehrer et al., 1978). Numerous systematic reviews, with or without meta-analysis, are available regarding the effects of Coffee on various disease entities, e.g., fertility (Ricci et al., 2017). In summary, they point to the conclusion that consumption of Coffee in amounts up to 2000 mg/d for 12 weeks is well tolerated in healthy men and women (Ottaviani et al., 2015). Epidemiological studies with prospective cohorts showed that Coffee intake is associated with reduced all-cause mortality independently of caffeine content (Je and Giovannucci, 2014; Li et al., 2019a) and higher Coffee intake might be modestly associated with reduced adiposity, particularly in men (Lee et al., 2019b). Indeed, these studies have shown that Coffee is not associated with overall cancer risk (Alicandro et al., 2017) but is addictive (Griffiths et al., 1986).

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Cohort and case–control studies showed an inverse association between Coffee consumption and the degree of liver fibrosis as well as the development of liver cancer (Alferink et al., 2018; Alicandro et al., 2017; Arauz et al., 2013; Bai et al., 2016; Bakhiya and Appel, 2010; Bøhn et al., 2014; Cornelis, 2019; de Melo Pereira et al., 2020; Furtado et al., 2012; 2014; Hasegawa et al., 1985; Higdon and Frei, 2006; Inoue and Tsugane, 2019; Kalthoff et al., 2017; Kennedy et al., 2016; Kolb et al., 2020; Marventano et al., 2016; O’Keefe et al., 2018; Pool et al., 2017; Salomone et al., 2017; Stalder et al., 1990; Tao et al., 2008; Watanabe et al., 2017; Zhao et al., 2020). In experimental models of fibrosis, caffeine was shown to inhibit hepatic stellate cell activation by blocking adenosine receptors, and emerging evidence indicates that caffeine may also favorably impact angiogenesis and hepatic hemodynamics (Bøhn et al., 2014). However, chlorogenic acids, potent phenolic antioxidants, suppress liver fibrogenesis and carcinogenesis by reducing oxidative stress and counteract steatogenesis through the modulation of glucose and lipid homeostasis in the liver (Salomone et al., 2017); it all depends on the dose. Habitual Coffee consumption is also associated with lower risks for cardiovascular (CV) mortality (Poole et al., 2017; Ribeiro et al., 2020; Whayne, 2015) and a variety of adverse CV outcomes (Nieber, 2017; Whayne, 2015), including coronary heart disease (CHD) (Ding et al., 2014a), congestive heart failure (HF) (Cano-Marquina et al., 2013; Mostofsky et al., 2012), controlled hypertension (Mesas et al., 2011; Nurminen et al., 1999), postcardiac myocardial infarction (Ribero et al., 2020) and stroke (Higashi, 2014; Shao et al., 2020) but Coffee’s effects on arrhythmias (Myers, 1991) and hypertension are neutral (Mesas et al., 2011). Coffee consumption is associated with improvements in some CV risk factors, as well as type 2 diabetes (Carlstro¨m and Larsson 2018; Ding et al., 2014b; Jiang et al., 2014; Tajik et al., 2017; van Dam 2006), depression (Tenore et al., 2015), suicide risk (Lucas et al., 2014) obesity (Santos and Lima, 2016) and urolithiasis (Wang et al., 2014). It does not have an effect on the frequency or severity of cardiac arrythmias (Myers, 1991). Excessive doses of caffeine, e.g., combination of Coffee and energy drinks (two energy drinks

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provides w400 mg/day) may result in adverse cardiovascular events (Beyer and Hixon, 2018). It is interesting to note that there are J-curve-type or U-curve-type dose–responses, as previously discussed, for Coffee consumption associated with cardiovascular events including myocardial infarction and stroke (Higashi, 2014), which is supported by evidence from a meta-analysis suggesting that Coffee consumption may be associated with an elevated risk for dyslipidemia and cardiovascular disease at high concentrations (greater than four cups/day) (Du et al., 2020). The human equivalent dose (HED) for the no observed adverse effect level (NOAEL) for cardiovascular effects is 260 mg caffeine (two to three cups of Coffee) for a single dose of caffeine for a 70-kg adult, while the lowest observed adverse effect level (LOAEL) is 770 mg (seven to eight cups of Coffee) for a 70-kg adult (Beyer and Hixon, 2018). There are studies that show negative cardiovascular effects (Higdon and Frei, 2006); however, the doses at which this happened were greater than six cups per day (Beyer and Hixon, 2018). Caffeine is a weak bronchodilator as it is chemically related to the drug theophylline, a drug that is used to treat asthma. It has been suggested that caffeine may reduce asthma symptoms and interest has been expressed in its potential role as an asthma treatment, especially since it reduces respiratory muscle fatigue; however, caffeine appears to improve airway function only modestly, for up to 4 hours, in people with asthma (Welsh et al., 2010). There are superior drugs for the treatment of asthma. Chronic Coffee consumption also appears to protect against some neurodegenerative diseases (Larsson and Orsini, 2018). There is decreased risk of dementia (Panza et al., 2015), in particular, Alzheimer’s disease (Butt and Sultan, 2011; Larrson and Orsini, 2018), and Parkinson’s disease (Costa et al., 2010; Grosso et al., 2017) associated with Coffee consumption. Coffee’s neuroprotective characteristics probably lie in its ability to reduce oxidative stress induced by reactive oxygen species (ROS) and the activation of mitogen-activated protein kinase (MAPKs) (Cho et al., 2009). A promising hypothesis is that caffeine controls the microglia-mediated neuroinflammatory response associated with the majority of

neurodegenerative conditions and is due to a predominant antiinflammatory action of Coffee but not of caffeine consumption (Paiva et al., 2019). Accordingly, Coffee modulates adenosine receptors, namely the A2A receptor, which affords neuroprotection through the control of microglia reactivity and neuroinflammation (Madeira et al., 2017). Secondly, caffeine interacts with the dopaminergic system and therefore the glutamatergic neurons. Coffee has been found to suppress the inhibitory (GABAergic) activity and modulate GABA receptors thereby having a beneficial effect on cognition and mood while protecting dopaminergic neurons (Alasmari, 2020); it is claimed to be highly neuroprotective (Aronowski et al., 2003). Coffee may reduce the risk of adult glioma (Holick et al., 2010) and neuropsychic symptoms (Kromhout et al., 2019). Quercetin appears to be the major neuroprotective component in Coffee’s activity against Parkinson’s and Alzheimer’s disease (Colombo and Papetti, 2020; Lee et al., 2016). According to the current state of knowledge, Coffee consumption is not associated with the majority of cancers although the results from some studies conflict, probably because of dose and confounding factors, e.g., smoking (La C et al., 1989). In case of ovarian (SalarMoghaddam et al., 2019), colorectal, liver, melanoma (Yew et al., 2016), and breast cancers, Coffee drinking may have a protective effect. Coffee contains numerous compounds with beneficial effects, e.g., polyphenols (antioxidants), as well as harmful effects, e.g., acrylamide (possibly carcinogenic) (Wierzejska 2015). However, Coffee is not associated with the overall cancer risk (Alicandro et al., 2017) in the liver and skin, where it is protective against basal cell carcinomas (Vaseghi et al., 2018) and others. For example, consumption of caffeinated Coffee and, to a lesser extent, decaffeinated Coffee is associated with reduced risk of hepatocellular carcinomas (Arab, 2010), even in those with preexisting liver disease (Kennedy et al., 2017). In fact, the effect of Coffee on hepatocellular and endometrial cancers is the strongest and most consistent protective association that has been documented (Arab, 2010; Bae et al., 2018). Endometrial cancer risk is also decreased in those consuming Coffee (Alicandro et al., 2017;

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Arab, 2010; Grosso et al., 2017; Je et al., 2011; Zhao et al., 2020; Zhou et al., 2015). A probable decreased risk was found in some studies for oral/pharyngeal cancer (Chen and Long, 2014); and for advanced prostate cancer (Akter et al., 2016; Cao et al., 2014; Grosso et al., 2017; Park et al., 2010; Vance et al., 2013), but these studies used doses above those known to be protective. Nonetheless, Coffee contains constituent(s) that are weakly estrogenic (Kitts, 1987). Coffee drinking is associated with a preventive effect on colorectal cancer (Bu1dak et al., 2018; Hu et al., 2017; Mori et al., 2000 Wierzejska, 2015), probable decrease (Bøhn et al., 2014; Grosso et al., 2017) or equivocal (Akter et al., 2016; Micek et al., 2018; Sartini et al., 2019; Tavani and Vecchia 2004) depending on the dose. Coffee has a positive effect on bowel movement in postoperative ileus (Dulskas et al., 2015; Eamudomkarn et al., 2018), without any effect on fluid balance (Maughan and Griffin, 2003). For bladder cancer, the results are not consistent; however, any possible direct association that was not dose- and duration-related might depend on a residual confounding effect of smoking and other factors (Morrison et al., 1982). Bladder cancer risk was elevated (Lina et al., 1993; Villaneva et al., 2009), equivalent (Alicandro et al., 2017; Tavani and Vecchia, 2000; Wierzejska, 2015), or decreased (Morrison et al., 1982) and there was no association made with chronic renal disease (Wijarnpreecha et al., 2016). There is no association of renal carcinoma with Coffee consumption (Alicandro et al., 2017; Arab, 2010), but there is a modest increased bladder cancer risk among Coffee drinkers (Morrison et al., 1982; Villanueva et al., 2009). Although the results of studies are mixed, the overall evidence suggests an uncertain association of Coffee intake with cancers of the stomach (Larrson et al., 2006) [increased risk {more than six cup per day} (Zeng et al., 2015) and reduced risk (Xie et al., 2016a)], pancreas (Heuch et al., 1983; MacMahon et al., 1981) [increased risk (Li et al., 2019a,b) and reduced risk (Stalder et al., 1990)], lung (Wierzejska, 2015) [increased risk (Xie et al., 2016b)], breast (Alicandro et al., 2017; Gierach et al., 2012) [increased risk [menopausal women (Jiang et al., 2013) and reduced risk (Grosso et al., 2017; Lubin et al., 1985)],

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ovary (Alicandro et al., 2017) and prostate (Arab, 2010; Park et al., 2010) [increased risk (not found) and reduced risk (Alicandro et al., 2017; Bøhn et al., 2014; Cao et al., 2014; Grosso et al., 2017)] overall. A few studies suggest an increased risk of childhood leukemia after maternal Coffee drinking during pregnancy, but data are limited and inconsistent (Alicandro et al., 2017) [increased risk (Arab, 2010) and reduced risk (not found)]. These results probably reflect the dose of caffeine and the concentrations of diterpenes cafestol and kahweol, caffeine, and chlorogenic acid. In a systematic review (Poole et al., 2017), Coffee consumption was more often associated with benefit than harm for a range of health outcomes across exposures including high versus low, any versus none, and one extra cup a day. There was evidence of a nonlinear association between consumption and some outcomes, with summary estimates indicating the largest relative risk reduction at intakes of three to four cups a day versus none, including all-cause mortality (relative risk 0.83 (95% confidence interval 0.79 to 0.88)), cardiovascular mortality (0.81, 0.72 to 0.90), and cardiovascular disease (0.85, 0.80 to 0.90). High versus low consumption was associated with an 18% lower risk of incident cancer (0.82, 0.74 to 0.89). Consumption was also associated with a lower risk of several specific cancers and neurological, metabolic, and liver conditions. Harmful associations were largely nullified by adequate adjustment for smoking, except in pregnancy, where high versus low/no consumption was associated with low birth weight (odds ratio 1.31, 95% confidence interval 1.03 to 1.67), preterm birth in the first (1.22, 1.00 to 1.49) and second (1.12, 1.02 to 1.22) trimester, and pregnancy loss (1.46, 1.06 to 1.99). There was also an association between Coffee drinking and risk of fracture in women but not in men. In another study it was found that the effect on male bones was reversed with Coffee when the authors concluded that Coffee was associated with a decreased risk of fracture (Lee et al., 2014). On a more humorous note, following a systematic review, it is not recommended to self-administer a Coffee enema as a complementary and alternative medicine modality and means of self-care, given the unsolved issues on its safety and insufficient evidence regarding the effectiveness (Son et al., 2020).

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Animal Studies Many of the laboratory prospective studies used pure caffeine and as such the results are only half of the story since polyphenols and other active compounds in Coffee were omitted. We will only discuss the studies that used either Coffee, or a Coffee extract. The acute toxicity of green Coffee oil enriched with cafestol and kahweol in rats gave an LD50 of greater than 2,000 mg/kg (de Oliviera et al., 2020) which, when compared to caffeine with an LD50 of 367 mg/kg dosed orally (Adamson, 2016), indicates that Coffee is less lethal than caffeine alone (Heimbach et al., 2010). This fact may influence the acceptance of caffeine laced energy drinks. Generally, the studies indicate that Coffee and Coffee extracts are slightly toxic, whereas caffeine can be considered moderately toxic. This assertion is supported by a 14-day rat study that did not show any effects up to 1000 mg/kg (Venkatakrishna et al., 2021). A Coffee extract significantly suppressed lipopolysaccharide (LPS)-induced hepatitis in D-galactosaminesensitized rats, as assessed by the plasma alanine and aspartate aminotransferase activities, when it was added to the diet (30 g/kg) and fed to rats for 14 days, whereas a decaffeinated Coffee extract had no significant effect. The hepatoprotective effect of caffeine was stronger than that of theobromine. These results indicate that Coffee can protect animals from LPSinduced hepatitis, and that the effect of Coffee might be mainly due to caffeine (He et al., 2001). In vitro studies revealed that chlorogenic acid protects PC12 neuronal hydrogen peroxideinduced cell death (apoptotic) by blocking the accumulation of intracellular ROS and the activation of MAPKs (Cho et al., 2009) and Coffee modifies sulfo-conjugation reactions within the human colon carcinoma cell line – Caco-2 (Okamura et al., 2005). Coffee also suppresses most of the changes that follow thioacetamide administration, a model for cirrhosis – a finding that indicates the beneficial effect of Coffee on the liver (Arauz et al., 2013). Even the borderline effect in the Ames assay was removed with metabolic activation (þS9) (Aeschbacher and Wu¨rzner 1980). Coffee extracts demonstrated improved redox status in myoblast and endothelial cells treated

in vitro where glutathione levels increased as an indicator that quenching molecules for ROS elements were preserved (Priftis et al., 2018). These findings may explain the neuro and other protective effects of Coffee (Miyazaki et al., 2019). From the above, Coffee is not genotoxic or mutagenic (Cavin et al., 2001; Majer et al., 2005; Nehlig and Derby 1994; Graf and Wu¨rgler, 1986; Nagpal et al., 2019) and has been shown to inhibit nitrosourea-induced DNA damage (Aeschbacher and Jaccaud, 1990), although this does not necessarily mean that it influences initiation and promotion (DMBA-induced mammary tumors in rats) in neoplasia (Welsch et al., 1988). For this reason, there were no increases in the numbers of malignant neoplasms in mice that had been fed a diet of up to 5% instant Coffee for 2 years (Wu¨rzner et al., 1997). There was no difference between caffeinated and decaffeinated Coffee with respect to genotoxicity and mutagenicity (Abraham and Singh, 1999). In a transplacental micronucleus test, Coffee effectively inhibits the genotoxic effects of cyclophosphamide, N-nitrosodiethylamine, N-nitroso-N-ethylurea, and mitomycin C (Abraham, 1995; 1989). Furthermore, it demonstrated significant protective effects against methyl-N-nitro-nitroguanidine-induced DNA damage in the comet assay, mutation at the Tk locus and chromosomal damage in the cytokinesis-block micronucleus test (Abraham and Stopper, 2004). In support, standard instant Coffee inhibited in vivo genotoxicity of 7,12dimethylbenz[a]anthracene, benzo[a]pyrene, aflatoxin B1, and urethane (Abraham, 1991), an effect that is postulated to be mediated via glutathione S-transferase activity to quench radical oxygen species (Schilter et al., 1996). Coffee roasting clearly affects the chemical diversity of the brew and gives the drink a different flavor depending on the roasting (Schouten et al., 2021). Fresh brewed Coffee given to rats for 2 years with up to 100% Coffee as drinking fluid induced significant mortality at the highest dose. No other significant doseresponse findings were seen (Palm et al., 1984). Reproduction and fetal development are only mildly affected at doses of 6000 mg/kg and above when given during gestation (Ajarem and Ahmad, 1996). In summary, Coffee is not genotoxic or mutagenic and actually is genoprotective in animals.

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Its toxicity lies in the caffeine component of the chemical mixture (Wendler et al., 2009). Mild reproductive effects occurred at 6000 mg/kg, which is w1050 times the dose advised to achieve the maximum benefit of Coffee exposure (400 mg/day for a 70 kg person – 5.7 mg/kg/ day), or approximately 250 cups of Coffee per day (Brent et al., 2011).

10.5. Cocoa – Theobroma cacao Chocolate’s 4000-year history began in ancient Mesoamerica, present day Mexico, where the Olmec were the first to turn the cacao plant into chocolate (Figure 4.6). They drank their chocolate during rituals and used it as medicine. Centuries later, the Mayans praised chocolate as the drink of the gods. Mayan chocolate was a revered brew made of roasted and ground cacao seeds mixed with chillies, water, and cornmeal. Mayans poured this mixture from one pot

FIGURE 4.6

Cocoa (Theobroma cacao).

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to another, creating a thick foamy beverage called “xocolatl,” meaning “bitter water.” By the 15th century, the Aztecs used Cocoa beans as currency. They believed that chocolate was a gift from the god Quetzalcoatl, and drank it as a refreshing beverage, an aphrodisiac, and even as preparation for war. No one knows for sure when chocolate came to Spain. Legend has it that explorer Herna´n Corte´s brought chocolate to his homeland in 1528, as Corte´s was believed to have discovered chocolate during an expedition to the Americas. In search of gold and riches, he instead found a cup of Cocoa given to him by the Aztec emperor. He introduced Cocoa seeds to the Spanish. Though still served as a drink, Spanish chocolate was mixed with sugar and honey to sweeten the naturally bitter taste. Chocolate quickly became popular among the rich and wealthy. Even Catholic monks loved chocolate and drank it to aid religious practices, and such it was called “Food of the Gods” (Rusconi and Conti, 2010). It was nearly a century before chocolate reached neighboring France, and then the rest of Europe. Following France’s lead, chocolate soon appeared in Britain at special “chocolate houses.” As the trend spread through Europe, many nations set up their own cacao plantations in countries along the equator. In 1828, the invention of the chocolate press revolutionized chocolate making. This device could squeeze Cocoa butter from roasted cacao beans, leaving a fine Cocoa powder behind. The powder was then mixed with liquids and poured into a mold, where it solidified into an edible bar of chocolate. Cocoa contains fat (40%–50% as Cocoa butter, with approximately 33% oleic acid, 25% palmitic acid, and 33% stearic acid). Polyphenols constitute about 10% of a whole bean’s dry weight, containing more phenolic antioxidants than most foods. Three groups of polyphenols are contained in Cocoa beans: catechins (37%), anthocyanidins (4%), and proanthocyanidins (58%). However, the bitterness caused by these polyphenols makes unprocessed Cocoa beans rather unpalatable (Magrone et al., 2017). Manufacturers have developed processing techniques for eliminating the bitterness. Such processes decrease the polyphenol content by up to 10-fold and add sugar and emulsifiers

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such as soy lecithin. Polyphenols are associated with beneficial effects; therefore, Cocoa (rich in polyphenols) and dark chocolate, with a high percentage of Cocoa and higher phenolic antioxidant compounds compared to the other chocolate varieties, has more of a benefit to health than does milk chocolate. The nitrogenous compounds of Cocoa include both proteins and methylxanthines (theobromine and caffeine). Cocoa is also rich in minerals: potassium, phosphorus, copper, iron, zinc, and magnesium (Montagna et al., 2019). Cocoa is especially abundant in flavanols, accounting for around 60% in nonfermented Cocoa beans. These flavanols comprise monomeric forms, (þ)-catechin and ()-epicatechin, which protects against myocardial ischemia (Li et al., 2018), and their oligomeric and polymeric forms, procyanidins. The types of Cocoa flavanols entail () – epicatechin, being the most abundant, (þ) catechin, procyanidin B1 and B2, and other flavanols at trace level, such as epigallocatechin, epigallocatechin-3-gallate, procyanidin B2–O-gallate, procyanidin B2-3,3-di-O-gallate, procyanidin B3, procyanidin B4, procyanidin B4–O-gallate, procyanidin C1, and procyanidin D. Other minor polyphenolic compounds include flavones (luteolin, luteolin-7-O-glucoside, orientin, isoorientin, apigenin, vitexin, and isovitexin), flavanones (naringenin, prunin, hesperidin, and eriodictyol), flavonols (quercetin, quercetin-3-O-arabinoside, isoquercitrin, and hyperoside), anthocyanidins (cyanidin, 3-a-l-arabinosidyl cyanidin, 3-b-darabinosidyl cyanidin, 3-b-D-galactosidyl cyanidin), and certain phenolic acids (Escobar et al., 2020; Sorrenti et al., 2020). Monomeric and polymeric flavanols are rapidly absorbed in the small intestine upon ingestion with a maximal plasma concentration (Cmax) after 2 h from intake (Actis-Goretta et al., 2013). Absorption not only depends on flavanol chemistry but also on their structural isomerism, stereoisomerism and the range of polymerization seems to determine their bioavailability (Bravo, 1998). Once absorbed as monomers, flavanols are transformed into metabolites detectable in plasma and urine, such as ()  epi as sulfate, glucuronides, or methyl conjugated forms (Ottaviani et al., 2012). Polymers and monomers of unabsorbed flavanols undergo colonic microbiota catabolism, where valero lactones and valeric acids

represent the first step of microbiota-derived catabolites (Monagas et al., 2010); a number of phenolic acids constitute intermediate and laststep catabolites (Monagas et al., 2010). These compounds also affect the microbiome reducing the percent of Bacteroides, Clostridium, and Staphylococcus and induce a significant increase of Lactobacillus species in feces and Bifidobacterium species in porcine proximal colon contents (Jang et al., 2016). A part of unabsorbed flavanols is excreted into the feces (Gu et al., 2011). Elimination of flavanols is completed after 6 h from ingestion giving an approximate half-life (t1/2) of 1.3 h (Holt et al., 2002). Cocoa is not genotoxic, mutagenic, carcinogenic, or inflammatory. In fact, Cocoa has antigenotoxic or geno-protective abilities (Leyva-Soto et al., 2018). Cocoa inhibits the growth of cells in vitro in breast, liver, colon, lung, and cervical cancer cell lines (Carnesecchi et al., 2002) and others in vivo. Cocoa polyphenols are known to possess anticarcinogenic properties, mainly because of their potential to reduce excessive oxidative stress that can damage DNA leading to mutation (Bravo, 1998). Flavanols and procyanidins from Cocoa are implicated in the regulation of different cancer-related signal transduction pathways relating to mutagenesis, tumorigenesis, angiogenesis, or metastasis, among others (Rojo-Poveda et al., 2020). Cocoa procyanidins also reduce vascular endothelial growth factor activity and angiogenic activity associated with tumors, thus impeding their growth (Magrone et al., 2017). There is growing evidence that polyphenols may play a role in regulating apoptosis. Apoptosis may be triggered intrinsically, through the mitochondrial pathway, or extrinsically by death ligands and receptors see Morphologic Manifestations of Toxic Cell Injury, Vol 1, Chap 6. It is the external pathway that may potentially be modulated by Cocoa. Flavanols found in Cocoa have also exhibited proapoptotic effects. For example, proanthocyanadins inhibit growth of human lung cancer cells in vitro and in vivo and epicatechin synergistically enhances apoptosis in lung cancer cells treated with epigallocatechin-3-gallate. Phenol-rich Cocoa extracts prevent apoptosis in MLP29 liver cells induced by Celecoxib, suggesting that upstream components of the apoptotic pathway (such as the Bax gene) are targets of Cocoa

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phytochemicals (Katz et al., 2011). Some authors demonstrated that Cocoa liquor procyanidins significantly reduced the incidence and multiplicity of lung carcinomas and decreased thyroid adenomas induced in male rats and inhibited mammary and pancreatic tumorigenesis in female rats (Rojo-Poveda et al., 2020). Regular intake of Cocoa-containing foods and beverages results in a number of beneficial effects, in rats, on the cardiovascular system including effects on blood pressure, insulin resistance, vascular and platelet function (Corti et al., 2009), lipid profile, and atherosclerosis (Guan et al., 2016) as well as downregulation of the systemic immune response (Pe´rez-Berezo et al., 2011). Polyphenols activate endothelial nitric oxide synthase leading to generation of nitric oxide (NO), which lowers blood pressure by promoting vasodilation (Fisher et al., 2003). Once released, NO also activates the prostacyclin synthesis pathway, which further causes vasodilation, thereby contributing to thrombosis protection. Furthermore, the antiinflammatory and vasoprotective properties of prostacyclin are enhanced by its ability to reduce plasma leukotrienes (Schwab et al., 1996). Practically, Cocoa ingestion effectively increases flow-mediated vasodilatation and reduces systolic and diastolic blood pressure, thus lowering the risk of myocardial infarction, ischemic heart disease, and stroke (Greenberg et al., 2018). Incorporating almonds, dark chocolate, and Cocoa into a diet without exceeding energy needs could reduce the risk of coronary heart disease. The preventive or therapeutic effects of Cocoa and Cocoa constituents against obesity and metabolic syndrome show promise (Gu et al., 2014a). Flavonoids can produce metabolic events that induce reduction of lipogenesis, induce lipolysis, and increase adiponectin secretion, thereby reducing lipid deposition and insulin resistance with subsequent weight loss (Tokede et al., 2011; Gu et al., 2014a). Dark chocolate has a positive effect on lipid profiles by significantly lowering total and LDL cholesterol levels, without effect on highdensity lipoprotein HDL and triglycerides (Onopa et al., 1999). It is unlikely that milk chocolate will be as effective. Cocoa components offer potential as antidiabetic agents, especially with type 2 diabetes mellitus. In vitro, Cocoa Shell aqueous phenolic extract preserves mitochondrial function and insulin sensitivity by attenuating inflammation

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between macrophages and adipocytes (RebolloHernamz et al., 2019). Cocoa flavonols also improve glucose homeostasis by slowing carbohydrate digestion and absorption in the gut (Martins et al., 2016) and dose-dependently inhibit pancreatic a-amylase, pancreatic lipase, and secreted phospholipase A2 (Gu et al., 2014b). They improve insulin sensitivity by regulating glucose transport and aid insulin signaling proteins in insulin-sensitive tissues (liver, adipose tissue, and skeletal muscle) preventing oxidative and inflammatory damage associated with the disease (Martı´n et al., 2016), an effect that is strongly dependent on the concentration of polyphenols in the Cocoa. Regular Cocoa consumption improves the immune imbalance induced by allergic processes by reducing release of mediators and restoring the balance of T-helper 1 and Thelper 2 cells with down-regulation of IgE production (Pe´rez-Berezo et al., 2011). In vivo and in vitro studies show that Cocoa regulates both arms of the immune system: innate and acquired immunity, probably via the activity of theobromine, a compound that is responsible for Cocoa’s toxicity (Wang et al., 1992; Wang and Waller, 1994). Dogs are particularly sensitive to this compound; hence, should not be given chocolate (Sutton 1981). Flavonoids, as with other herbal remedies, in Cocoa are neuroprotective. By increasing cerebral blood flow, angiogenesis and new nerve cell growth occurs in the hippocampus, which may provide insights to possible protective effects against dementia and stroke. Certainly, Cocoa reduces neural inflammation, the common factor to many neurodegenerative disorders such as Parkinson’s, Alzheimer’s diseases, and stroke (Katz et al., 2011). It probably partly acts via attenuating alpha-synuclein toxicity due to caffeine content of the mixture (Kardini and Roy, 2015). Cocoa extract protects against early alcoholinduced liver injury in the rat (McKim et al., 2002) and a number of studies have identified a role for Cocoa flavanols in protecting skin from UV light damage, believed to be mediated through factors such as generation of ROS and inflammatory prostaglandins, NO, leukotrienes, and histamine (Ramos-Romero et al., 2012). The antioxidant actions of Cocoa flavanols are one possible mechanism by which skin protection could be conferred in conjunction with providing increased nutrient delivery and better thermoregulation (Katz et al., 2011).

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Finally, continuous Cocoa powder consumption by rats at levels as high as 5.0% of the diet did not have any effect on reproductive capacity under the conditions of a standard threegeneration evaluation (Hostetler et al., 1990). Similarly, Cocoa powder given at 0, 2.5, 5.0, or 7.5% in the diet throughout gestation and lactation (postnatal day 21) showed no fetal or reproduction anomalies (Tarka et al., 1986). Female Sprague–Dawley rats receiving 0.0 (control), 1.5%, 3.5% and 5.0% Cocoa powder for 104 weeks did not show an effect on reproduction. No evidence of carcinogenicity from dietary Cocoa was found in either sex (Tarka et al., 1991).

10.6. Echinacea – Echinacea purpurea Echinacea is a genus of herbaceous flowering plants in the daisy family and has 10 species, which are commonly called coneflowers. They are found only in eastern and central North America, where they grow in moist to dry prairies and open wooded areas Echinacea purpurea is used in traditional medicine (Manayi et al., 2015) (Figure 4.7). Echinacea is most commonly used for treatment of the common cold. This was supported by the findings of a systematic review that concluded that there is some evidence of a possible benefit from Echinacea purpurea for treatment of the common cold, though the results are not consistent. A subsequent large, high-quality randomized controlled trial found no benefit of Echinacea angustifolia for the treatment of experimentally induced rhinovirus infection. The herb is believed to be safe, with prior studies showing rates of side effects similar in Echinacea and placebo groups (Brent, 2008). Acute human intoxication has not been reported and on the basis of the animal experimental data are not expected. Acute lethality, LD50 value was calculated as 2500 mg/kg following ip injection. LD50 values of oral and intravenous administration of the plant juice evaluated more than 30 g/kg and 10 g/kg in mice, and 15 g/kg and 5 g/kg in rats, respectively. The lowest dose would classify the Echinacea acute experiment as slightly toxic based on the LD50 results. Administration of expressed juice from Echinacea purpurea to rats and mice in doses of up to 8000 mg/kg/day showed no biologically significant findings after 4 weeks (Mengs et al.,

FIGURE 4.7 Echinacea (Echinacea purpurea).

1991); single intravenous doses gave similar results. Necropsy findings gave no evidence of toxic effects in mice. Echinacea is not genotoxic; nor is it a mutagen, carcinogen, or a developmental toxicant (Perri et al., 2006).

10.7. Ephedra – Ephedra sinica Ephedra is an herb (Ephedra sinica) – known as ma huang or Mormon tea. American Ephedra comes from Ephedra nevadensis. The branches and tops are most commonly used to make medicine, but the root or whole plant can also be used. Mormon tea lacks ephedrine, which gives Ephedra its effects and potentially serious side effects. Ephedra is banned in the United States due to safety concerns (Figure 4.8). Ephedra is used for weight loss and obesity and to enhance athletic performance. It is also used for allergies, hay fever, nasal congestion and respiratory tract conditions such as bronchospasm, asthma, and bronchitis. To complicate the issue, Ephedra is often used in conjunction with caffeine.

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FIGURE 4.8 Ephedra (Ephedra sinica).

Ephedrine produces indirect stimulation of adrenoreceptors due to its ability to release norepinephrine and epinephrine from adrenergic nerve terminals. Release after binding of ephedrine to a/b adrenergic membrane receptors leads to calcium release and changes in electrical and contraction properties of the heart. The combination of ephedrine and caffeine (sometimes used in energy drinks) has been linked to cardiotoxicity in rats and humans, including an increase in blood pressure, heart rate, with interstitial hemorrhage and myocardial degeneration in the subendocardial myocardium of the ventricles and interventricular septum. The combination of Ma Huang (ephedrine) with caffeine enhances the cardiotoxicity (Dunnick et al., 2007). Reported adverse reactions to Ephedra sinica have included insomnia, nervousness, tremor, headaches, hypertension, seizures, arrhythmias, heart attack, stroke, and death. In addition to its cardiovascular effects, the use of ephedrine causes insomnia, restlessness, and anxiety; on occasion, ephedrine may induce psychosis (Kalix

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1991). The most common adverse effects and toxicity observed in patients are liver problems, renal failure, and cardiac failure (Alsaeed et al., 2019). Some adverse effects following the use of Ephedra are reversible, whereas others are not. The mean lethal dose (LD50) of Ephedra alkaloids is 610 mg/kg, and the maximum tolerated dose (MTD) of oral Ephedra nonalkaloids in mice is 367.5-fold larger than the clinical dosage in humans (Fan et al., 2015). The lowest dose in acute experiments would classify Ephedra as slightly toxic based on the LD50 results. Acute studies on Ma Huang in mice and rats showed obvious cardiac changes where myofiber coagulative necrosis was seen mostly in the interventricular septum and left ventricle (Dunnick et al., 2007; National Toxicology Program, 1986; Nyska et al., 2005) (Figure 4.9). Nasal septal perforation and turbinate atrophy along with olfactory epithelial lesions of respiratory metaplasia, fibrosis, and nerve atrophy was also seen. In contrast, subchronic toxicity (13-week) studies in rats and mice and lifetime exposure (102 weeks) using an Ephedra Herbal aqueous extract did not show a dose–response in relation to cardiomyopathy. Additional histopathological changes include renal tubular basophilia and acinar hypertrophy of the salivary glands in the 13-week studies and additionally in the 103week studies bone marrow hyperplasia and increased myeloid/erythroid ratio, lymphoid hyperplasia, adrenal cortical hypertrophy, and pancreatic islet hyperplasia and extramedullary hemopoiesis in the high dose male mice (National Toxicology Program, 1986). Lifetime exposure studies failed to demonstrate any evidence of carcinogenicity in rats and mice (National Toxicology Program, 1986). Incidence of hepatocellular adenoma (single and multiple) or carcinoma with accompanying inflammation was significantly less than those in the vehicle control groups. An NOAEL of 125 mg/kg/day was determined (Han et al., 2018). Based on the pathology findings of these studies and the known properties of ephedrine and caffeine, it has been suggested that the cardiac toxicity is the result of catecholamine release after binding of ephedrine to a/b adrenergic membrane receptors, leading to calcium release and changes in electrical and contraction properties of the heart (Figure 4.10). These effects result in myocardial ischemia, necrosis, and

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FIGURE 4.9 Treatment-related cardiotoxic lesions. (A) Cardiomyopathy, minimal (25 mg/kg ephedrine and 7.25 mg/kg caffeine). This change is considered to be an incidental background change. Note (arrows) a single small focus of mononuclear cell (lymphocytes and histiocytes) infiltration, associated with variable myofiber degeneration, necrosis, and loss. H&E, X20. (B) Degeneration, minimal (12.5 mg/kg ephedrine and 15 mg/kg caffeine), 20. Note a focus of myocardial fibers with clear, vacuolated cytoplasm (arrows). H&E, X20. (C) Hemorrhage, minimal (25 mg/ kg). Note small foci in which free red blood cells filling the spaces between adjacent myocardial fibers (arrows). H&E, X20. (D) Necrosis, minimal (25 mg/kg ephedrine and 30 mg/kg caffeine). Note the presence of numerous minute clusters of deeply basophilic fragments of nuclear debris (arrows), mixed with some macrophages. H&E, 40. Reproduced with permission from Dunnick et al., Toxicol Pathol 35(5):657–664, 2007. H&E X40 [Page 661].

apoptosis, which either culminates in hemorrhage and sudden death, or resolves with inflammation and fibrosis.

10.8. Garlic – Allium sativum Garlic (Allium sativum) is used for many purported medicinal properties, but the most substantial body of research examines the effect

on cholesterol as it is an oxidant (Banerjee et al., 2003) (Figure 4.11). The most recent systematic review concluded that Garlic lowers cholesterol levels by 4–6%, which is a modest effect in comparison to the 17–32% reduction achieved with the use of statin drugs. Randomized, double-blind, placebo-controlled trials showed that 10.8 mg/ day of Garlic powder tablet for 12 weeks reduces the triacylglycerol concentration in healthy

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FIGURE 4.10 Proposed mechanism of ephedrine cardiotoxicity (cardiotoxicity at 25 mg/kg ephedrine and 30 mg/ kg caffeine is depicted in figure). Reproduced with permission from Dunnick et al., Toxicol Pathol 35(5):657–664, 2007.

FIGURE 4.11

Garlic (Allium sativum).

volunteers. Furthermore, an enteric-coated Garlic powder tablet containing 400 mg garlic and 1 mg allicin two times daily reduced cholesterol and LDL levels further (Zeng et al., 2013) showing protective effects in a single-blind study with 150 hypercholesterolemic patients (Ansary et al., 2020). The most common side effects are gastrointestinal problems and halitosis (Garlic breath) (Chen et al., 2019; Rana et al., 2006, 2011). Case reports suggest a possible increase in the risk of bleeding following Garlic use (Zeng et al., 2013). Raw Garlic homogenate has been reported to be toxic to the heart, liver, and kidney (Banerjee et al., 2003). Acute lesions in rat liver and lungs at 250, 500, and 1000 mg/kg per day (equivalent to a human dose of 70 g/day) led to suggested dose-related toxicity (El-Saber et al., 2020). Garlic is generally regarded as safe (GRAS).

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Rat oral LD50 values of crushed garlic are 173.8 mL/kg and 635.08 mg/kg in mice and rabbits 3304 mg/kg (European Medicines Agency, 2016). The LD50 of a polyherbal formulation including garlic was estimated to be more than 2000 mg/kg (Sholikhah et al., 2020), so garlic is slightly toxic based on the LD50 results. The acute toxicity of garlic extract in rats and mice did not show adverse effects (Nakagawa et al., 1984). In subacute studies (21- and 28-day) garlic extract caused toxic effects affecting weight gain, clinical pathology parameters and histological altered morphology (not detailed) (Fehri et al., 1991). In another study, deterioration in hepatic function was seen after 21, 14, and 7 days, respectively, suggesting that garlic given at a high dose has the potential to induce hepatic damage while low doses (0.1 or 0.25 g/kg body weight/day) are safe (Rana et al., 2006), a high dose finding supported by mortality in rats given 5.0 mg/ kg/day for 21 days (Nakagawa et al., 1980). There were no symptoms of toxicity or morphological alterations even at the highest dose level of 2000 mg/kg in Wistar rats treated daily with a garlic extract for 6 months (Sumiyoshi et al., 1984). Rats treated for 91 days orally with a polyherbal formulation of garlic constituents did not show significant treatment related effects on clinical signs and symptoms, weight gain, food intake, hematological parameters, biochemical parameters, and macroscopic and microscopic examination of organs (Sholikhah et al., 2020). In another study using rats, garlic (50 mg/kg), given either orally or intraperitoneally, had little effect on lung and liver as compared to control animals while high doses (500 mg/kg) resulted in profound changes in lung and liver (Alnaqeeb et al., 1996). Garlic is not mutagenic, cytotoxic, and is in fact probably antimutagenic (Morales-Gonzalez et al., 2019). There are conflicting reports regarding dose dependent increases of micronucleated cells and polychromatocytes in the bone marrow cells of mice and morphological change in Chinese hamster embryo cells treated with garlic juice at doses beyond those recommended (Yoshida et al., 1984). No reproduction and fetal development studies were found. Garlic can be considered moderately toxic based on median lethal dose but does not appear to be genotoxic or mutagenic. It is however

hepatotoxic and probably pneumotoxic unusually high concentrations.

at

10.9. Ginkgo Biloba – Ginkgo biloba Ginkgo is one of the oldest living tree species in the world, often referred to as a “living fossil.” Ginkgo biloba extract (GBE) is an herbal supplement that has been used worldwide for decades and is part of a complex mixture, which includes flavanol glycosides and terpene lactones as the major components. The major flavanol glycosides are quercetin, kaempferol, and isorhamnetin, which were identified not to be mutagenic at usual exposures (Mei et al., 2017) (Figure 4.12).

FIGURE 4.12

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Ginkgo (Ginkgo biloba).

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Ginkgo extracts provide inconsistent results, but Ginkgo is likely effective for dementia, Alzheimer’s disease, traumatic brain injury (TBI), stroke, normal aging, edema, tinnitus, and macular degeneration (Diamond et al., 2000), given at an effective, not toxic dose. Ginkgo was not effective for improving cognitive function in elderly patients without dementia. The active ingredients in Ginkgo biloba extract account for its antioxidant properties and its ability to inhibit platelet aggregation. Consequently, this herbal product is promoted for use in improving cognitive function and blood flow, but the downside is spontaneous bleeding and its interaction with warfarin (Coumadin) (Mei et al., 2017; National Toxicology Program, 2013b). Several mechanisms of action, identified in in vitro and in vivo studies, are hypothesized to play a role in the hemostatic adverse effect showing antagonism of platelet activating factor (PAF) by the ginkgolides and the antioxidant action of the flavonoids (National Toxicology Program, 2013b; Smith and Luo, 2004). The precise pharmacokinetic profile remains undetermined. Ginkgo biloba is considered generally regarded as safe (GRAS). Unsurprisingly, human reports detail spontaneous bleeding (Naderi et al., 2010), toxicity in an infant (Hasegawa et al., 2006), and acute convulsions (Kosaki et al., 2020) as exceptions to the rule. The LD50 of a standardized Ginkgo biloba extract (GBE) orally administered to mice was 7730 mg/ kg and intravenously 1100 for rats and mice (Cosmetic Ingredient Review Panel, 2007). Gingko biloba is slightly toxic based on the LD50 results. No acute toxicity was found in mice following dosing with 5, 7.5, 10, 15, and 21.5 g/kg/day GBE for 14 days (Wang et al., 2015). Further, an Ames toxicity assessment was carried out by plate incorporation assay on spontaneous revertant colonies of TA97, TA98, TA100 and TA102, with GBE doses of 0, 8, 40, 200, 1000, and 5000 mg/dish, and subchronic toxicity was evaluated in rats for 91 days given GBE doses of 500, 1000, and 2000 mg/kg/day. No mutagenicity effects were produced by Ginkgo (mutation rate .05) in the Ames toxicity test. Furthermore, the no observed adverse effect level (NOAEL) of Ginkgo was 2000 mg/kg for 91 days feeding of rats in the subchronic toxicity tests (Wang et al., 2015).

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In another 14-week study in rats, hepatic, thyroid, and nasal pathology was observed following administration of 0, 62.5, 125, 250, 500, or 1000 mg/kg/day GBE. All rats survived to the end of the study and histologically showed midzonal hepatocellular hypertrophy and fatty change (Figure 4.13), thyroid follicular cell hypertrophy, and pigmented olfactory epithelium with mild atrophy at an LOAEL of 500 mg/kg/day (National Toxicology Program, 2013b). Lifetime exposure revealed hepatotoxicity and hepatic neoplasia in rats and mice given 0, 200, 400, or 2000 mg GBE/kg 5 days per week for 104 weeks (Rider et al., 2014). Rats developed a dose-related increase of hepatocellular adenomas in females. Nonencapsulated follicular cell adenomas, and thyroid follicular cell hypertrophy and hyperplasia were observed in all treated groups of rats (Figure 4.14) as was olfactory epithelial atrophy and metaplasia – increased goblet cells – as well as atrophy of nerves in the olfactory epithelium (Figure 4.15) (Rider et al., 2014). Hepatocellular carcinoma, hepatoblastoma, and hepatocellular adenoma and thyroid follicular

FIGURE 4.13 Effect of Ginkgo biloba extract on rat liver. Fatty changes in the liver of a female F344/N rat administered 1000 mg/kg GBE by gavage for 2 years. Note the hepatocytes displaying microvesicular and macrovesicular fatty changes associated with microgranulomas (arrows) scattered throughout the lesion. The microgranulomas are composed predominantly of macrophages with fewer lymphocytes, plasma cells, and occasional neutrophils. The macrophages often contained fine, acicular clefts (cholesterol clefts). H&E stain. Reproduced with permission from Rider et al., Toxicol Pathol 42(5):830–843, 2014 [Page 836].

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FIGURE 4.14 Effect of Ginkgo biloba extract on thyroid gland from 2-year F344/N rat study of GBE. Rider et al.: Toxicol Pathol 42(5):830–843, 2014. 2A: Normal thyroid gland follicles from a male vehicle control F344/N rat. Most of the follicles are lined by flattened epithelium (arrows) and the follicles are distended with homogeneous colloid [Page 838]. 2B: Thyroid gland follicular cell hypertrophy in a female F344/N rat administered 1000 mg/kg GBE. Most of the follicles are lined by cuboidal epithelium (arrows), and there is a decreased amount of colloid as compared to Figure 2A [Page 838]. 2C: Thyroid gland follicular cell hyperplasia in a male F344/N rat administered 1000 mg/kg GBE. Note the focal enlargement of follicles that typically compress the surrounding parenchyma (arrows). The follicles are lined by epithelial-lined septae, and papillary projections frequently project into the follicular colloid [Page 837].2D: Thyroid gland follicular cell adenoma in a male F344/N rat administered 1000 mg/ kg GBE. The adenoma tends to be larger with more compression of the adjacent parenchyma and more complex epithelial infoldings than in hyperplasia (arrows) [Page 837]. 2E: Thyroid gland follicular cell carcinoma in a female F344/N rat administered 300 mg/kg GBE. The neoplasm is highly cellular (arrows) with local invasion. H&E stain.

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FIGURE 4.15 Effect of GBE on nasal tissue. 2-year F344/N rat study of GBE. Rider et al.: Toxicol Pathol, 42(5), 830–843, 2014. (A): Chronic active inflammation in the nose of a female F344/N rat administered 1000 mg/kg GBE. Note the presence of mixed inflammatory infiltrate that involves the submucosa and the lumen of the nasal cavity. There was also respiratory metaplasia of the olfactory epithelium (arrows) [Page 838]. (B): Atrophy of the olfactory epithelium in the nose (Level III) of a female F344/N rat administered 1000 mg/kg GBE. Note the thinning and disorganization of the olfactory epithelial layer (arrows). H&E stain. Reproduced with permission from Rider et al., Toxicity and carcinogenicity studies of Ginkgo biloba extract in rat and mouse: liver, thyroid, and nose are targets [Page 838].

cell hyperplasia were observed in another study. Nonneoplastic lesions included hepatocellular hypertrophy, inflammation, erythrophagocytosis, cytoplasmic vacuolization and necrosis, and hematopoietic hyperplasia (Hoenerhoff et al., 2013). Gingko biloba has a negative impact on reproduction and development in animal models. Significant changes were seen in the weight of the caudae epididymis, prostate, chromosomal aberrations, rate of pregnancy, and preimplantation loss in mice treated with Ginkgo at 0, 25, 50, and 100 mg/kg/day for 90 days (Al-Yahya et al., 2006). However, the effect on rodent development depended on the experiment – no effect was seen at a dose of 1225 mg/kg/day (Koch et al., 2013), and at 14.0 mg/kg/day (Fernandes et al., 2010). Malformations, including round shaped eye and orbits, syndactyly, malformed pinnae, nostrils, lips, and jaws led the authors to conclude that Ginkgo was teratogenic in mice (Zehra et al., 2010). In the rat, maternal toxicity was observed at 100 mg/kg/day with corresponding developmental toxic effects (decreased fetal and placental weights, increased incidences of skeletal variations, and delay in fetal ossification) (Li et al., 2018). There was maternal toxicity in rabbits given 60 mg/kg/day of

dimethylaminoethyl ginkgolide B and fetal growth and development were affected (Li et al., 2018). In vitro, cytotoxicity tests demonstrated that Ginkgo reduced cell viability in a dosedependent manner in human renal tubular epithelial cells (HK-2) and human normal hepatocytes (L-02), indicating there might be potential liver and kidney toxicity (Li et al., 2019a,b). Similar findings were documented in human keratinocyte cell line HaCaT and the rhesus monkey kidney tubular epithelial cell line LLCMK(2) (Hecker et al., 2002). However, no remarkable increases in gpt or Spi() mutation frequencies were observed in DNA extracted from the livers of gpt delta mice that had been exposed to GBE up to 2000 mg/kg/day. In the comet and micronucleus assays, no statistically significant increases in positive cells were reported at doses up to 2000 mg/kg/day of GBE providing evidence that GBE is not genotoxic in vivo. Therefore, Ginkgo biloba-induced hepatocarcinogenesis in mice probably occurs through a nongenotoxic mode of action (Maeda et al., 2014). The major flavanol glycosides of Ginkgo biloba are quercetin, kaempferol, and isorhamnetin, which are mutagenic (Mei et al., 2017); however, even though Ames assays have failed to demonstrate mutagenicity of GBE (Wang et al., 2015), GBE

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does cause DNA damage in human hepatic HepG2 cells (Zhou et al., 2015) a finding attributed to quercetin and kaempferol (Lin et al., 2014).

10.10. Ginger – Zingiberis rhizome Ginger (Zingiberis officinale) is a member of a plant family that includes cardamom and turmeric. Its spicy aroma is mainly due to presence of ketones, especially the gingerols, which appear to be the primary component of Ginger studied in the laboratory. The rhizome is the main portion that is consumed (Figure 4.16). Ginger has been purported to exert a variety of therapeutic and preventive effects to treat hundreds of ailments from colds to cancer. Like many medicinal herbs, much of the information has been handed down by word of mouth as empirical knowledge with little controlled scientific evidence to support the numerous claims. However, by using modern techniques and equipment, Ginger has shown in vitro antioxidative, antitumorigenic and immunomodulatory effects. It is an effective antimicrobial and antiviral agent (Bode and Dong 2011; Chrubasik et al., 2005). Although clinical and experimental studies suggest that Ginger has some antiemetic properties, clinical evidence only supports an effect on pregnancy-related nausea and vomiting. Other indications for Ginger, including seasickness,

FIGURE 4.16

Ginger (Zingiberis rhizome).

morning sickness, and chemotherapy-induced nausea, offer preliminary, although inconclusive, evidence of efficacy. Whether Ginger preparations are clinically useful to alleviate osteoarthritic or other pain requires confirmation. Adverse effects after ingestion of Ginger are uncommon, but they can include mild gastrointestinal effects such as heartburn, diarrhea, and irritation of the mouth (Chrubasik et al., 2005). Because there is a possibility that Ginger may affect fibrinolytic activity, patients taking anticoagulants such as warfarin (Coumadin) should exercise caution (White 2007). Cardiac arrhythmias may occur, probably as a result of Ginger’s inotropic properties (Verma and Bordia, 2001). Ginger has an LD50 of 1551 mg/kg ip in mice (European Medical Agency, 2010); hence, Ginger is slightly toxic based on the LD50 results. No mortality or abnormal clinical signs, growth, and food and water consumption were observed in rats treated daily with Ginger powder at 500, 1000, and 2000 mg/kg by gavage for 35 days. However, altered morphology was present on gross and histopathology (Rong et al., 2009). Similarly, no significant clinical signs or lesions were seen in rats dosed with 0, 100, 250, and 500 mg/kg Ginger oil per day for 13 weeks (Jeena et al., 2011) or 26 weeks (Li et al., 2020). In mice fed 0, 1000 and 2000 Ginger mg/kg/day for 30 days, there was significant anticholangiocarcinoma activity (as determined using the CCAxenografted mouse model) illustrated by tumor growth inhibition and obvious prolongation of survival time at 2000 mg/kg body weight, an effect that was moderate in the 1000 mg/kg group (Plengsuiyakarn and Na-Bangchang 2020). No other carcinogenicity studies were found. Minimal but positive effects as compared to controls of Ginger on fertility of male diabetic rats given methanolic extract (100 and 200 mg/ kg), or water extract (150 and 300 mg/kg), for 65 days included increased fertility index, sexual organ weights, serum testosterone level and sperm motility and count. Ginger extracts also reduced the degree of histopathological changes in the testes of diabetic rats showing mild to moderate degenerative changes of spermatogenic cells, diffuse edema, and incomplete arrest of spermatogenesis (Shalby and Hamowieh, 2010), which may be a result of enhanced masculine testosterone production (Banihani, 2018). Conversely, Ginger given 0, 250, 500, 1000, or 2000 mg/kg/day orally for 90, 35, 20, days caused maternal toxicity at 2000 mg/kg/day, significantly

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reducing the number of implantation sites and live fetuses and increasing fetal death and resorptions. At the dose of 2000 mg/kg/day (35 days), Ginger prolonged the length of the estrous cycle with a significant decrease in the duration of diestrus-metestrus (luteal) phase, prolonged proestrus-estrus (ovulatory) phase and reduced the number of cycles. This indicates that Ginger impairs the normal growth of the corpus luteum via progesterone insufficiency and may cause failure of blastocyst implantation without teratogenesis (El Mazoudy and Attia, 2018).

10.11. Ginseng – Panax ginseng Little scientific evidence shows that Ginseng (Panax ginseng, C.A. Meyer) is effective for any purpose. Nonetheless, this herb has been purported to strengthen normal body functions, increase resistance to stress and improve sexual function. Ginseng is generally well tolerated, but a probable interaction between the herb and warfarin has been reported. Ginseng is primarily marketed to improve energy and physical or cognitive performance (Rotblatt and Ziment, 2002) so it is present in many drinks and tonics. Ginseng is believed to be safe in humans, although there are some case reports of excessive arousal and hyperactivity (Volger et al., 1999). Regardless, Ginseng is generally regarded as safe (GRAS) (Figure 4.17). Due to its antioxidative and potential neuromodulating effects, it has become a popular supplement in neurodegenerative diseases such as Alzheimer disease, Parkinson disease, Huntington disease, and brain ischemia. Additional claims are for its antihypertensive, cardioprotective, and anticancer effects. However, there are few efficacy studies, or studies are inadequate, showing improvements in cognitive function in Alzheimer’s disease, chronic obstructive pulmonary disease, influenza, fatigue related to multiple sclerosis, erectile dysfunction, premature ejaculation, and sexual arousal. Indeed, there is little compelling evidence for efficacy for improved physical performance, psychomotor performance, cognitive function, or immunomodulation (Vogler et al., 1999). Some limited cardiac effects include amelioration of unstable angina indicated by an improved electrocardiogram, decreased frequency, and duration of angina episodes (Cambria et al., 2021).

FIGURE 4.17

Ginseng (Panax ginseng).

While some epidemiological or clinical studies have reported efficacy for specific health benefits, there are an equal number of studies that provide contradictory evidence. Regardless, data from clinical trials suggest that the incidence of adverse events with Ginseng is similar to that with placebo (Kitts and Hu, 2000). Headache, and sleep and gastrointestinal disruption were the most commonly experienced adverse events, with spontaneous reporting of more serious adverse events confined to isolated case reports making assessment of causality difficult. Combination products containing Ginseng as one of several constituents have been associated with serious adverse events and even fatalities, indicating that ingredients other than Ginseng may have caused the problems. Collectively, these data suggest that ginseng monopreparations are rarely associated with adverse events, and if so, they are usually mild and transient (Coon and Ernst, 2002). No serious reactions occurred following administration of P. ginseng extract (1.0 g/day or 2.0 g/day) or placebo over a 4-week period (Lee et al., 2012). Ginseng interacts with other herbal products and prescribed medications, including bloodthinners, HIV treatments, diabetic medications, immunosuppressants, antidepressants, bitter orange, Ephedra, bitter mallow, caffeine, and alcohol (Ernst, 1998).

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The LD50 of Ginseng >1600 mg/k in rats dose orally (Li et al., 2020). Ginseng is slightly toxic based on the LD50 results. Acute animal studies showed no mortality or abnormal necropsy findings in rats treated with 5000 mg/kg ginseng (Park et al., 2019). No deaths, changes in the body condition, body weights, hematological and biochemistry tests, and pathology were seen in rats treated intramuscularly and intravenously with 0.1, 0.5 and 1.0 mL of a Ginseng extract (Yu et al., 2015), although another study reported an LD50 of 1.0 mL (Yu et al., 2015). There was no Ginsengrelated gross or microscopic finding attributed to Ginseng at doses of 0, 125, 250, 500, 1000, or 2000 mg/kg, 5 days per week for 16 days (National Toxicology Program 2011a). Subacute animal toxicity studies show a lack of toxicity, e.g., there was a lack of clinical signs, changes in body weight and food consumption, normal hematology, and clinical chemistry, as well as no gross or histopathological findings, giving an NOAEL of 2000 mg/kg/day in rats dosed with 0, 500, 1000, or 2000 mg/kg/day orally by gavage for 28 days with a Ginseng formulation (Seo et al., 2019). Similarly, chronic toxicity studies failed to reveal significant findings (Aphale et al., 1998; Kim et al., 2020; Lu et al., 2012; Li et al., 2018; National Toxicology Program 2011a; Park et al., 2018; Suh et al., 2019). Ginseng was not mutagenic in the Ames assay, micronucleus mouse assay (Park et al., 2019; Seo et al., 2019) and lifetime studies failed to provide evidence of carcinogenicity (Chan et al., 2011). No effect on reproduction and fetal development has been reported, e.g., Ginseng did not affect parameters for fertility, early embryonic development, maternal function, and embryofetal development, giving an NOAEL 2000 mg/ kg/day (Lee et al., 2019a).

10.12. Goldenseal – Hydrastis canadensis Goldenseal (Hydrastis canadensis), a member of the plant family Ranunculaceae, is a plant native to North America. It has been traditionally used by Native Americans as a coloring agent. The five major alkaloid constituents in Goldenseal are berberine, palmatine, hydrastine, hydrastinine, and canadine (Chen et al., 2013) (Figure 4.18).

FIGURE 4.18

Goldenseal (Hydrastis canadensis).

Goldenseal is a commonly used herbal product to treat ulcers, wounds, skin, mouth and eye infections, urinary disorders, and gastrointestinal disturbances. Berberine is reported to have antifungal activity against Candida species, antimicrobial activity against Staphylococcus aureus, and also against various infectious agents such as cholera, amebiasis, and Leishmania and Plasmodium. Berberine has also been reported to have antiinflammatory, antiproliferative, cytostatic, and antioxidative effects, probably mediated by inhibition of enzymes such as CYP450 and topoisomerases, or effects on cell signaling, receptors, and transporters (Jahnke et al., 2006). Goldenseal extract is possibly neurotoxic, hepatotoxic, and phototoxic (Mandal et al., 2020). Overdoses cause cardiopathy, death, depression, hypotension, nausea and vomiting, nervousness, paralysis, respiratory failure, seizures, shortness of breath, bradycardia, or spasms (Haller et al., 2002). The oral LD50 dose for berberine is 329 mg/kg (0.98 mmol/kg) in mice and the sc LD50 dose is

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18 mg/kg (0.054 mmol/kg). The ip LD50 for berberine sulfate in mice is 24.3 mg/kg (0.056 mmol/kg). In rats, the ip LD50 dose for berberine is greater than 500 mg/kg (>1.49 mmol/kg), while the ip LD50 for berberine sulfate is 88.5 or 205 mg/kg (0.20 or 0.47 mmol/ kg). Also, for rats, the LD50 doses for berberine sulfate are 14.5 mg/kg (0.033 mmol/kg) when administered im, and greater than 1000 mg/kg (>2.31 mmol/kg) when administered orally. In rabbits, the sc LDLo dose for berberine is 100 mg/kg (0.30 mmol/kg). Goldenseal is moderately toxic based on these results. Mild hypertrophy of centrilobular hepatocytes was the only lesion described in rats and mice fed diets containing 0, 1560, 3121, 6250, 12,500, 25,000, or 50,000 ppm Goldenseal root powder for 15 days (National Toxicology Program, 2010b). Hepatocellular hypertrophy was also seen in chronic studies in rats and mice fed diets containing 0, 3121, 6250, 12,500, 25,000, or 50,000 ppm Goldenseal root powder for 14 weeks. Minimal to moderate hepatocellular hypertrophy occurred in three male and all female rats exposed to 25,000 ppm and in all 50,000 ppm males and females. The incidences of hepatocyte hypertrophy were significantly increased in male and female mice exposed to 12,500 ppm or greater in mice. Hepatocellular hypertrophy, hepatocellular adenomas, hepatoblastomas, and eosinophilic foci were seen in rats and mice fed diet containing 0, 3000, 9000, or 25,000 ppm Goldenseal root powder for 105–106 weeks. The incidences of hepatocellular adenoma were significantly increased in male and female rats exposed to 25,000 ppm, and the incidence of hepatocellular adenoma or carcinoma (combined) was significantly increased in 25,000 ppm males. All exposed groups of males and females had significantly increased incidences of hepatocyte hypertrophy. The incidences of hepatocyte degeneration were significantly increased in all exposed groups of males and in 9000 and 25,000 ppm females. The incidences of eosinophilic focus were significantly increased in 9000 and 25,000 ppm males and all exposed groups of females. The incidence of hepatocellular adenoma in mice occurred with a positive trend in males, and the incidence of multiple hepatocellular adenomas were significantly increased in 9000 and 25,000 ppm males. The incidence of

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hepatoblastoma occurred with a positive trend in males with a marginal increase in the 25,000 ppm group. Significantly increased incidence of eosinophilic focus or mixed cell focus occurred in all exposed groups of males (National Toxicology Program, 2010b; Dunnick et al., 2011). No reproductive or developmental effects were seen in rats given 65 times the human dose (Yao et al., 2005). Goldenseal is not mutagenic in Salmonella typhimurium strains TA100 or TA98 or Escherichia coli strain WP2 uvrA pKM101 with or without rat liver S9 metabolic activation enzymes, nor the mouse micronucleus test (National Toxicology Program 2010b).

10.13. Green Tea – Camellia sinensis Green tea extract is obtained from leaves of the plant Camellia sinensis of the Theaceae family. Approximately 10% of the dry weight of Green tea is made up of catechins, mainly epicatechin, epicatechin-3-gallate, epigallocatechin, and epigallocatechin-3-gallate (EGCG), with EGCG being the highest in concentration. The catechins are considered to be the active compounds in Green tea (Figure 4.19). Green tea has antioxidative, antiinflammatory, antidementia, anticarcinogenic, thermogenic, probiotic, and antimicrobial properties. It may be consumed as a dilute beverage, concentrated supplement, or as a topical application. The health promoting effects of are mainly attributed to its polyphenol content, including catechins, which are powerful antioxidants (Xing et al., 2019). The tea flower extract does not possess

FIGURE 4.19

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Green tea (Camellia sinensis).

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mutagenic potential, and little effect in acute and subchronic rat toxicity studies. An NOAEL for tea flower extract was set at 4.0 g/kg/day for rats (Li et al., 2011). Consumption of Green tea has been associated with a statistically significant reduction in LDL cholesterol and fewer prostate cancer cases have been reported in men who consume Green tea. While studies have been performed to determine whether Green tea has effects on pancreatic, esophageal, ovarian, breast, bladder, and colorectal cancer, the evidence remains inadequate (Chacko et al., 2010). Consumers who are taking anticoagulation drugs, such as warfarin, should avoid Green tea due to its vitamin K content. Pregnant or breastfeeding women, those with heart problems or high blood pressure, kidney or liver problems, stomach ulcers, or anxiety disorders should use caution consuming Green tea (Hirose et al., 1993). Meta-analysis and systematic reviews concluded that three or more cups of tea per day was associated with a lower risk for diabetes (Yang et al., 2014; Asbaghi et al., 2020), reduced risk for coronary heart disease, cardiac death, stroke, and total mortality (Oketch-Rabah 2020). Conversely, Green tea was associated with increased lung cancer risk and teratogenicity (neural tube defects and anencephaly) and potentially with hepatotoxicity. LD50 for a Green tea extract containing 74.5% (w/w) total catechins to be 3300 and 5000 mg/ kg in female and male ddY mice, respectively, via oral gavage administration. An LD50 value was greater than 12.0 g/kg body weight (Yamane et al., 1996) in rats. Green tea is slightly toxic based on the LD50 results. Chronic toxicity studies failed to show significant effects in rats given 0, 1.0, 2.0, and 4.0 g/kg daily for 13 weeks (National Toxicology Program, 2016). In contrast, hepatic necrosis, hepatocellular hypertrophy (Figure 4.20A [1–2], olfactory lesions (Figure 4.20A [3–8], Figure 4.20B [9–12]), and splenic atrophy were seen in mice given 0, 62.5, 125, 250, 500, or 1000 mg Green tea extract/kg in deionized water by gavage, 5 days per week for 14 weeks (Chan et al., 2010). Rats following the same treatment protocol also had thyroid follicular cell hyperplasia (Figure 4.20B [13–14], and thymic atrophy (National Toxicology Program, 2016).

Lifetime exposure (2-year) studies in rats and mice given 0, 30 [100 mouse only], or 300 mg Green tea extract/kg by gavage, 5 days per week for 105 weeks resulted in no toxicity. Necrosis of the gastrointestinal tract of 1000 mg/kg treated male and female rats was the prominent lesion, which corresponds to reports of hepatic necrosis and rectal bleeding in humans. Hepatic necrosis was noted in the 3-month studies where female rats appeared to be more susceptible to the liver toxicity of Green tea extract in the 3-month and 2-year studies (National Toxicology Program, 2016).

10.14. Indole-3-Carbinol – Brassica sp. Glucosinolates Indole-3-carbinol (I3C) is a breakdown product of glucosinolate glucobrassicin. It is found in cruciferous vegetables of the Brassica genus, including brussels sprouts, cauliflower, cabbage, and broccoli. Cruciferous vegetables consumption has been associated with a decreased risk of cancer in humans (Figure 4.21). I3C induces the expression and activity of cytochrome P450 (CYP)1A1 in liver, mammary gland, and colon; and 1A2, 1B1, 2B1/2, and 3A in the liver. I3C induction of CYP enzymes thus may activate carcinogens. CYP-mediated estrogen metabolism is believed to be a protective effect of I3C against estrogen-responsive tumors. Several carcinogenicity studies have been conducted in various animal models to assess I3C inhibitory and promotional effect. I3C promoted aflatoxin B1-initiated hepatocarcinogenesis but there were fewer endometrial adenocarcinomas and mammary gland tumors in the higher treatment groups. I3C is believed to be nonmutagenic (National Toxicology Program, 2017). Subcutaneous rat LD50 is 500 mg/kg (Nishie and Daxenbichler, 1980). Indole-3-carbinol is moderately toxic based on the LD50 results. The intestine appeared to be the target of I3C toxicity (Figure 4.22). I3C significantly alters the number and width of intestinal villi and reduces cell proliferation and increases apoptosis in a dosedependent manner (Fletcher et al., 2017). The rodent model of immune-deficient male BALB/c nu/nu athymic mice were given diets

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FIGURE 4.20A [1] Effect of green tea extract on rat liver. Chronic inflammation (arrows) bordered by vacuolated hepatocytes (fatty change), in a female rat administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months. H&E stain. [2] Prussian Blue positive (detecting ferritin) pigment accumulation in Kupffer cells in the liver of a female rat administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months (arrows). The pigment was also positive for ceroid lipofuscin (Schmorl’s staining) and glycoproteins (PAS staining). Prussian Blue, X20. [3] Effect of green tea extract on olfactory epithelium of rat. Olfactory epithelium necrosis (thin arrow),

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supplemented with 0–100 mmoles I3C/g diet for 4 weeks. All mice died after 3 days given 100 mmoles I3C/g supplemented diet. Mice fed with 10–50 mmoles I3C/g supplemented diet survived but showed concentration-dependent adverse effects (Fletcher et al., 2017). No histopathologic lesions in rats were attributed to the administration of Indole-3-carbinol dosed with 0, 18.75, 37.5, 75, 150, or 300 mg Indole-3-carbinol/kg in corn oil by gavage, 5 days per week for 14 weeks (Boyle et al., 2012; National Toxicology Program, 2017). In cancer bioassays (2-year), there was no evidence of carcinogenic activity of Indole-3carbinol in male Harlan Sprague Dawley rats administered 75, 150, or 300 mg/kg by gavage, but there was some evidence of carcinogenic activity of Indole-3-carbinol in female Harlan Sprague–Dawley rats based on an increased number of malignant uterine neoplasms (primarily adenocarcinoma), dermal fibroma and fibrosarcoma. There was clear evidence of carcinogenic activity of Indole-3-carbinol in male B6C3F1/N mice based on increased incidence of liver neoplasms (hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma) but there was no evidence of carcinogenic activity of Indole-3-carbinol in female B6C3F1/N mice administered 62.5, 125, or 250 mg/kg (National Toxicology Program, 2017). Perinatal exposure to I3C did not affect the total number of sperm in the epididymis (1 mg/kg) but reduced daily sperm production/testis (100 mg/kg) although total epididymal transit time was significantly increased

by 31% male Sprague–Dawley rats given I3C at 1.0 or 100 mg/kg Pregnant rats did not show significant alterations in reproductive and developmental parameters (Wilker et al., 1996) given either 200 or 300 mg Indole-3-carbinol/kg on GDs 8 and 9 (Nishie and Daxenbichler 1980).

10.15. Kava kava – Piper methysticum Kava kava (Piper methysticum) has been cultivated in the South Pacific for its rootstock, also referred to as “the stump.” Kava kava is traditionally used in the islands of the South Pacific as a sedative and relaxant and a beverage in ceremonies or religious occasions. Prior clinical studies suggest a small benefit of Kava kava for the treatment of anxiety (Clayton et al., 2007; Pittler and Ernst, 2003). Kava has been used for treatment of skin disorders, asthma, lung disorders, and urologic problems in Hawaii and Germany. Before penicillin was discovered, Kava kava was used to treat gonorrhea (Fu et al., 2008) (Figure 4.23). The active principles of Kava kava rootstock are mostly contained in the lipid-soluble resin. Avalactones (alpha-pyrones) of the Kave kava compounds have the highest pharmacological activity. The six major kavalactones include yangonin, methysticin, dihydromethysticin, dihydrokawain, kawain, and demethoxyyangonin. When tested individually, kavalactones did not exhibit biological activity similar to that found in the whole Kava kava extract but they did once the kavalactones were recombined (Singh 1992).

=atrophy, nerve fiber atrophy (thick arrows), and pigmented histiocytes (arrowhead) in the dorsal meatus of level II of the nasal cavity of a male rat administered with 1000 mg/kg green tea extract (GTE) by gavage for 3 months. [4] Olfactory epithelium in the dorsal meatus of level II of the nasal cavity of a control rat from the 3-month green tea extract (GTE) gavage study. Note normal size and number of the olfactory nerves in the lamina propria (arrows). H&E, X32. [5] Glycoproteins (PAS)-positive pigment accumulation in olfactory epithelium (arrows) of nasal cavity of a male rat administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months. The pigment was also positive for ceroid lipofuscin (Schmorl’s staining). PAS, X40. [6] Basal cell hyperplasia (arrows) and Bowman’s gland hyperplasia (arrowheads) in the olfactory epithelium lining the dorsal meatus of level III of the nasal cavity of a male rat administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months. The basal cell hyperplasia is characterized by minimal proliferation (two to three cells thick) of basal cells. The Bowman’s gland hyperplasia is characterized by nodular proliferation of cells that compressed and occasionally filled glandular lumina. H&E, X40. [7] Nasopharyngeal duct degeneration (arrow) located at level III of the nasal cavity of a male rat administered 500 mg/kg GTE by gavage for 3 months. The degeneration is characterized by decreased goblet cell numbers and transformation of tall columnar ciliated epithelial cells to more attenuated or cuboidal cells. H&E, X4. [8] Tall columnar, ciliated epithelium lining the nasopharyngeal duct (arrow) in level III of the nasal cavity of a male control rat from the 3-month green tea extract (GTE) gavage study. H&E, X40. Reproduced with permission from Chan et al., Toxicol Pathol 38(7):1070–1084, 2010. [Page 1076].

9

10

11

12

13

14

FIGURE 4.20B [9] Centrilobular hepatocellular inflammation (arrowheads) and fatty change (arrows) in a male mouse administered 1000 mg/kg green tea extract (GTE) by gavage. H&E, X20. [10] Hepatocyte karyomegaly (arrowhead) and trinucleated hepatocyte (arrows) in a male mouse administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months. H&E, X60. [11] Unilateral olfactory epithelium atrophy (arrowhead), olfactory nerve atrophy (thin arrows), and metaplasia of olfactory epithelium to respiratory epithelium in the dorsal meatus of level III in a male mouse administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months. Note the normal aspect of the olfactory nerves in the lamina propria of the unaffected side (thick arrows). H&E, X16. [12] Basal cell hyperplasia (arrows) and Bowman’s gland hyperplasia (arrowhead) in the olfactory epithelium in the dorsal meatus of level III in a male mouse administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months. Note the increased number of basal cells. Instead of a single layer of basophilic nuclei, the nuclei are piled up or crowded and extend up into the olfactory cells. The Bowman’s gland hyperplasia is characterized by nodular proliferation of cells that compressed and occasionally filled glandular lumina in the lamina propria underlying or continuous with the olfactory epithelium. H&E, X40. [13] Splenic white pulp atrophy. The arrows indicate reduced lymphoid cells in the periarteriolar lymphoid sheath corona and marginal zone of the white pulp in a female mouse administered 1000 mg/kg green tea extract (GTE) by gavage for 3 months. H&E, X10. [14] Normal aspect of the splenic white pulp, and in particular the periarteriolar lymphoid sheaths (arrows), in a control female mouse from the 3-month green tea extract (GTE) gavage. H&E, X4. Reproduced with permission from Chan et al., Toxicol Pathol 38(7):1070–1084, 2010. [Page 1080].

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Brassica sp. [contains Indole-3-carbinol].

Similar to methenesin, Kava kava can cause muscle relaxation without depressing CNS function. Both yangonin and desmethoxy-yangonin possess weaker central nervous activity than Kava kava extract. The other alpha-pyrones exhibit markedly enhanced activity (Bent, 2008). The main concern with the use of Kava kava is hepatotoxicity, characterized by dose-related functional disturbances, hepatitis, cirrhosis, and liver failure (Tesche, 2010). This has led to a ban on sale of ethanolic and acetonic Kava kava extracts in Germany in 2002 (Fu et al., 2008). Toxicity of Kava kava has been attributed to CYP2D6 deficiency seen in 7%–9% of Caucasian, 5.5% of Western European, almost 1% of Asian, and less than 1% of Polynesian populations (Centers for Disease Control and Prevention (CDC), 2002). Kava kava is photocytotoxic and photogenotoxic, both mediated by free radicals (ROS) generated during photoirradiation (Fu et al., 2013; Xia et al., 2012). Male Balb/c mice had a LD50 of 700 mg/kg and above, with death

due to respiratory failure (Jamieson and Duffield, 1991). Kava kava is slightly toxic based on the LD50 results. Most rodent studies suggest mild hepatotoxicity (National Toxicology Program, 2012). For example, minimal hepatocellular hypertrophy was seen in mice and rats given 0, 0.125, 0.25, 0.5, 1, and 2 g/kg Kava kava extract by gavage after 2 weeks of observation (Behl et al., 2011) and minimal hepatocellular hypertrophy was seen in rats and mice given 0, 0.125, 0.25, 0.5, 1, and 2 g/kg Kava kava extract by gavage (National Toxicology Program, 2012). A dose–response increase of neoplastic lesions was observed in the livers of mice: hepatoblastomas (males) (Figure 4.24), hepatocellular adenomas (males and females) and hepatocellular carcinomas (females) (Figure 4.25). Groups of 50 male and 50 female mice were dosed with Kava kava extract by gavage at concentrations of 0, 0.25, 0.5, 1.0 g/kg, with males treated with 0, 0.3, 1.0 g/kg extract. In another experiment, rats were dosed with Kava kava extract by gavage at concentrations of 0, 0.3, 1.0 g/kg. There was a dose-related increase in number of testicular interstitial (Leydig) cell adenomas with an increase in bilateral interstitial cell adenomas in treated groups. Centrilobular hepatocellular hypertrophy and metaplasia of pancreatic acinar cells to a hepatocytic morphology was characterized by small clusters of normal hepatocytes adjacent to islets of Langerhans. The finding was present in both male and female treated rats, but not in controls animals. Additional findings related to the Kava-kava treatment included inflammation, ulceration, and epithelial hyperplasia in the forestomach; nephropathy and transitional epithelial hyperplasia of the pelvis of the kidney; and retinal degeneration in the eye. There were decreased numbers of pars distalis adenomas of the pituitary gland and fibroadenoma of the mammary gland (National Toxicology Program, 2012). Kava kava is considered a Group 2B carcinogen (IARC, 2002). Kava kava was mutagenic in S. typhimurium strains TA98 and TA100 in the presence of induced rat liver S9; no mutagenicity was observed in these strains without S9 or in the E. coli strain WP2 uvrA/pKM101, with or without S9. In vivo, no increases in the frequencies of micronucleated erythrocytes were seen in

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FIGURE 4.22 Representative photomicrographs of lesions in the small intestine and mesenteric lymph nodes of rats exposed to a 3-month treatment of 300 mg/kg indole-3-carbinol via oral gavage. (A) Duodenum; moderate ectasia of villous lacteals (lymphangiectasis) (arrows). Original objective. H&E, 4X. (B) Duodenum; lymphangiectasis (stars). Note vacuolated macrophages within the lamina propria (arrows). Original objective. H&E, X40. (C) Mesenteric lymph node; moderate lymphangiectasis of the subcapsular sinus (arrows). Original objective. H&E, X4. (D) Mesenteric lymph node; lymphangiectasis of the subcapsular sinus (stars). Note vacuolated multinucleated giant cells (arrow). H&E, X40. (E and F) Mesenteric lymph node; large aggregates and pools of red staining material within subcapsular sinuses (arrows) are consistent with excessive lipid accumulation (lipidosis). Note grade 4 lymphangiectasis. H&E X2 (E) and X40 (F), Oil Red O. Reproduced with permission from Boyle et al., Toxicol Pathol 40, 561–576, 2012. [Page 567].

peripheral blood of male or female B6C3F1/N mice in the 3-month study (National Toxicology Program, 2012).

No data on the reproductive and developmental toxicity of kava kava in experimental animals or humans were found in the literature.

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FIGURE 4.23 Kava kava (Piper methysticum).

10.16. Milk Thistle – Silybum marianum Milk thistle, Silybum marianum, a member of the Asteraceae (Aster) family, is a tall edible plant with a 2000-year history of use. The fruits of this plant contain the relatively water-insoluble flavonolignans, known as silymarin complex (silybin, isosilybin, silydianin, silychristin, and isosilychristin). This complex constitutes 70% of the Milk thistle extract. Silymarin possesses an antiproliferative and antioxidant effects, by reducing ROS production and lipid peroxidation (Dunnick et al., 2011; National Toxicology Program, 2011a) (Figure 4.26).

Milk thistle has been used in herbal remedies for the treatment of liver diseases (alcoholic and viral hepatitis, cirrhosis) and gallbladder disorders. Other potentially beneficial effects include reduction of breast, cervical, and prostate cancer cell growth, reduction of insulin resistance, and lowering of cholesterol levels. Indeed, studies have shown other possible therapeutic effects on Alzheimer’s and Parkinson’s disease (Dunnick et al., 2011). Milk thistle has no major toxicity in animals and is generally regarded as safe (GRAS). Silymarin is mutagenic in Salmonella typhimurium strains in the presence of metabolic enzymes, but silybin, silydianin, and silychristin are not cytotoxic and genotoxic at concentration of 100 mM. Silymarin is safe in humans at therapeutic doses and is well tolerated even at a high dose of 700 mg three times a day for 24 weeks. Silymarin has few conventional drug interactions, and it does not have major effects on cytochromes P-450 (Soleimani et al., 2019). Silymarin also has antifibrotic activity and may act as a toxin blockade agent by inhibiting binding of toxins to the hepatocyte cell membrane receptors (Feher and Kengyel, 2012). In animals, silymarin reduces liver injury caused by acetaminophen, carbon tetrachloride (Aslan and Can, 2014), radiation, iron overload, phenylhydrazine, alcohol, cold ischemia, and Amanita phalloides (Abenavoli et al., 2018).

FIGURE 4.24 Photomicrograph of hepatoblastoma from a male mouse treated with 1 mg/kg Kava kava for 2 years (arrow). Note the hyperbasophilic appearance of the mass due to presence of basophilic fusiform cells with a high nucleus to cytoplasmic (N:C) ratio. Figure (A): H&E X2. Figure (B): note the presence of basophilic fusiform cells with a high nucleus to cytoplasmic. H&E X10. Reproduced with permission from Behl et al., Food Chem Toxicol 49, 2820–29, 2011. [Page 20]. II. SELECTED TOXICANT CLASSES

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FIGURE 4.25 Photomicrographs of hepatocellular carcinoma from a male mouse treated with 0.25 mg/kg Kava kava for 2 years. Figure (A): Note (arrow), margins of the carcinoma compressing the adjacent normal tissue. H&E X2. Figure (B): Higher magnification of Figure A. Note the trabecular architecture displayed by the neoplastic cells. H&E X10. Reproduced with permission from Behl et al., Food Chem Toxicol 49, 2820–2829, 2011. [Page 19].

FIGURE 4.26

Milk thistle (Silybum marianum).

In humans, clinical trials report laxative side effects and allergic reactions (Dunnick et al., 2011) but these studies are largely heterogeneous and contradictory. In sum, aside from mild gastrointestinal distress and allergic reactions, side effects are rare, and serious toxicity has not been reported (Rainone, 2005). A systematic review concluded that the clinical evidence of a therapeutic effect of silymarin in toxic liver diseases is scarce and there is no evidence of a favorable influence on the evolution of viral hepatitis, particularly hepatitis C (de Avelar et al., 2017). Milk Thistle can be considered moderately toxic based on the LD50 results of 140 mg/kg iv but

can be considered practically nontoxic when given orally at 10 g/kg. Chronic toxicity studies failed to show any adverse effects up to 50,000 ppm Milk thistle in feed (National Toxicology Program, 2011a) and lifetime exposure of up to 50,000 ppm Milk thistle extract in male mice significantly decreased numbers of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined). Similarly, lifetime exposure of rats to the extract decreased numbers of mammary gland fibroadenomas (consisting of both ductular and/or alveolar epithelium and fibrous connective tissue), adenomas, or carcinomas (combined) in female rats were decreased (Dunnick et al., 2011). Milk thistle does not appear to be genotoxic, mutagenic, or show reproductive toxicity (National Toxicology Program, 2011a).

10.17. Mint Mentha sp. [Contains Pulegone] [R-(þ)-pulegone], a colorless, oily liquid with an odor between peppermint and camphor, is used for flavoring foods, drinks, and dental products as well as a fragrance agent. It has been also used as an abortifacient (Figure 4.27). Pulegone is found naturally in food products but may also be produced synthetically. Essential oils containing Pulegone are derived from many

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FIGURE 4.27

4. HERBAL REMEDIES

Mint (Mentha sp.) [contains pulegone].

plant species, including Hedeoma pulegioides (American pennyroyal), species of the genus Bystropogon (evergreen shrubs), and species of the genus Mentha [e.g., European pennyroyal (M. pulegium), corn mint, Biblical mint, etc.]. Peppermint, containing Pulegone, is a common ingredient in herbal products marketed to treat irritable bowel syndrome. A Cochrane systematic review concluded that peppermint appears to be beneficial for IBS-related symptoms and pain, bloating, gas, and diarrhea, which was supported by a recent multicenter, randomized, placebo-controlled, double-blind study. The study found that 43.3% of subjects improved with a peppermint-caraway oil combination after 8 weeks, compared with 3.5% receiving placebo, revealing decreased colonic spasm, itching and skin irritation, musculoskeletal pain, carpal tunnel syndrome and chronic neuropathic pain (Malone and Tsai, 2018). Pulegone compounds are nontoxic and nonirritant in humans (Raza et al., 2016). Pulegone is moderately toxic based on the LD50 results: rats 150 mg/kg and mice 1709 mg/kg sc, which was supported by the findings of hepatic cytoplasmic vacuolization, diffuse fatty change, necrosis, hemorrhage, inflammation, bile duct hyperplasia, and mineralization (National Toxicology Program, 2011b). It appears that a phenobarbital-induced cytochrome P-450 catalyzed reactive metabolite(s) may be responsible for the hepatotoxicity caused by Pulegone (Moorthy et al., 1989). The NOAEL for short-term exposure Pulegone via the oral route is 20 mg/kg (Thorup et al., 1983).

Three-month exposure at 150 mg/kg showed no significant findings in mice and treatmentrelated findings of bile duct hyperplasia, hepatocyte hypertrophy, focal hepatic necrosis, oval cell hyperplasia, and periportal fibrosis in rats. Bone marrow hyperplasia, heart mineralization, glandular stomach mineralization, cellular histiocytic infiltration in the lung and ovarian cysts were nonneoplastic changes (National Toxicology Program, 2011b). Lifetime exposure up to 150 mg/kg/day orally resulted in treatment-related increase in hepatocellular adenomas and hepatoblastoma in mice. In rats, urinary bladder papilloma and of papilloma or carcinoma (combined) were significantly increased in females. Nephropathy was severe in most high-dose rats, particularly in males, consistent with end-stage renal disease. Ovarian atrophy, pancreatic acinar atrophy, olfactory epithelial degeneration, metaplasia, and inflammation were treatment-related as was ovarian atrophy. Pulegone is genotoxic but not mutagenic, teratogenic or a reproductive toxicant (da Silva et al., 2012). However, it is carcinogenic causing cancer of the urinary bladder and liver at high doses. There is clear evidence of carcinogenic activity in male and female B6C3F1 mice based on hepatocellular neoplasms (National Toxicology Program, 2011b) at doses two orders of magnitude greater concentration of than that seen in herbal remedies (Table 4.20).

10.18. Rattlepods, Yellow Burrweed, and GroundseldCrotalaria, Amsinckia, and Senecio Containing [Contains Riddelliine a Pyrrolizidine Alkaloid] Riddelliine is a pyrrolizidine alkaloid (PA) found in plants of the genera Crotalaria, Amsinckia, and Senecio, which maybe contaminants of herbal remedies. Indeed, Comfrey (Symphytum officinale) contains PAs and has inherent toxicity if consumed in excessive amounts. Pyrrolizidine alkaloids (PAs) constitute a class of plant secondary metabolites that primarily cause hepatotoxicity in humans and animals (pulmonary toxicity also occurs). They are found globally, and it is estimated that w3% of the world’s flowering plants contain toxic PAs. The wide distribution of plants containing PAs

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around the globe makes it difficult to prevent human and animal exposure, e.g., Riddelliine can be found as a contaminant in foods such as meat, grains, seeds, milk, herbal tea, and honey (Song et al., 2020) as well as herbal remedies (Figure 4.28). Liver toxicity, specifically in the form of hepatic veno-occlusive disease (HVOD), which is now called sinusoidal obstruction syndrome (SOS), is known from reports on human poisonings following chronic ingestion of herbs containing 1,2-unsaturated pyrrolizidine alkaloids. Food supplements, e.g., comfrey tea may significantly contribute to PA exposure (Dusemund et al., 2018). LD50 data obtained following intraperitoneal administration to male rats are available for some PAs and are given in Table 4.19. Deaths, which occurred 3–7 days after administration, were associated with severe hemorrhagic liver necrosis. The LD50 was variable depending on the specific alkaloid. Riddelliine can be considered highly toxic based on the LD50 results. In acute and chronic animal studies (Chan et al., 1994), Riddelliine causes dose-related hemorrhagic centrilobular hepatic necrosis and hepatocytic karyomegaly. Pyrrolizidine alkaloids (plant, crude extracts of the plant, or the alkaloids) have been administered to rats via varying routes (gavage, drinking water, subcutaneous, and intraperitoneal) to study their carcinogenicity (Chan et al., 2003b).

FIGURE 4.28 Crotalaria (Crotalaria juncea) [contains riddelliine a pyrrolizidine alkaloid].

TABLE 4.19

Reported Intraperitoneal LD50 Values for Pyrrolizidines for the Male Rat

Alkaloid

LD50 (mg/kg Body Weight)

Retrorsine

34

Senecionine 50

50 also quoted as 85

Heliosupine 60

60

Lasiocarpine 72

72

Seneciphylline

77

Jacobine

77 (mouse)

Riddelliine

105 (mouse)

Symphytine

130, also quoted as 300

Heleurine

140

Jaconine

168 (female rat)

Monocrotaline

175

Echimidine

200

Spectabiline

220

Senkirkine

220

Heliotrine

300

Echinatine

350

Supinine

450

Europine

>1000

Heliotridine

1200

Intermedine

1500

Lycopsamine

1500

Female rats were given Riddelliine by gavage at doses of 0.01, 0.033, 0.1, 0.33, or 1 mg riddelliine/ kg for 2 years; male mice were given gavage doses of 0.1, 0.3, 1.0, or 3.0 mg/kg by gavage at doses of 0.01, 0.033, 0.1, 0.33, or 1.0 mg Riddelliine/kg; and four groups of male mice received gavage doses of 0.1, 0.3, 1.0, or 3.0 mg Riddelliine/kg. Treatment related neoplasia included a dose-response increase in hemangiosarcoma (Figure 4.29), hepatocellular adenomas, alveolar-bronchiolar neoplasms, and mononuclear cell leukemia (National Toxicology Program, 2003). When administered sc, rats

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FIGURE 4.29 Female rat administered 1.0 mg/kg per day riddelliine by gavage for 2 years. Liver hemangiosarcoma in multiple hemorrhagic cavities are surrounded by a variably sized rim of neoplastic tissue (H&E, low magnification). Reproduced with permission from Chan et al., Toxicol Lett 144:295–311, 2003. [Page 301 panel 3A].

developed rhabdomyosarcomas at the site of injection. Tumors in the liver, lungs, intestines, and other organs occurred when administered ip (Nyska et al., 2002). These studies support the International Agency for Research on Cancer (IARC, 2016, 2018) placing Riddelliine in category Group 2B (possibly carcinogenic to humans), or Group 3 (not classifiable as to its carcinogenicity to humans). Riddelliine is genotoxic and mutagenic and there is clear evidence of carcinogenesis (IARC, 1983). Although Riddelliine has not been studied systematically for potential reproductive effects, the developmental toxicity of pyrrolizidine alkaloids has been evaluated (IARC, 2002; 2016) and they are considered teratogenic and fetotoxic.

10.19. Saw Palmetto – Serenoa repens Saw palmetto extract (SPE) is an extract of the fruit of the Saw palmetto (Serenoa repens) used in the treatment of symptoms related to benign prostatic hyperplasia. The active component is found in the fruit of the American dwarf palm tree (Figure 4.30). Studies have demonstrated the effectiveness of Saw palmetto in reducing symptoms associated with benign prostatic hyperplasia and suggest a use for the treatment of chronic prostatitis. Its

FIGURE 4.30

Saw Palmetto – (Serenoa repens).

efficacy is similar to that of medications like finasteride, a 5a reductase inhibitor, but it is better tolerated and less expensive. There are no known drug interactions with Saw palmetto, and any reported side effects are minor and rare. No data on its long-term usage are available (Gordon and Shaughnessy, 2003). Numerous mechanisms of action have been proposed for Saw palmetto, including the inhibition of 5a-reductase (Wadsworth et al., 2007). The improvement of voiding symptoms in patients taking SPE may arise from its binding to pharmacologically relevant receptors in the lower urinary tract, such as a1-adrenoceptors, muscarinic cholinoceptors, 1,4-dihyropyridine receptors and vanilloid receptors. Oral administration of SPE attenuates the up-regulation of a1adrenoceptors in the rat prostate induced by testosterone. Saw palmetto does not interact with coadministered conventional drugs (Suzuki et al., 2009), nor does it have a detrimental effect on reproduction (Ferna´ndez et al., 2008).

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Saw Palmetto can be considered relatively harmless: LD50 > 50 g/kg. An LD50 could not be calculated (USP, 2010). In animal toxicity studies, rats showed increased creatinine and potassium when given 300 mg/kg/day for 30 days (Duborija-Kovacevic et al., 2011) but other studies are unremarkable. Saw Palmetto is not a mutagen, teratogen, or reproductive toxicant.

10.20. Senna – Senna alexandrina Senna is a leaf of Senna alexandrina P. Mill (Leguminosae) and it is available over the counter as a laxative stimulant and tea. A high dose of Senna may be used for bowel preparation prior to colonoscopy or surgery. The bacterial enzymes break down sennosides in the intestines and release the active form, rhein-9anthrone, which possesses a laxative effect. Some case studies reported allergic reactions and hepatitis associated with the excessive use of Senna in humans (Figure 4.31). Senna is practically nontoxic based on acute lethality (Vashishtha et al., 2009). The median lethal dose (LD50) was higher than 5 g/kg (Mengs 1988; Silva et al., 2011). It is not cytotoxic (Vitalone et al., 2011), genotoxic (Sandnes et al., 1992), mutagenic (Silva et al., 2008), or carcinogenic (Mitchell et al., 2006; Surh et al., 2013; Lyde´n-Sokolowski 1993). Rodent toxicity studies have failed to show toxic effects after short-term exposures (Silva et al., 2011; Surh et al., 2013). Subacute exposure of rabbits to Senna caused

FIGURE 4.31

Senna – (Senna alexandrina).

271

myocardial necrosis, hepatic centrilobular degeneration (Tasaka et al., 2000) and an altered myeloid erythroid series (Teles et al., 2015). Following chronic exposure, hyperplastic (reversible) changes in the forestomach and large intestine were observed in rats (Mengs et al., 2004; National Toxicology Program, 2013c). Lifetime exposure studies failed to demonstrate carcinogenesis in rats (Lyde´nSokolowski, 1993; Mitchell et al., 2006; Surh et al., 2013). Most genotoxicity studies showed negative results (Heidemann et al., 2002; Mengs et al., 1999; Silva et al., 2008); however, some in vitro studies reported positive results in the occasional bacterial strain gene mutation and in mammalian cells (Sandnes et al., 1992). After intragastric administration to pregnant rats and rabbits, there is no evidence of any embryolethal, teratogenic or fetotoxic action. Furthermore, sennosides had no effect on the postnatal development of young animals, on the rearing behavior of mother animals or on male or female fertility (Mengs, 1986; Samavati et al., 2017).

10.21. St. John’s Wort – Hypericum perforatum St. John’s wort is the common name for Hypericum perforatum, a yellow flower with a long and rich history. It was originally harvested for the feast of St. John the Baptist. The word ‘wort’ is the Old English name for plant. St. John’s wort has become one of the most popular herbal supplements for the treatment of depression. Hypericin, a purported monoamine oxidase (MAO) inhibitor is the active ingredient in the herb, but pure hypericin does not bind to MAO while the crude extract does. St. John’s wort extract inhibits serotonin, dopamine, and norepinephrine reuptake in vitro. Hyperforin is also thought to be responsible for the induction of the cytochrome P450 enzymes CYP3A4 and CYP2C9 by binding to and activating the pregnane X receptor (PXR) (Chen et al., 2002) (Figure 4.32). Prior clinical trials of St. John’s wort present conflicting and confusing evidence, but in sum the herb is likely effective for the treatment of patients with mild-to-moderate depression, but

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FIGURE 4.32

4. HERBAL REMEDIES

St. John’s Wort – (Hypericum perforatum).

ineffective for patients with severe depression. The many well-documented conventional drug interactions with St. John’s wort are of concern (Hammerness and Basch, 2003; Nicolussi et al., 2020). In addition, pyrrolizidine plant contamination should be considered as a potential risk (Habs et al., 2018). Normal dosages of St. John’s wort have relatively few side effects. In general, the most common adverse effects are gastrointestinal symptoms, allergic reactions, dizziness, confusion, restlessness, lethargy, and dryness of the mouth, effects that are generally mild, moderate, or transient. Meta-analyses of clinical trials to treat depression revealed that adverse effects and dropout rates were less than 2.5% for patients receiving St. John’s wort, far lower than for those receiving conventional antidepressants (Ernst et al., 1998). Other systematic reviews, with or without meta-analysis, reveal that adverse events reported in RCTs were comparable to placebo and fewer compared with antidepressants (Apaydin et al., 2016). St. John’s wort has similar efficacy to conventional antidepressants (AbdelSalam, 2005; Gaster and Holroyd, 2000; Hoban et al., 2015; Linde et al., 2008) and is potentially beneficial for those with dementia (Hofrichter et al., 2013; Go´mez del Rio et al., 2013). St. John’s wort extracts have also been found to lack genotoxic potential (Peron et al., 2013) and mutagenic activity, based on in vivo and in vitro studies. However, St. John’s wort has shown antimutagenic activity against the clastogenic action of cyclophosphamide (Peron et al., 2013). Interestingly, the increase in hepatic

apoptosis (Schempp et al., 2005) and necrosis following a 48-h exposure to cytokines can be fully prevented by St. John’s wort and partially by hyperforin (Novelli et al., 2014). Hypericin has a unique phototoxic effect that can result in photodermatitis when taken in high doses (He et al., 2004). The toxic effects are attributed to an acidification of the surrounding environment caused by the transfer of hydrogen between hydroxyl groups on receiving light energy. Excessive cutaneous phototoxicity occurs at a high dose of 0.5 mg/ kg but lower doses of St. John’s wort taken for mild depression should not have significant associated phototoxic effects (Ernst et al., 1998). St. John’s wort is virtually nontoxic with oral LD50 (rat): 5628 mg/kg; dermal LD50 (rabbit): 15,800 mg/kg; sc LD50 (mouse): 9800 mg/kg; and ip LD50 (rabbit): 1826 mg/kg St John’s wort extract. The only toxicity study to report severe hepatic degeneration and necrosis (Gregoretti et al., 2004) was in rats given up to 1 g/ kg over 21 days. Prenatal exposure to a therapeutic dose of hypericum does not have a major impact on certain cognitive tasks in mice offspring (Rayburn et al., 2001) and is not toxic to the mother, nor is hypericum teratogenic (Borges et al., 2005). Maternal exposure to St. John’s wort does not interfere with reproductive parameters in adult male rats (Viera et al., 2013).

10.22. Tobacco – Nicotiana tabacum Tobacco is derived from the leaves of the Nicotiana tabacum, a plant from the night-shade family, indigenous to North and South America. Archeological studies suggest the use of Tobacco can be traced back to the first century BCE, when Maya people of Central America used Tobacco leaves for smoking in sacred and religious ceremonies. Its use started spreading as far as the Mississippi Valley between 470 and 630 CE. Gradually, it was adopted by neighboring and native tribes (Figure 4.33). Native American “Shamans” developed Tobacco use for religious rites. Simultaneously, people practicing medicine also started using Tobacco in different forms to cure certain illnesses such as asthma, earaches, bowel problems, fever, sore eyes, depression, insect bites, and burns (Mishra and Mishra, 2013). The nascent colonies of the Americas survival

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10. TOXICOLOGIC PATHOLOGY OF SELECT HERBAL REMEDIES

FIGURE 4.33 Tobacco – (Nicotiana tabacum).

depended upon the growth of Virginia Tobacco. Tobacco did not reach Europe until the 1500s at which time it was seen as a medicine (Charlton, 2004). The current major Tobacco-growing and consuming countries are China, United States, the Former Soviet States, India, and Brazil. In South and Southeast Asia, it is incorporated into existing traditional customs, in the form of betel quid (paan) chewing Tobacco (Mishra and Mishra, 2013). Tobacco smoke is a complex mixture of toxicants, and the chemical properties changed rapidly in some casesdas smoke ages (Institute of Medicine, 2001). Toxicants measured at one point in time may not be what the smoker actually experiences. It is estimated that there are more than 2000 chemical constituents of Tobacco. Tobacco smoking has been shown to cause cancer of the lung, urinary bladder, renal pelvis, oral cavity, pharynx, larynx, esophagus, lip, and pancreas in humans (IARC, 1986, 2012, 2018).

273

The carcinogenic effects of Tobacco smoke are increased in individuals with certain predisposing genetic polymorphisms that code for different forms of the metabolic enzyme CYP450. The International Agency for Research on Cancer has reevaluated the evidence for the carcinogenicity of Tobacco smoking and Tobacco smoke and concluded that there was sufficient evidence in humans that cigarette smoking caused myeloid leukemia and cancer of the nasal cavities and nasal sinus, stomach, liver, kidney (renal-cell carcinoma), and uterine cervix, in addition to the tissue sites mentioned above. It is listed as a Group 1 carcinogen (IARC, 2004). Tobacco smoke has been shown to cause cancer in several species of experimental animals, e.g., inhalation exposure to cigarette smoke causes cancer of the larynx in hamsters and increases the incidence of benign and/or malignant lung tumors in rats. Dermal exposure to cigarette-smoke condensates causes skin tumors in mice and rabbits, and topical application of cigarette-smoke condensates to the lining of the mouth (oral mucosa) results in lung tumors and lymphoma in mice. Intrapulmonary injection of cigarette-smoke condensate causes lung tumors in rats (IARC, 1986, 1987). Tobacco smoke or Tobacco-smoke condensates caused cell transformation, mutations, or other genetic damage in a variety of in vitro and in vivo assays. The urine of smokers has been shown to be mutagenic, and there is evidence that the somatic cells of smokers contain more chromosomal damage than those of nonsmokers (Devesa et al., 2005; IARC, 1986). Lung tumors from smokers contained a higher frequency of mutations in the p53 tumor-suppressor gene and the K-ras proto-oncogene than do tumors from nonsmokers; most of the mutations were G to T transversions (IARC, 2004; Vineis et al., 1995). Additionally, an association between cigarette smoking and bronchioalveolar carcinoma has also been found in several studies (Falk et al., 1992; Morabia and Wynder, 1992). Correspondingly, active smokers have significant excess lung cancer risks among Japanese men for both squamous cell carcinoma (relative risk (RR), 11.7) and adenocarcinoma (RR, 2.30). Among women the risks were 11.3 for squamous cell carcinoma and 1.37 for adenocarcinoma (Wakai et al., 2006).

II. SELECTED TOXICANT CLASSES

274

4. HERBAL REMEDIES

Lung tumors of smokers contain a high frequency and unique spectrum of TP53 and KRAS mutations, reflective of the polyaromatic hydrocarbons (PAHs) and possibly other compounds in the smoke. Polycyclic aromatic hydrocarbons in Tobacco smoke are believed to be responsible for the induction of cytochrome P450 (CYP) 1A1, CYP1A2 and possibly CYP2E1, CYP1A1. These data support a model of Tobacco smoke carcinogenesis in which the components of Tobacco smoke induce mutations that accumulate in a field of tissue that, through selection, drive the carcinogenic process (DeMarini, 2004). Induced metabolism because of cigarette smoking may have clinical consequences for the following drugs: theophylline, caffeine, tacrine, imipramine, haloperidol, pentazocine, propranolol, flecainide, and estradiol. Cigarette smoking results in faster clearance of heparin, possibly related to smoking-related activation of thrombosis with enhanced heparin binding to antithrombin III. Pharmacodynamic interactions have also been described. Cigarette smoking is associated with a lesser magnitude of blood pressure and heart rate lowering during treatment with betablockers, less sedation from benzodiazepines and less analgesia from some opioids, most likely reflecting the effects of the stimulant actions of nicotine (Zevin and Benowitz, 1999). The histopathological findings in rats exposed to cigarette smoke or electronic cigarette vapor can be found in (Wawryk et al., 2020).

10.23. Turmeric Oleoresin – Curcuma longa Turmeric is derived from the plant Curcuma longa, related to the Ginger family (Zingiberoside), which originated from India, and is currently grown in several other parts of the world, including Southeast Asia, China, and Latin America. It is available as the whole rhizome or bulb from the plant, as a ground powder, which contains the oleoresin. The major component in all Turmeric oleoresins is curcuminoid, primarily curcumin (Kotha and Luthria, 2019) (Figure 4.34). Turmeric has potential benefits as an anticancer (Zhou et al., 2013b), antioxidant,

FIGURE 4.34

Turmeric oleoresin – (Curcuma longa).

antibiotic, antimutagenic, antiinflammatory (Menon and Sudheer, 2007), antiobesity, hypolipidemic, cardioprotective, neuroprotective effects, and antiaging agent as suggested by several in vitro and in vivo studies and clinical trials (Ahmad et al., 2020). In animals, Turmeric is practically nontoxic based on acute lethality (LD50 > 5 g/kg) (Aggarwal et al., 2016; Majeed et al., 2019; Soleimani et al., 2018) and other studies – rat and mouse (National Toxicology Program, 1993; Liju et al., 2013) but other toxicity studies done in laboratory animals did show a hepatotoxic effect at high doses – pig (Bille et al., 1985) and rat (Deshpande et al., 1998). An NOAEL of 760 mg/kg/day was suggested (rat) (Dadhaniya et al., 2011). Turmeric effectively reduces proinflammatory cytokine/chemokine expression (Kloesch et al., 2016). It is not genotoxic (Soudamini et al., 1995), mutagenic (Aggarwal et al., 2016), nor causes DNA damage (Liju et al., 2013), nor cancer (National Toxicology Program, 1993). Turmeric does not seem to be toxic to the

II. SELECTED TOXICANT CLASSES

11. INTERNATIONAL REGULATORY OVERVIEW

mother nor to interfere with the progress of gestation during organogenesis (Ganigier et al., 2007; Majeed et al., 2019), in animals and humans (Joshi et al., 2003).

11. INTERNATIONAL REGULATORY OVERVIEW To obtain a license to market a therapeutic product, regulatory authorities expect sufficient proof that the chemical is efficacious, safe, and of a high quality. Critical review of experimental preclinical and clinical exposure, along with chemistry and manufacturing documentation of the chemical and drug product is required before a marketing authorization can be obtained. As such, safety and efficacy of herbal remedies, as well as quality control, have become important concerns for both health authorities and the public. Regulations regarding herbal remedies have been developed by individual countries without a parallel development of international standards and appropriate methods for evaluating the registration package (Rousseaux and Schachter 2003). Countries face major challenges in the development and implementation of the regulation of traditional and complementary/alternative medicine, as well as herbal remedies. These challenges are related to regulatory status, assessment of safety and efficacy, quality control, safety monitoring, and lack of knowledge about herbal remedies within national drug regulatory authorities. In many countries or economic regions, a single medicinal plant may be defined as a food, a functional food, a dietary supplement, or an herbal remedy, depending on the regulations applying to foods and medicines in each country. This makes it difficult to define the concept of herbal remedies for the purposes of national drug regulation, and also confuses patients and consumers. Requirements and methods for research and evaluation of the safety and efficacy of herbal remedies are more complex than those for conventional pharmaceuticals. A single medicinal plant may contain hundreds of natural constituents, and a mixed herbal medicinal

275

product may contain several times that number. If every active ingredient were to be isolated from every herb, the time and resources required would be tremendous. Such an analysis may actually be impossible in practice, particularly in the case of mixed herbal remedies. The safety and efficacy of herbal remedies is closely correlated with the quality of the source materials used in their production, as previously described, and as such it is a challenge to perform quality controls on the raw materials of herbal remedies. Good Manufacturing Practice (GMP) specifies many requirements for quality control of starting materials, including correct identification of species of medicinal plants, special storage and special sanitation and cleaning methods for various materials. In the quality control of finished herbal medicinal products, particularly mixed herbal products, it is more difficult to determine whether all the plants or starting materials have been included and there are no other foreign plants in the mix. Adverse events arising from consumption of herbal remedies may be due to any one of a number of factors. These include the use of the wrong species of plant by mistake, adulteration of herbal products with other undeclared medicines, contamination with toxic or hazardous substances, overdosage, misuse of herbal remedies by either healthcare providers or consumers, and use of herbal remedies concomitantly with other medicines. Therefore, analysis of adverse events related to the use of herbal remedies is more complicated than in the case of conventional pharmaceuticals. The general lack of knowledge about herbal remedies within national drug authorities and the lack of appropriate evaluation methods are factors that delay the creation or updating of national policies, laws and regulations for traditional medicines, contemporary/alternative medicines, and herbal remedies (Bhat et al., 2019; WHO, 2019; 2021). Herbal remedies can be classified into four categories as per WHO, based on their origin, evolution, and the forms of current usage. While these are not always mutually exclusive, these categories have sufficient distinguishing features for a constructive examination of the ways in which safety, efficacy, and quality can be determined and improved.

II. SELECTED TOXICANT CLASSES

11.1. Select List of Countries and Their Regulatory Requirements Australia In Australia, medicinal products containing such ingredients as herbs, vitamins, minerals, nutritional supplements, homoeopathic and certain aromatherapy preparations, are referred to as “complementary medicines”, and are regulated as medicines under the Therapeutic Goods Act 1989. Therapeutic Goods Administration, the regulatory agency of Australia, regulates herbal products under the category of complementary medicine. Ayurvedic medicine, traditional Chinese medicine, and Australian indigenous medicines are all covered under this category. A complementary medicine is defined in the Therapeutic Goods Regulations 1990 as a therapeutic goods consisting principally of one or more designated APIs mentioned in Schedule 14 of the Regulations, each of which has a clearly established identity and traditional use: Complementary medicines which do not require medical supervision are permitted and have to be entered on the Australian Register for Therapeutic Goods (ARTG) before marketing. The low-risk medicines require to be listed while the medicines for comparatively higher risk therapeutic conditions require registration on the ARTG. Only evidence-based claims which are entered on the ARTG are allowed. (https://www.tga.gov.au/ overview-regulation-complementary-medicines-a ustralia [Accessed April 1, 2021]) Canada Since January 1, 2004, Health Canada regulates the sale of herbal remedies and traditional medicines such as Ayurvedic medicine, under the Natural Health Products Regulations under the Food and Drugs Act. The regulations mandate that a manufacturer, packer, labeler, or importer need to have a prior registration with Health Canada before commencing any commercial activity. Before obtaining a Natural Health Products Number as a marketing authorization, registration of the manufacturing site/s is required. Complete data on product composition, standardization, stability, microbial and chemical contaminant testing methods and tolerance limits, safety, and efficacy along with ingredient characterization, quantification by assay or by input needs to be submitted to Natural Health Product Directorate (NHPD). The authority mandates that NHPs must comply with the contaminant limits and must be manufactured as per the GMP norms.

Health Canada used a risk-based approach to safety and efficacy. Risks related to safety and efficacy includes potential risks due to an ingredient’s physical or chemical form; the seriousness of the health claim and the conditions of use implied; and the health impact from lowerthan-expected performance of the product. A risk-based assessment approach is used to categorize evidence recommendations into three levels of risk: low, medium, and high. These levels are proportionate to the standard of evidence necessary to support safety and efficacy of a product (https://www.canada.ca/en/healthcanada/services/drugs-health-products/naturalnon-prescription.html [Accessed April 1, 2021]). China According to the Chinese Medicine Ordinance, regulatory measures relating to Chinese medicines are divided into two areas: First, Chinese medicines traders require a license. Chinese medicines traders who wish to engage in the business of retail and wholesale of Chinese herbal remedies as well as the wholesale and manufacturing business of proprietary Chinese medicines must first apply for the relevant license from the Chinese Medicines Board (http://english. nmpa.gov.cn). They may continue operating their own business only after they have obtained the license. Second, proprietary Chinese medicines must be registered. All proprietary Chinese medicines must first be registered by the Chinese Medicines Board before they can be imported, manufactured, and distributed in China. According to the Chinese Medicine Ordinance, Chinese herbal remedies mean the Chinese herbal remedies specified in Schedule 1 of the Ordinance and the Chinese herbal remedies specified in Schedule 2 of the Ordinance. And “proprietary Chinese medicine” means any proprietary product composed solely of the following as active ingredients: any Chinese herbal remedies; any materials of herbal, animal or mineral origin customarily used by the Chinese; formulated in a finished dose form; and known or claimed to be used for the diagnosis, treatment, prevention or alleviation of any disease or any symptom of a disease in human beings, or for the regulation of the functional states of the human body. The Chinese Medicines Board has formulated the Guidance Notes on How to Classify Products as proprietary Chinese Medicines. According to the Import and Export Ordinance (Cap.60 of the Laws of Hong Kong), any person who wishes to import or export any of the

11. INTERNATIONAL REGULATORY OVERVIEW

Chinese herbal remedies specified in Schedule 1 or the five types of the Chinese herbal remedies specified in Schedule 2 as well as any proprietary Chinese medicines must first apply for an import or export license. The Protection of Endangered Species of Animals and Plants Ordinance (Cap. 586) imposes regulation on proprietary Chinese medicines containing ingredients of endangered species. The following legislation should be taken as the standards for regulation of Chinese medicines: Chinese Medicine Ordinance, Chinese Medicines Regulation, Chinese Medicines (Fees) Regulation, and Chinese Medicines Traders (Regulatory) Regulation (Salmon and Liu, 2010). European Union The European Medicine Agency have laid down two ways of registration of herbal medicinal products: First, a full marketing authorization by submission of a dossier, which provides the information on quality, safety and efficacy of the medicinal products including the physicochemical, biological, or microbial tests and pharmacological, toxicological, and clinical trials data; under directive 2001/83/EC. Second, for traditional herbal medicinal products which do not require medical supervision, where evidence of long traditional use of medicinal products exists, and adequate scientific literature to demonstrate a well-established medicinal use cannot be provided, a simplified procedure under directive 2004/24/EC exists. The evidence of traditional use is accepted as evidence of efficacy of the product. However, authorities may still ask for evidence to support safety. Quality control requirements require physicochemical and microbiological tests to be included in the product specifications. The product should comply with the quality standards in relevant pharmacopoeias of the member state or European Pharmacopoeia. The bibliographic evidence should support that the product has been in medicinal use for at least 30 years out including at least 15 years within the European community. The application for traditional use registration is referred to the Committee for Herbal Medicinal Products, if the product has been in the community for less than 15 years, but otherwise qualifies for the simplified registration procedure under the directive (https://www.ema.europa.eu/en/ human-regulatory/herbal-medicinal-products [Accessed April 1, 2021]).

277

India Herbal drugs are regulated under the Drug and Cosmetic Act (D and C) 1940 and Rules 1945 in India, where regulatory provisions for Ayurveda, Unani, Siddha medicine are clearly laid down. Department of Ayush is the regulatory authority and mandates that any manufacture or marketing of herbal drugs have to be done after obtaining a manufacturing license, as applicable (Kumar, 2017). The D and C Act extends the control over licensing, formulation composition, manufacture, labeling, packing, quality, and export. Schedule “T” of the act lays down the good manufacturing practice (GMP) requirements to be followed for the manufacture of herbal remedies. The official pharmacopoeias and formularies are available for the quality standards of the medicines. First schedule of the D and C Act has listed authorized texts, which have to be followed for licensing any herbal product under the two categories: ASU drugs and Patent or Proprietary Medicines. Japan In Japan, two overlapping types of traditional herbal remedies coexisted for centuries. The first one was the traditional Japanese and Chinese medicine. These medical systems were damaged by the first Medical Care Law in 1874 that proclaimed the abrogation of traditional Japanese medicine. The second type of herbal remedy used in Japan originated in Europe and Southeast Asia and became popular after the law in 1874 was announced and some of those products are still used today as prescription drugs. Although the renaissance of the traditional medicines has been on the rise since approximately 1960, dietary supplements have added to the confusion and decline of the traditional Japanese medicines. Regulation of herbal remedies, except “Kampo” formulas is the same as the approval for both prescription and over the counter (OTC) drugs. Regulation of quality standards of those herbal remedies was established in Japanese Pharmacopoeia for more than 90% products (Saito 2000). In the early 1970s, The Internal Assignments on the Review for Approval of OTC Kampo Products, known as 210 OTC Kampo Formulae, was published by the Ministry of Health and Welfare (currently the Ministry of Health, Labour and Welfare). In 2008, 210 OTC Kampo Formulae was revised and

278

4. HERBAL REMEDIES

presented as The Approval Standards for OTC Kampo Products and now 294 Kampo formulae are listed in the standards. Both the Approval Standards and The Quality Standards play a key role in regulation of Kampo products. The Application Guideline for Western Traditional Herbal remedies as OTC Drugs was published in 2007. Other ethnopharmaceuticals mostly from Europe could be approved as OTC drugs in Japan. Korea The Republic of Korea has legally adopted two Medicare systems, Western Medicine and Oriental Medicine. Traditional medicine in Korea is based on both traditional Chinese medicine and Korean folk medicine. The empirical folk medicine has passed on from generation to generation and is not prescribed by Korean oriental physicians. The Composite Pharmacy Law governs all activities concerning pharmacies, pharmaceutical industries, and suppliers of medicines including herbal raw materials. The two official drug compendia are the fifth edition of the Korean Pharmacopoeia and the Korean Natural Drug Standards. Since 1993, only standardized medicinal plants can legally be distributed. For herbal remedies produced by domestic pharmaceutical companies, the government imposes strict regulations on these companies, so that they follow the GMP standard in manufacturing herbal remedies. The Ministry of Public Health and Social Affairs has published a notification under which a new license will be issued to the manufacturer of standardized herbs. Herbal drugs and preparations thereof have to be standardized and controlled according to the requirements of the Korean Pharmacopoeia, the National Institute of Health and the Ministry of Public Health and Social Affairs. The information required for these products includes taxonomic status, parts of plants, morphology, qualitative examination, purity, content of essential oil or extract, and grade of quality. The Medical Act and the Drug Administrative Act stipulate that only certified oriental medical doctors or pharmacies with oriental medical doctors’ prescriptions can provide patients with any of the herbal remedies listed in the Korean Pharmacopoeia https://www.mfds.go.kr/ eng/brd/m_18/list.do [Accessed April 1, 2021]) Malaysia Herbal products in Malaysia fall under the category of regulated products. Any marketer intending to place the herbal products in the market is required to register the product first.

The applicant is required to be registered with the Malaysia Registrar of Business or Suruhanjaya Syarikat Malaysia under two classifications: traditional products and health supplements. These categories of medicines generally fall under the purview of various regulations such as the Control of Drugs and Cosmetics Regulations (1984), Sale of Drug Act (1952), Poison Act (1952), and the Advertisement and Sale Act (1956). While the authorities mandate only labeling “traditionally used for” in front of any claim made on the traditional product, only those functional claims, which are listed by the authority, are allowed in supplements (Ismail et al., 2020). Philippines Herbal remedies are regulated in the Philippines as traditionally used herbal products. The regulators require that the preparations from plant materials, whose claimed application is based only on traditional experience of long usage, which should be at least five or more decades as documented in medical, historical, and ethnological literature are permitted to be marketed under this category. The Bureau of Food and Drugs (BFAD), which is the regulating body in the country, mandates registration of the traditionally used herbal products before manufacture, import, or market. The extent of control of BFAD includes the brand names of the traditional herbal products as well, and their prior clearance is required, before filing for product registration. Authentication of the plant specimen needs to be obtained from the Philippine National Museum or any BFAD recognized taxonomist, and for imported products, the certificate of authenticity of the plants from the authorized government agency of the country of origin is accepted. The quality control requirements further lay down the pharmacopeial standards. BFAD further mandates that product indications should not require supervision by a physician. United States of America Botanical products are classified as a drug, food or a dietary supplement by the United States Food and Drug Administration on the basis of the claims or end use. A product that is used to prevent, diagnose, mitigate, treat, or cure a disease would fall under the category of drug. If the intended use of a botanical product is to affect the structure or function of the human body, it may be classified as

12. DISCUSSION

either a drug or a dietary supplement. As per FDA, the drug must be marketed under an approved New Drug Application (NDA). FDA regulates the dietary supplements under the Dietary Supplement Health and Education Act of 1994. These do not require premarket approval and it’s the responsibility of the marketer to ensure the safety and labeling compliance of their products with the regulations. The claims need to comply with the regulatory guidelines issued by the FDA. The manufacturing of dietary supplements should be done as per the current GMP for dietary supplements (Wu et al., 2008; https://www.fda.gov/food/dietary-supplements [Accessed April 1, 2021]). Health claims for herbal remedies must contain the elements of a substance and a disease or health-related condition; are limited to claims about disease risk reduction; cannot be claims about the diagnosis, cure, mitigation, or treatment of disease; and are required to be reviewed and evaluated by FDA prior to use. There are two types of claims: “authorized” and “qualified” health claims. Authorized claims must have significant scientific agreement (SSA) among qualified experts that the claim is supported by the totality of publicly available scientific evidence for a substance/disease relationship and qualified health claims are supported by some scientific evidence, but do not meet the significant scientific agreement standard.

12. DISCUSSION The target organ of many herbal remedy overdoses is the liver. In the case of carcinogenesis, Goldenseal root power [Hydrastis canadensis] (National Toxicology Program, 2010b), gingko biloba extract [Ginkgo biloba] (National Toxicology Program, 2013b), riddelliine (pyrrolizidine alkaloid) (National Toxicology Program, 2003), Pulegone [Mentha sp.] (National Toxicology Program, 2011b), Kava kava [Piper methysticum] (National Toxicology Program, 2012), and Indole-3-carbinol [Brassica sp] (National Toxicology Program, 2017) are hepatotoxic at the doses given experimentally. The International Agency for Research on Cancer (IARC, 2002, 2016) has reviewed the evidence for carcinogenic activity for four of these herbal remedies Goldenseal, Gingko, Kava kava, and Pulegone), and has classified these herbals as “possibly carcinogenic to humans” (Group 2B)

279

because of evidence for carcinogenic activity in two species or both sexes of the same rodent species (IARC, 2016). Other target organs damaged during excessive exposure include: the gastrointestinal tractdAloe vera nondecolorized extract [Aloe barbadensis] (National Toxicology Program, 2013a) and Senna [Senna alexandrina] (National Toxicology Program, 2013c); kidney – Aloe vera [Aloe barbadensis], raw garlic [Allium sativum], Ginkgo [Ginkgo biloba], Kava kava [Piper methysticum], Pulegone [Mentha sp.], St John’s wort [Hypericum perforatum, and Tobacco [Nicotiana tabacum]; heartdEphedra [Ephedra sinica]; and the vascular system – [riddelliine pyrrolizidine alkaloid]. For Milk thistle [Silybum marianum] (National Toxicology Program, 2011a), Turmeric oleoresin [Curcuma longa] (National Toxicology Program, 1993), Green tea extract [Camellia sinensis] (National Toxicology Program, 2016), studies has not provided clear evidence for carcinogenic activity in rodent model systems nor for Marijuana [Cannabis sativa and Cannabis indica], Chamomile tea [Chamomilla recutita], Coffee [Coffea arabica], Cocoa [Theobroma cacao], Echinacea [Echinacea purpura], Ephedra [Ephedra sinica], Ginger [Zingiberis rhizome], Ginseng [Panax ginseng], Milk thistle [Silybum maranum], St. John’s wort [Hypericum perforatum], and Turmeric[Curcuma longa]. It is usual to undertake dose range finding studies to obtain the highest dose of a drug that does not cause unacceptable side effects or overt toxicity in a specific period of exposure, the maximum tolerated dose (MTD), for use in a 2year rodent bioassay. This may result in genotoxic, mutagenic, and carcinogenic signals at a dose that may be difficult to achieve in real life. For example, the mild reproductive effects (LOAEL) occurred at 6000 mg/kg Coffee oral dosing in rodents, which is w1050 times the dose advised to achieve the maximum benefit of Coffee exposure (400 mg/day for a 70 kg person – 5.7 mg/kg/day), or approximately 250 cups of Coffee per day (Brent et al., 2011). Generally, three to five cups a day maximizes the health benefits (Rodrı´guez-Artalejo and Lo´pez-Garcı´a, 2018). The classical dose–response curve does not model effectively the adverse effects that may occur near the human recommended dose e.g., Table 4.20. Herbal remedies are used at doses orders of magnitude less than those used for determining toxicity and their therapeutic

Relative Lowest Observable Adverse Effect Level Experimental Versus Recommended Dosing for the Herbal Remedya

Herbal Remedy

LOAEL Mg/Kg (Experimental) Feeding Studies Percent wt./wt.b

Aloe vera (Aloe barbadensis) [gel]

2-week rat: 3.0% males; 3.0% females

Human Dose (mg/ kg)d

200

2.9

Ratio Recommended/ experimental Dose

Reference

Male ¼ 0.01 Female ¼ 0.01

Aloe vera, NTP TR 577

3-month rat: 1.0% males; 2.0% females

Male ¼ 0.03 Female ¼ 0.02

2-year rat: >1.5% males; 1.0% females

Male ¼ ND Female ¼ 0.03

2-week mouse: >3.0% males; >3.0% females

ND

3-month mouse: >3.0% males; >3.0% females

ND

2-year mouse: >3.0% males; >3.0% females

ND

2-week rat [water]: >1200 males; >1200 females

150

2.2

ND

2-week rat: [Feed] >1500 males; >1500 females

ND

3-month rat: 500 males; 2000 females

Male ¼ 0.004 Female ¼ 0.001

2-year rat: >250 males; 125 females

Male ¼ ND Female ¼ 0.02

2-week mouse [water]: 5000 males; >5000 females

Male ¼ 0.0004 Female ¼ ND

Ephedrine sulfate, NTP TR 307

4. HERBAL REMEDIES

II. SELECTED TOXICANT CLASSES

Ephedra (Ephedra sinica) [ephedrine sulfate]

Recommended Dose Human (Total mg)c

280

TABLE 4.20

Ginkgo (Ginkgo biloba)

Goldenseal (Hydrastis canadensis)

Male ¼ 0.0004 Female ¼ 0.0004

3-month mouse: 1250 males; 310 females

Male ¼ 0.002 Female ¼ 0.007

2-year mouse: 250 males; 125 females

Male ¼ 0.009 Female ¼ 0.02

3-month rat: > 1000 males and females

240

3.4

ND

2-year rat: 300 males and females

Male ¼ 0.01 Female ¼ 0.01

3-month mouse: > 2000 males and females

ND

2-year mouse: 600 males and 2000 females

Male ¼ 0.006 Female ¼ 0.002

3-month rat: >2000 males; >2000 females

200

2.9

ND

2-year rat: >2000 males; >2000 females

ND

3-month mouse: >2000 males; >2000 females

ND

2-year mouse: >5000 males; >5000 females

ND

3-month rat: >25,000 males; >25,000 females 2-year rat: >25,000 males; >25,000 females

300

4.3

ND

Ginkgo biloba extract, NTP TR 578

Ginseng, NTP TR 567

12. DISCUSSION

II. SELECTED TOXICANT CLASSES

Ginseng e American (Panax quinquefolium)

2-week mouse [feed]: 5000 males; 5000 females

Goldenseal root powder, NTP TR 562

ND

(Continued)

281

Relative Lowest Observable Adverse Effect Level Experimental Versus Recommended Dosing for the Herbal Remedyadcont’d

Herbal Remedy

Indole-3-carbinol

Kava kava (Piper methysticum)

Recommended Dose Human (Total mg)c

Human Dose (mg/ kg)d

Ratio Recommended/ experimental Dose

13-week mouse: >25,000 males; >25,000 females

ND

2-year mouse: >25,000 males; >25,000 females

ND

3-month rat: 250 males; >1000 females

300

4.3

Male ¼ 0.02 Female ¼ ND

2-year rat: 300 males; 100 females

Male ¼ 0.01 Female ¼ 0.04

3-month mouse: 250 males; 125 females

Male ¼ 0.02 Female ¼ 0.03

2-year mouse: 100 males; 100 females

Male ¼ 0.04 Female ¼ 0.04

3-month rat: >300 males; >300 females

112

1.6

ND

2-year rat: >300 males; 150 females

Male ¼ ND Female ¼ 0.01

3-month mouse: >250 males; >250 females

ND

2-year mouse: >250 males; >250 females

ND

2-week rat: >2000 males; >2000 females 3-month rat: 2000 males; >2000 females

250

3.6

ND Male ¼ 0.002 Female ¼ ND

Reference

Green tea extract, NTP TR 585

Indole-3-carbinol, NTP TR 584

Kava kava extract, NTP TR 571

4. HERBAL REMEDIES

II. SELECTED TOXICANT CLASSES

Green tea (Camellia sinensis)

LOAEL Mg/Kg (Experimental) Feeding Studies Percent wt./wt.b

282

TABLE 4.20

Pulegone (Nepeta cataria, Mentha piperita)

Male ¼ 0.004 Female ¼ 0.004

2-week mouse: >2000 males; >2000 females

ND

3-month mouse: >2000 males; >2000 females

ND

2-year mouse: 1000 males; >1000 females

Male ¼ 0.004 Female ¼ ND

3-month rat: >50,000 males; >50,000 females

420

6

ND

2-year rat: >50,000 males; 50,000 females

Male ¼ ND Female ¼ 0.0001

3-month mouse: 25,000 males; 25,000 females

Male ¼ 0.0001 Female ¼ 0.0001

2-year mouse: 50,000 males; 25,000 females

Male ¼ 0.0001 Female ¼ 0.001

2-week rat: 150 males; 300 females

140

2.0

Male ¼ 0.01 Female ¼ 0.01

3-month rat: 150 males; 150 females

Male ¼ 0.01 Female ¼ 0.01

2-year rat: 75 males; 75 females

Male ¼ 0.003 Female ¼ 0.003

2-week mouse: >300 males; >300 females

Male ¼ ND Female ¼ ND

3-month mouse: >150 males; >150 females

Male ¼ ND Female ¼ ND

Milk thistle extract, NTP TR 565 12. DISCUSSION

II. SELECTED TOXICANT CLASSES

Milk thistle (Silybum marianum)

2-year rat: 1000 males; 1000 females

Pulegone, NTP TR 563

283

(Continued)

284

TABLE 4.20

Relative Lowest Observable Adverse Effect Level Experimental Versus Recommended Dosing for the Herbal Remedyadcont’d

Herbal Remedy

LOAEL Mg/Kg (Experimental) Feeding Studies Percent wt./wt.b

Recommended Dose Human (Total mg)c

Human Dose (mg/ kg)d

Turmeric (Curcuma longa)

a

2.5

Male ¼ 0.04 Female ¼ 0.04

2-year mouse: 3.0 males; 3.0 females

Male ¼ 0.01 Female ¼ 0.01

5 weeks mouse: >10,000 males; >10,000 females

ND

40 weeks mouse: >1000 males; >1000 females

ND

3-month rat: >50,000 males; >50,000 females

500

7.1

ND

2-year rat: >50,000 males; 50,000 females

Male ¼ ND Female ¼ 0.0001

13-week mouse: >50,000 males; >50,000 females

ND

2-year mouse: 50,000 males; 25,000 femalse

Male ¼ 0.0001 Female ¼ 0.0001

National Toxicology Program, National Institute of Health Science, USA. LOAEL was a decrease in body weight of greater than or equal to 10%. c Recommended dose by manufacturer. d Assume average human weight of 70 kg. ND, not determined. b

0.04

Riddelliine, NTP TR 508

Senna, NTP GMM 15

Turmeric oleosin, NTPTR 427

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Senna (Senna alexandrina P. Mill)

2-year rat: 1.0 males; 1.0 females

Reference

Male ¼ 0.01 Female ¼ 0.01

2-year mouse: 150 males; 150 females Riddelliine

Ratio Recommended/ experimental Dose

13. SUMMARY

potential lies in the “Goldilocks dose,” the dose that is “just right” (works) not too little (doesn’t work) and not too much (doesn’t work). It is only when there is way too much that we start to see toxicity. In terms of therapeutic index, herbal remedies have a much larger window than many conventional pharmaceuticals. This being the case, how does one determine benefit: risk? The benefit is based on traditions and years of accumulated experience and public belief. There are some herbal remedies that have evidence of efficacy. For example, Saw Palmetto [Serenoa repens] provides an equivalent clinical response when compared to finasteride for benign prostatic hyperplasia; however, finasteride is superior in decreasing the prostate volume (Carraro et al., 1996). How can one determine health risks for an herbal remedy given the unlikelihood of reaching a toxic dose? We are left with the tools of toxicologic pathology and, unsurprisingly, we see carcinogenic effects enough for IARC to classify some herbs as Group 2 carcinogens (apart from Tobacco – Group 1). Should we be looking into further detail as to what is in the extract apart from the assumed APIs? What is the potency of the remedy? Is there evidence of inorganic and organic material added to elevate the potential toxicity of the remedy? Herbal remedies and conventional pharmaceuticals are different and should be placed in context with respect to health. The concept of maintaining and improving wellness (herbal remedies) and targeted therapies for specific disease entities (conventional pharmaceuticals) is germane. Both have a role in optimizing health. Quality is probably the most important aspect of herbal remedy regulation. Even if caveat emptor holds true, it is essential for the patient to know what they are ingesting. They are taking the risk and they must have information regarding what they ingest. Even those well versed in the interactions and limitations need to be sure that what is in the bottle is really reflected on the label.

13. SUMMARY Since prehistoric times, humans have used natural products, such as plants, animals,

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microorganisms, and marine organisms in medicines to alleviate and treat illness. These remedies are often prescribed as part of traditional medicine, a discipline that has been built over the ages based on empirical knowledge that was passed down through the generations. Herbal remedies are aimed at wellness and general disease states, whereas conventional pharmaceuticals are directed at specific molecular disease targets. Herbal remedies come from various sources where growth, harvesting and storage conditions can alter the amount of the active ingredients substantially. In addition, fungal and other organic and inorganic contaminants add to the complexity of safety assessment. Sometimes adulterants are intentionally added for the purpose of efficacy. Herbal remedies are labeled as mg of the plant, an extract (in mg or percent), or the active ingredient in mg, e.g., capsules containing 80 mg of the herb [Ginger root 750 mg], or capsules containing 80 mg of the active [Saint John’s Wort 300 mg–0.3% hypericin], or as a straight concentration [CBD oil 2.5%] for the purpose of dosage. The therapeutic index for herbal remedies is large: the recommended doses for herbs are usually in milligrams per 70 kg person compared to toxicity experiments that require grams per kilogram for an effect. This does not mean that herbal remedies are innocuous, as most users are aware of the intrinsic hazard of some herbal remedies and risk of exposure, cigarette smoking, for example. In toxicologic pathology, the organ of impact following herbal remedy overdose, like conventional pharmaceuticals, is usually the liver. However, toxicity can be seen in other organs such as the heart following Digitalis exposure. Goldenseal root power [Hydrastis canadensis]), Gingko biloba extract [Ginkgo biloba], Riddelliine (pyrrolizidine alkaloid), Pulegone [Mentha sp.], Kava kava [Piper methysticum], and Indole-3carbinol [Brassica sp] are hepatotoxic at the doses given experimentally. In addition, some herbs have been classified as carcinogens based on experimental evidence. The International Agency for Research on Cancer (IARC) has reviewed the evidence for carcinogenic activity for four of these herbal remedies (Goldenseal, Gingko, Kava kava, and Pulegone), and

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classified them as “possibly carcinogenic to humans” (Group 2B) because of evidence for carcinogenic activity in two species or both sexes of the same rodent species. Other target organs damaged during high exposure to herbal remedies include the gastrointestinal tract (Aloe vera and Senna); kidney: (Aloe vera, raw Garlic, Ginkgo, Kava kava, Pulegone, St John’s wort, and Tobacco); heart: (Ephedra); and the vascular system: Riddelliine pyrrolizidine alkaloid and Tobacco). Milk thistle, Turmeric oleoresin, Green tea extract studies have not provided clear evidence for carcinogenic activity in rodent model systems nor did Marijuana, Chamomile, Coffee, Cocoa, Echinacea, Ephedra, Ginger, Ginseng, Milk thistle, St. John’s wort, and Turmeric. The dose or exposure is critical to understanding the toxicity and helps explain the conflicting results of so many studies. It is the dose that differentiates a therapy from a poison as illustrated in their dose–response curves. Regardless, given enough of the remedy, hepato-, nephron-, cardio-, and neurotoxicity is likely.

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C H A P T E R

5 Phycotoxins Val Beasley1, Wayne Carmichael2, Wanda M. Haschek1, Kathleen M. Colegrove1, Philip Solter1 1

University of Illinois at Urbana-Champaign, Urbana, IL, United States, 2Wright State University, Dayton, OH, United States

O U T L I N E 1. Introduction 1.1. Harmful Algal Blooms 1.2. Aquatic Hypoxia 1.3. Some Important Marine and Freshwater Toxins 1.4. The Need for Greater Access to and Reliance on Diagnostic Expertise and Instrumentation 1.5. A Future with Fewer Harmful Algal Blooms and Phycotoxin Poisonings 1.6. Rationale for the Subsequent Discussions of Phycotoxins

306 306 308

2. Saxitoxins 2.1. Source/Occurrence 2.2. Toxicology 2.3. Clinical Signs and Pathology 2.4. Human Exposure and Disease 2.5. Diagnosis, Treatment, and Control

314 314 315 315 315 316

3. Cyclic Imines 3.1. Source/Occurrence 3.2. Toxicology 3.3. Clinical Signs and Pathology 3.4. Human Exposure and Disease 3.5. Diagnosis, Treatment, and Control

317 317 318 318 319 319

4. Domoic Acid 4.1. Occurrence and Species Susceptibility 4.2. Toxicology 4.3. Clinical Signs and Pathology 4.4. Human Exposure and Disease 4.5. Diagnosis, Treatment, and Control

319 319 321 321 324 325

5. Brevetoxins 5.1. Source/Occurrence 5.2. Toxicology

325 325 326

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00005-8

5.3. 5.4. 5.5. 5.6.

309 310 311 311

Clinical Signs Gross and Histologic Findings Human Exposure and Disease Diagnosis, Treatment, and Control

326 327 328 329

6. Ciguatoxins 6.1. Source/Occurrence 6.2. Toxicology 6.3. Maitotoxins 6.4. Clinical Signs and Pathology 6.5. Human Exposure and Disease 6.6. Diagnosis, Treatment, and Control

330 330 331 331 331 331 332

7. Okadaic Acid and Dinophysistoxins 7.1. Source/Occurrence 7.2. Toxicology 7.3. Clinical Signs and Pathology 7.4. Human Exposure and Disease 7.5. Diagnosis, Treatment, and Control

333 333 333 334 334 335

8. Azaspiracid Toxins 8.1. Source/Occurrence 8.2. Toxicology 8.3. Clinical Signs and Pathology 8.4. Human Exposure and Disease 8.5. Diagnosis, Treatment, and Control

335 335 336 336 338 338

9. Cylindrospermopsins 9.1. Source/Occurrence 9.2. Toxicology 9.3. Clinical Signs and Pathology 9.4. Human Exposure and Disease 9.5. Diagnosis, Treatment, and Control

339 339 340 341 344 344

10. Microcystins and Nodularins 10.1. Source/Occurrence

305

345 345

Copyright Ó 2023 Elsevier Inc. All rights reserved.

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10.2. 10.3. 10.4. 10.5.

Toxicology Clinical Signs and Pathology Human Exposure and Disease Diagnosis, Treatment, and Control

347 348 351 354

11. Anatoxins 11.1. Source/Occurrence 11.2. Toxicology 11.3. Clinical Signs and Pathology 11.4. Human Exposure and Disease 11.5. Diagnosis, Treatment, and Control

355 355 356 356 357 358

12. Guanitoxin [Formerly Anatoxin-A(S)] 12.1. Source/Occurrence 12.2. Toxicology 12.3. Clinical Signs and Pathology 12.4. Human Exposure and Disease 12.5. Diagnosis and Treatment

358 358 359 359 360 361

13. Lyngbyatoxins and Aplysiatoxins 13.1. Source/Occurrence 13.2. Toxicology 13.3. Clinical Signs and Pathology

361 361 362 363

1. INTRODUCTION Phycology is the study of algae and cyanobacteria, and phycotoxins are toxicologically potent organic compounds produced by these species. The word, algae, is an informal one that is most often used to refer to simple photosynthetic eukaryotic plants that lack flowers, leaves, stems, vascular tissue, and roots (Beasley, 2020). Algae include myriad unicellular plants, with nonmotile examples such as Chlorella spp.; motile forms including Chlamydomonas spp. and many genera of dinoflagellates and silica-encased diatoms; simple multicellular organisms, such as Volvox spp.; and many kinds of kelp (Wehr and Sheath, 2003; Umen, 2014). Dinoflagellates and diatoms produce the most important marine phycotoxins. Cyanobacteria are prokaryotes. They dominated the planet for most of its existence, generating its oxygen-rich atmosphere. They continue to produce much of today’s atmospheric oxygen, and fix nitrogen and produce polymers that enrich soils and prevent erosion. Cyanobacteria were also incorporated into plants

13.4. Human Exposure and Disease 13.5. Diagnosis, Treatment, and Control 14. b-Methylaminoalanine 14.1. Introduction 14.2. Sources/Occurrences/Exposures 14.3. Toxicology 14.4. Animal Studies 14.5. Mechanism of Action 14.6. Human Exposure and Disease 14.7. Analytical Methods for Detection and Quantification 14.8. Conclusion

363 364 365 365 365 365 366 366 367 367 368

15. Emerging Phycotoxins 368 15.1. Vacuolar Myelinopathy and Aetokthonotoxin 368 15.2. Palytoxins 370 15.3. Yessotoxins 372 16. Conclusions and Future Needs

373

References

374

as chloroplasts, making their photosynthesis possible. Cyanobacteria produce most of the known freshwater phycotoxins, which are referred to as cyanotoxins.

1.1. Harmful Algal Blooms The term harmful algal bloom (HAB) is used to describe dense aquatic growths of algae or cyanobacteria that cause depletion of dissolved oxygen or produce phycotoxins at concentrations that poison invertebrate and/or vertebrate animals. HABs have also triggered the collapse of food webs, reduced the productivity, economic sustainability, and nutritional benefits of fisheries, undermined the public’ valuation and recreational use of aquatic ecosystems, and lowered real estate prices. Marine, estuarine, and coastal algae can color the waters, and some of them produce what are known as “red tides.” Cyanobacteria have often been called blue-green algae, but they may also be green, white, yellow, red, brown, purple, or black (Figure 5.1). Nontoxigenic blooms of algae or cyanobacteria may have the same colors as

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1. INTRODUCTION

307

FIGURE 5.1 Bloom of Microcystis on the Copco Reservoir, California. Eutrophication is a major cause of cyanobacterial blooms in lakes and other water bodies. Courtesy of Amber Roegner. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 3, Academic Press, 2013, Fig. 38.1, p. 1156, with permission.

those that are highly toxic, and even clear water may harbor hazardous levels of phycotoxins. Moreover, epiphytic (growing on the surfaces of plants), periphytic (growing on a range of submerged surfaces) and benthic (growing on or within the sediment) species of toxigenic algae and cyanobacteria have repeatedly caused mild to lethal poisonings (Quiblier et al., 2013). Accordingly, discoloration of water should trigger collection and study of water specimens to identify phycotoxin producers and phycotoxins, but risks of direct phycotoxin poisonings as well as relay poisoning through ingestion of water, shellfish, and finfish can be important, even when waters appear normal. In addition, aerosolization of toxins from marine harmful algal blooms (HABs) has been well documented, and aerosolization of cyanotoxins in freshwater systems has recently been demonstrated (Plaas and Paerl, 2021). Finally, dermal absorption of some of the marine phycotoxins and cyanotoxins is another potentially important route of exposure (Osborne et al., 2001; Nielsen and Jiang, 2020). Blooms of algae and cyanobacteria often result from human actions that produce excessive aquatic concentrations of free nutrients, especially nitrogen and phosphorus (Watson et al., 2016). High nutrient concentrations in ponds, lakes, streams, estuaries, and coastal zones

may result from inadequate public or private sewage systems; runoff from surface-applied fertilizers on turf grasses or fertilizers and liquid manure on farm fields; poorly designed or managed animal feeding operations; absence of cover crops; insufficient buffering of water bodies by adjacent plants; deforestation; atmospheric deposition of nitrogen oxides and trace elements downwind and downstream from burning of trees, crop residues, other plants, and fossil fuels (e.g., in vehicles, refineries, power plants, factories, businesses, and homes); and altered hydrology such as from dams and water abstraction that reduce dilution, and increase nutrient retention and stagnation. High numbers of grazing livestock and herbivorous wildlife that rely on limited surface sources of drinking water are often at high risk of both driving cyanobacterial overgrowth and suffering the effects of cyanotoxin poisonings. In some places, a long history of nutrient overloading of soils and/or thousands of small millassociated dams have produced a legacy of excessive nutrients in sediments and stream banks that are now eroding and being carried downstream, a process that will persist for some time, even if significant new inputs of nutrients are curtailed (Walter and Merritts, 2008; Niemitz et al., 2013; Ho and Michalak, 2020). Herbicides kill other algae and aquatic

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macrophytes, so that more ultraviolet (UV) and nutrients are available to cyanobacteria. Moreover, the widely used herbicide, glyphosate, also serves as a phosphate source that can increase proliferation of toxigenic cyanobacteria, such as Microcystis (Wang et al., 2021). Climate change, with resulting warmer temperatures, contributes to shorter periods of ice cover, warmer waters, and more intensive droughts and storms. Droughts stress terrestrial plants so that they have less capacity for nutrient uptake and incorporation into complex biomolecules. Intense storms create pulses of nutrient runoff. There is a growing consensus that both nutrient pollution and climate change are contributing to increasingly frequent, longer lasting, and more expansive HABs (Paerl, 2008; Paerl and Scott, 2010; Gobler et al., 2017; Burford et al., 2020; Gobler, 2020; Griffith and Gobler, 2020).

1.2. Aquatic Hypoxia During periods of adequate sunlight, the photosynthesis of algal and cyanobacterial species adds dissolved oxygen to the water and raises its pH, but at night their respiration consumes dissolved oxygen and aquatic pH drops. This process continues so that dissolved oxygen concentrations and aquatic pH continue declining until adequate sunlight penetrates the water early the following day. Cloudy weather limits daily UV penetration, photosynthesis, and related oxygenation and buffering of water. Moreover, when cyanobacterial, algal, and/or aquatic macrophyte biomass are high enough to limit light penetration, photosynthesis by submerged plants or cyanobacteria may be insufficient to effectively oxygenate and buffer the water. Also, warm water simply cannot hold as much dissolved oxygen as cold water, and warmer conditions increase metabolic rates and thus oxygen demand in aquatic animals. Accordingly, early mornings and warm, cloudy weather can interact with blooms of cyanobacteria or algae to create hypoxic conditions that readily harm or kill aquatic animals. In stocked aquaculture ponds that have high animal biomass, the inputs of feed and wastes from the fish readily set the stage for cyanobacterial blooms. Although the cyanobacteria serve to limit toxic nitrite and nitrate concentrations, in the absence of adequate aeration, they may also cause aquatic hypoxia, resulting in massive losses of fish and other aquatic species. Other nutrient-rich water bodies that are less carefully monitored than aquaculture ponds commonly

experience similar problems, such as after periods of hot weather and minimal rainfall. Low dissolved oxygen may also trigger the liberation of phosphorus from sediments, which may favor a subsequent HAB (Zilius et al., 2014). Hypoxic waters can disrupt food webs (Roman et al., 2019). When algae or cyanobacteria of a bloom run low on a limiting nutrient and suddenly die, aerobic species that feed on the decaying organisms or their nutrients may rapidly deplete dissolved oxygen, creating severe hypoxia or anoxia (Altieri et al., 2017). Historically, dead zones, which result from severe oxygen depletion, were rare, but they markedly increased, beginning in the 1950s. Applications of industrially produced fertilizers have consistently preceded the increase in dead zones. In recent decades, dead zones have been recognized in many rivers, lakes, reservoirs, and coastal waters, and today, hundreds of coastal dead zones develop each summer around the world (Nu¨rnberg, 2004; Diaz and Rosenberg, 2008). Highly mobile species, such as many species of finfish, may leave areas of hypoxia. In smaller bodies of hypoxic water or in the presence of large hypoxic areas of coastal oceans, they may be unable to escape. Controlled studies indicate that fish surviving, but stressed by hypoxic waters: expend more energy on respiration and on moving their fins to pass more water over their developing early embryos; eat less and grow more slowly; have impaired innate and adaptive immunity potentially resulting in more infections; develop gill hyperplasia, degeneration, and necrosis; experience hypoxic liver injury; and/or change from efficient aerobic to inefficient anaerobic metabolism so that they have less adenosine triphosphate (ATP) (AbdelTawwab et al., 2019). They may also experience endocrine disruption (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). For example, carp (Cyprinus carpio) held in hypoxic water had reduced serum testosterone, estradiol and triiodothyronine, retarded gonadal development, and reductions in sperm motility, spawning, fertilization, hatching, and larval survival (Wu et al., 2003). A series of studies of male and female Atlantic croakers (Micropogonias undulatus) revealed additional insights into how stress from hypoxic waters may be manifested as endocrine disruption in fish (Thomas and Rahman, 2009, 2010; Murphy et al., 2009). Effects of hypoxic conditions included: (i) reduced concentrations of the progestin that

1. INTRODUCTION

stimulates gamete maturation, (ii) impaired oocyte maturation and sperm motility, (iii) marked reductions in fertilization, and (iv) when fertilization was successful, reduced hatching and larval survival. Moreover, female Atlantic croakers in hypoxic water in a lab setting, or in hypoxic waters of the northwest coast of Florida, had low estradiol-17b, vitellogenin, gonadal-somatic index, and fecundity. Male Atlantic croakers from hypoxic sites in Gulf of Mexico had 74% lower sperm production when compared to males of the species from reference normoxic sites. Also, when zebrafish (Danio rerio) were held in hypoxic water, they experienced downregulation of genes, 3ß-Hsd, Cyp11A, Cyp19A, and Cyp19B, which are important in regulating sex hormones (Shang et al., 2006). The outcome was an increase in the ratio of testosterone to estradiol, and a maledominated population. Additional studies of captive and free-ranging fish in hypoxic environments are needed, including those that assess the potential for multigenerational impacts.

1.3. Some Important Marine and Freshwater Toxins Tubaro et al. (2012) and Cifuentes et al. (2015) have provided useful overviews of the occurrence, mechanisms, and manifestations of a wide range of marine phycotoxins. Also, Fire and Van Dolah (2012) and Broadwater et al. (2018) have published helpful reviews that focus on major phycotoxin poisonings of marine mammals. Highly important marine phycotoxins are produced by diatoms or dinoflagellates and include saxitoxins, domoic acid, brevetoxins, and ciguatoxins. Marine phycotoxin poisonings in man, other mammals, and birds can occur from: (i) direct exposure of eyes and skin; (ii) inhalation of aerosols; (iii) ingestion of producing organisms or lysates; or (iv) ingestion of contaminated shellfish, finfish, or other aquatic animals. Although tetrodotoxin is produced by certain dinoflagellates and cyanobacteria, its production has most often been associated with nonphotosynthetic bacteria, some of which are endosymbiotic or components of intestinal microbiomes (Lago et al., 2015). Accordingly, it will be discussed only briefly here (see Bacterial Toxins, Vol 3, Chap 9, and Animal Toxins, Vol 3, Chap 8, for more information). Tetrodotoxin is an extremely toxic alkaloid that works by blocking sodium channels. Several derivatives of

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tetrodotoxin are found in contaminated specimens. They account for some of the most important toxic effects of freshwater, marine, and terrestrial animals, including starfish, mussels, gastropods, blue-ringed octopuses, crustaceans, finfish including puffer fish, and amphibians (Tubaro et al., 2012). For some of these species, tetrodotoxin affords protection from predation. Eating tetrodotoxin-contaminated fish is a frequent cause of lethal poisonings in humans, and dogs are likely at risk from tetrodotoxin when they ingest contaminated fish and sea slugs (Yasumoto and Murata, 1993; Magarlamov et al., 2017). Tetrodotoxins are concentrated in the skin, gonads, and viscera of fish and amphibians. Research is needed on the exposures and effects of tetrodotoxin in aquatic invertebrates, fish, water birds, and marine, freshwater, and terrestrial mammals. Among the most important cyanotoxins are microcystins, nodularins, cylindrospermopsins, lyngbyatoxins, aplysiatoxins, anatoxins, guanitoxin, and saxitoxins (Loftin et al., 2016). Recent reviews have addressed a number of important acute effects of cyanotoxins in humans and other animals (Chorus and Bartram, 1999; van Apeldoorn et al., 2007; Backer et al., 2013; Hilborn and Beasley, 2015; Wood, 2016; Nowruzi and Porzani, 2021; Chorus and Welker, 2021). Poisonings of domestic and wild birds and mammals by cyanobacterial toxins from fresh or brackish water most often result from direct consumption of contaminated water. When terrestrial animals enter water bodies, they may disturb benthic and periphytic cyanobacteria and free up dissolved toxins, increasing risks of poisonings. In humans, exposure to contaminated dialysate solution, drinking contaminated water, food web contamination, inhalation, and skin absorption can lead to significant cyanotoxin exposures and, in some cases, life-threatening toxicoses. As discussed below, the importance of b-methylamino-L-alanine (BMAA) from ingestion of contaminated cycads, bats, and aquatic animals is currently a focus of much debate (Lance et al., 2018). The effects of different phycotoxins range from peracute lethality due to respiratory paralysis or cardiotoxicity to chronic neurologic disability, and from acute liver and sometimes kidney failure and death, to skin irritation, tumor promotion, and increased cancer risk. Some toxigenic organisms produce multiple phycotoxins, blooms may harbor more than one toxigenic species, and their

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populations change exponentially over time. Indeed, studies on sick or dead marine mammals and their environments have sometimes revealed more than one phycotoxin from diatoms and/or dinoflagellates (Twiner et al., 2011). In addition, investigators have sometimes found unexpected freshwater cyanotoxins in estuarine and marine waters (Metcalf et al., 2021). For example, a coastal zone of California was the site of lethal poisonings of southern sea otters (Enhydra lutris neresis) after microcystins in cyanobacteria were transported downstream from a nearby eutrophic freshwater body (Miller et al., 2010). The diversity of blooms, the dynamics of reproduction and death of phycotoxin producers, their transport driven by currents and wind, their active and passive rise and fall in the water column including when aquatic stratification is disrupted, their elaboration and release of toxins, and the dilution, modifications, and breakdown of toxins in the environment interact to necessitate sequential sampling of both water and exposed animals.

1.4. The Need for Greater Access to and Reliance on Diagnostic Expertise and Instrumentation Insufficient access to experts capable of comprehensive identifications of phycotoxin producers and analytical quantifications of the broad array of phycotoxins impedes surveillance efforts and reduces the probability of timely diagnoses. The ongoing reliance on mouse bioassays to identify phycotoxin effects causes animal pain and suffering, yet such tests provide a more comprehensive assessment of acute toxicity than any analytical method available to date. The development of bioassays that do not involve such sentient animals is an important need. Analytical methods, such as immunoassays, including enzyme-linked immunosorbent assay (ELISA) kits, and high-performance liquid chromatography–mass spectrometry (HPLC-MS/ MS), often have value in determining the presence and concentrations of one or more phycotoxins in water, bloom materials, seafood, stomach and intestinal contents, and tissues. Also, over the past 20 years, molecular approaches, including polymerase chain reaction (PCR) assays, have been developed to identify target genes in cyanobacteria and various algal species

to confirm, not only their identity, but also their capacity to produce phycotoxins. The report of Nowruzi and Porzani (2021) and the handbook edited by Meriluoto et al. (2017) list these and other techniques that have been used to characterize cyanotoxin producers and cyanotoxins, including previously unknown compounds, some of which are being considered as potential pharmaceuticals and many of which are not discussed elsewhere in this chapter. Unfortunately, the limited availability of analytical standards for a large number of phycotoxins and the specialization of laboratories so that they focus only on subsets of phycotoxins without attention to others can result in a failure to detect potentially important phycotoxins in diagnostic cases as well as research studies. Presumptive diagnoses of phycotoxin poisonings are frequently based on a combination of a history of consumption or other exposure to a suspected source of toxin, clinical signs, hematologic and serum biochemistry data, and in lethal cases, compatible gross and microscopic findings. However, the integration of such observations with analytical studies that confirm and quantify phycotoxins in source materials, digestive tract contents, and appropriate tissues is necessary to have confidence in diagnoses of specific phycotoxin poisonings since morphologic findings are usually not pathognomonic. Phycotoxins may be present at concentrations that are too low to be responsible for clinical signs, at threshold concentrations for toxicity, or at higher levels, some of which have been associated with major mortality events. The knowledge base needed to correlate cause and effect is growing over time based on a combination of field data and laboratory studies of cells, invertebrates, fish, birds, rodents, and other mammals. Diagnostic work and research that involve collaborations with laboratories with the sophisticated instrumentation and expertise needed for phycotoxin analyses and interpretations are extremely important. Recognizing the many phycotoxin producers in the environment, that a given water body may have more than one toxigenic HAB, that a single phycotoxin producing species can elaborate structurally diverse phycotoxins (e.g., Colas et al., 2021), and considering the diversity of animals exposed, the various matrices assayed, and the rapid evolution in analytical technologies, there is and will remain

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1. INTRODUCTION

a need for a “living,” i.e., continuously updatable, database of diagnostically relevant criteria including residues of phycotoxins. There is also a need for more assessments of the impacts of HABs and phycotoxins in ecological contexts. For example, recent observations from field studies have suggested that poor body condition in dolphins may be a result of phycotoxin-induced reductions in the populations of prey species (Landsberg et al., 2009; Fire and Van Dolah, 2012). Dolphins lacking their usual prey in association with phycotoxin-induced die-offs may also experience a greater risk of trauma when they turn to eating fish that were hooked by fishermen or when they eat a stingray and are “barbed” (Colegrove, personal observation). Also, evidence of starvation of Florida manatees in the Indian River Lagoon (IRL) along the East Coast of Florida due to shifts in aquatic communities from predominantly seagrasses to phytoplankton were noted as early as 2013, but record high mortalities from this problem began and have persisted since 2020 (Owen et al., 2018; de Wit et al., 2021; Herren et al., 2021). After decades of development, septic systems now account for more than 50% of the total wastewater disposal in the central part of the lagoon. The nutrients from septic systems enter groundwater that flows into the IRL, creating widespread eutrophication (Lapointe et al., 2020; Herren et al., 2021). The resultant dense blooms of phytoplankton block sunlight and produce aquatic hypoxia, which interact to kill the seagrasses. In a vicious cycle, the depleted seagrasses are less able to stabilize the sediments, which aggravates the turbidity. Along the west coast of Florida, St. Joseph Sound, Clearwater Harbor, Tampa Bay, Sarasota Bay, and Charlotte Harbor previously experienced phytoplankton-dominated waters that resulted in seagrass depletion (Lapointe et al., 2020). Nutrient control via regional wastewater treatment enabled substantial recovery of seagrass communities in those areas. Accordingly, a region-wide nutrient control program is needed to conserve seagrasses and manatee populations in the IRL, although the challenge is substantial because the lagoon is over 240 km long and mixing with ocean waters is limited.

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1.5. A Future with Fewer Harmful Algal Blooms and Phycotoxin Poisonings Optimally, large-scale, site-specific, transdisciplinary studies of hydrology, nutrient sources and concentrations, historic and current land and water management practices, climate, weather, runoff, hypoxia, phycotoxin producers, food chain effects, toxin residues, and comprehensive clinical and pathological assessments will be integrated in cumulative data sets. Analyses of such assessments along with data on economic incentives and disincentives should point to societal drivers of HABs as well as effective countermeasures for risk reduction and ecological recovery.

1.6. Rationale for the Subsequent Discussions of Phycotoxins A number of important phycotoxins are presented below in a sequence that will hopefully assist in understanding the spatial distributions of documented poisonings, mechanisms of action, and predominant clinical and pathologic effects. The first phycotoxins listed are from marine dinoflagellates and diatoms, including the neurotoxic saxitoxins, cyclic imine phycotoxins, and domoic acid; then the brevetoxins and ciguatoxins, which affect the nervous system and multiple other organ systems as well; and okadaic acid and azapiracid toxins that have often been associated with digestive system problems. Subsequently, cyanotoxins from freshwater, estuarine, and marine sources are discussed: beginning with cylindrospermopsin, which is known for its oxidative injury, genotoxicity, hepatotoxicity, and nephrotoxicity; then the potently hepatotoxic microcystins and nodularins; the neurotoxic anatoxins and guanitoxin; the dermatotoxic lyngbyatoxins and aplysiatoxins; and the controversial BMAA. Lastly, are succinct discussions of three groups of emerging phycotoxins, including one from freshwater and two from marine systems: the freshwater cyanotoxin, aetokthonotoxin, which causes vacuolar myelinopathy; the neurotoxic, cardiotoxic, and hemolytic palytoxins; and the cytotoxic and cardiotoxic yessotoxins. Tables 5.1 and 5.2 provide summary information on sources, disease conditions, major sites of action (targets), and mechanisms of action.

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TABLE 5.1 Toxins of Marine Dinoflagellates and Diatoms, Associated Disease Conditions, Documented Major Targets, and Mechanisms of Action Toxins

Diseases

Major Targets

Mechanisms of Action

Saxitoxins (STXs)a

PSP

Myelinated and nonmyelinated nerves, skeletal muscle, vascular smooth muscle, cardiac muscle

Nondepolarizing blockade of voltage-regulated sodium channels

Cyclic imines (CIs)

Experimental CI toxicosis

Central and peripheral nerves and neuromuscular junctions

Blockade of nicotinic and muscarinic acetylcholine receptors

Domoic acid (DA)

DA toxicosis; ASP of humans

Brain neurons and astrocytes, cardiomyocytes

Stimulates postsynaptic Nmethyl-D-aspartate (NMDA) and glutamate receptor subtypes: Opens sodium-, potassium-, and calciumselective ion-gated channels

Brevetoxins (BTXs)

Red tide poisoning; NSP of humans

Gastrointestinal, nervous, and respiratory systems

Binds to voltage-sensitive sodium channels, causing sodium ion influx, spontaneous depolarization of membranes

Ciguatoxins (CTXs)

CFP of humans

Gastrointestinal, nervous, and cardiovascular systems

Binds to voltage-sensitive sodium channels, causing sodium ion influx, spontaneous depolarization of membranes

Okadaic acid (OA) and dinophysistoxins (DTXs)

DSP of humans

Gastrointestinal tract

Inhibition of serine/threonine protein phosphatase types 1 (PP1) and 2A (PP2A)

Azapiracid toxins (AZAs)

AZA poisoning in humans

Gastrointestinal tract

Undetermined

Palytoxins (PLTXs) dinoflagellates of genus Ostreopsis (see Table 5.2 as well)

PLTX toxicosis

Respiratory, nervous, gastrointestinal, renal, and cardiovascular systems

Dysfunction of Na/K-ATPase pumps resulting in increased intracellular sodium and decreased intracellular potassium, cell depolarization, and secondary increases in intracellular calcium

Yessotoxins (YTXs)

Experimental YTX toxicosis

Heart

Undetermined

a

Saxitoxins are also produced by several genera of fresh and brackish water cyanobacteria, and drinking the contaminated water has poisoned animals. ASP, amnesic shellfish poisoning; CFP, ciguatera fish poisoning; DSP, diarrheic (alternatively diarrhetic or diarrheal) shellfish poisoning; NSP, neurotoxic shellfish poisoning; PSP, paralytic shellfish poisoning.

II. SELECTED TOXICANT CLASSES

TABLE 5.2

Toxins From Freshwater, Estuarine, and Marine Cyanobacteria (and Sometimes Other Organisms), Associated Disease Conditions, Documented Major Targets, and Mechanisms of Action Major Targets

Mechanisms of Action

Cylindrospermopsin (CYN)

Palm island mystery disease

Hepatocytes and renal epithelium

Dose-dependent toxicity: Impacts on pathways that regulate cell proliferation and cell survival; inhibition of protein synthesis; cytochrome P450 induction that increases toxicity, glutathione depletion, oxidative damage; and genotoxicity

Microcystins (MCs), nodularins (NDs)

MC and ND toxicoses, netpen liver disease of salmon

Hepatocytes

Selective uptake by the liver and to a lesser extent the central nervous system and kidneys; inhibition of serine/threonine protein phosphatase 2A (PP2A)

Anatoxins (ANTXs)

ANTX toxicosis

Neuromuscular junctions, brain, autonomic ganglia

Postsynaptic depolarization and paralysis of muscles of respiration

Guanitoxin [formerly ANTX-a(s)]

Guanitoxin toxicosis

Gastrointestinal tract, neuromuscular junctions including in respiratory muscles

Peripheral inhibition of acetylcholinesterase and paralysis of muscles of respiration

Lyngbyatoxins and aplysiatoxins

Swimmer’s itch

Skin, ocular, and respiratory systems

Bind to phorbol ester receptors and activate protein kinase C, resulting in excessive phosphorylation of regulatory proteins

b-methylaminoalanine (BMAA)

Experimental BMAA toxicosis

Neurons

Glutamate receptor agonist, depolarization of postsynaptic neurons

Aetokthonotoxin

Vacuolar myelinopathy (VM)

Central nervous system

Undetermined

Palytoxins (PLTXs) cyanobacteria of genus Trichodesmium, and corals of genus Palythoa (see Table 5.1 as well)

PLTX toxicosis

Respiratory, nervous, gastrointestinal, renal, and cardiovascular systems

Dysfunction of Na/K-ATPase pumps resulting in increased intracellular sodium and decreased intracellular potassium, cell depolarization, and secondary increases in intracellular calcium

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Disease

1. INTRODUCTION

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Toxin

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FIGURE 5.2 Structures of saxitoxin and neosaxitoxin. Created by Ed (Edgar181), Own work, Public Domain,https://en.wikipedia.org/wiki/Saxitoxin#/media/File: Saxitoxin_neutral.svg and https://en.wikipedia.org/wiki/Neos axitoxin#/media/File:Neosaxitoxin.svg. Retrieved May 15, 2022.

2. SAXITOXINS 2.1. Source/Occurrence The prototype member of this group, saxitoxin (STX), was first isolated from Saxidomus giganteus, the butter clam, after a number of individuals who ate them became poisoned (Etheridge, 2010). STXs include around 60 structurally related tricyclic guanidium alkaloids, some of which are termed gonyautoxins (Figure 5.2) (Wiese et al., 2010). STXs are also called paralytic shellfish poisons (PSPs) or paralytic shellfish toxins (PSTs). Although STXs were first identified from marine environments, in the 1970s, it was revealed that freshwater cyanobacteria also produce certain members of this group (D’Agostino et al., 2019). Marine producers of STXs include dinoflagellates of the genera Alexandrium (Gonyaulax), Gymnodinium, and Pyrodinium. Cyanobacterial genera that produce STXs in fresh and brackish water include Dolichospermum (formerly Anabaena), Aphanizomenon, Cylindrospermopsis, Moorea (formerly Lyngbya), and Planktothrix (Carmichael et al., 1997; Weise et al., 2010). Additional genera of dinoflagellates and cyanobacteria that also produce STXs were listed by the

World Health Organization (WHO, 2020d). Significant taxonomic changes, including genus and species names, have been made for toxigenic cyanobacteria. A review and update of these can be found in Vidal et al. (2021, Table 3.2). In marine waters, STXs accumulate and are transferred through food chains. They are heat stable, and their ingestion has caused significant human illness and mortality (Morse, 1977; Gessner and Middaugh, 1995; Etheridge, 2010; van der Merwe, 2014). In Alaskan waters, regular blooms of toxin producing Alexandrium sp. have been linked to increases in sea surface and air temperatures, as well as freshwater nutrient runoff. In that region and elsewhere, STX contamination causes significant economic impacts to the shellfish industry (Tobin et al., 2019). Nonfilter feeding gastropods, crustaceans, and certain fish can also contain the toxins (Etheridge, 2010). Consumption of puffer fish is an important source of human exposure to not only tetrodotoxins, as discussed above, but also STXs, which accumulate in fish muscle (Landsberg et al., 2006; Etheridge, 2010). Massive fish kills from toxic blooms have repeatedly been linked to STXs (Etheridge, 2010). STX poisoning was associated with deaths of sea otters in Alaska, as well as the loss of 67% of the local population of highly endangered Mediterranean monk seals (Monachus monachus) off the coast of Cap Blanc, western North Africa, in 1997 (Reyero et al., 1999; Fire and Van Dolah, 2012). The deaths in the monk seals were also blamed on morbillivirus infections (Osterhaus et al., 1998). It is possible that both were affecting the monk seals in the same time frame. Exposure to STXs was implicated in deaths of several humpback whales (Megaptera novaeangliae) on the coast of Massachusetts in late 1987 and early 1988. This association was based on the findings of: (i) good body condition in the whales; (ii) stomachs full of planktivorous Atlantic mackerel (Scomber scombrus) and similar fish contained high levels of STX; (iii) the likely distribution of the water-soluble toxin into the heart and nervous system rather than blubber; (iv) reductions in blood flow to organs of detoxification during diving; and (v) extrapolations based on knowledge of lethal oral doses in exposed humans (Fire and Van Dolah, 2012). More recently, STX was found in bottlenose dolphins

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2. SAXITOXINS

stranded along the eastern coast of Florida, and it was detected in Alaskan marine mammals, although effects were unclear (Lefebvre et al., 2016; Fire et al., 2020a). STX poisoning was also identified in dogs that consumed various marine organisms along the shoreline in England following a winter storm (Turner et al., 2018). Two of the affected dogs died. Documented STX poisonings in freshwater systems are not as common as in marine waters, but, as noted above, the number of cyanobacteria known to produce these toxins is expanding. The main freshwater STXs found to date are STX and neosaxitoxin (Carmichael, 1997; Pereira et al., 2004). Research in Australia has shown the widespread occurrence of STXs in blooms of Dolichospermum circinale (formerly, Anabaena circinalis) in rivers and reservoirs (Humpage et al., 1994). In 1994, a drought in a sparsely populated agricultural region slowed and concentrated nutrients in the waters of Australia’s Darling River, and around 100 km of the river developed a dense bloom of STX-producing D. circinale (Stewart et al., 2008). Livestock in the area were poisoned by the bloom over a period of 6 weeks, including an episode involving one producer who lost over 1100 sheep. Because of the toxic bloom, recreational uses of the river were curtailed, and losses to the region’s tourist industry totaled around $1.5M. The freshwater mat-forming cyanobacteria, Moorea wollei (formerly, Lyngbya wollei) can produce STX analogs (Onodera et al., 1997b). M. wollei has been found in several lakes and reservoirs of the southern and south-central United States as well as the St. Lawrence River in Canada (Onodera et al., 1997b; Poirier-Larabie et al., 2020). Cylindrospermopsis, collected from Brazil, can also produce STXs (Lagos et al., 1999). Like the marine-sourced STXs, these sodium channel blocking agents inhibit transmission of nervous impulses and cause respiratory arrest.

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susceptible after oral exposure (WHO, 2020d). In orally dosed mice, lethal doses of gonyautoxins 1–4 were comparable to those associated with STX, whereas neosaxitoxin was almost three times more toxic than STX. The toxic effects of STXs are primarily due to nondepolarizing blockade of voltage-regulated sodium channels in myelinated and nonmyelinated nerves, inhibition of axonal impulse transmission to skeletal muscles, relaxation of vascular smooth muscle, and depression of cardiac muscle action potentials. Sensory nerves are apparently more susceptible to sodium channel blockade from STXs than motor nerves. As a consequence, sensory abnormalities generally precede paresis and paralysis (Wiese et al., 2010; Etheridge, 2010). STXs may also block calcium and potassium channels in the heart, potentially interfering with cardiac function (WHO, 2020d). Hypotension has been seen in animal studies of STX toxicity. In humans with PSP, hypotension is very rare, but hypertension has repeatedly been encountered. Whether the hypertension is due to direct central nervous system toxicity or a compensatory response to aspects of neurotoxicity is unknown.

2.3. Clinical Signs and Pathology Manifestations of STX poisonings in animals include incoordination, ataxia, trembling, recumbency, crawling, paralysis, and death from respiratory failure (Negri et al., 1995; Andrinolo et al., 1999; Turner et al., 2018). Some of the humpback whales mentioned above were noted to have been displaying normal behaviors 90 min before they died (Fire and Van Dolah, 2012). Diving whales and dolphins must contract muscles to open their blowholes; thus paralysis could potentially lead to suffocation.

2.4. Human Exposure and Disease 2.2. Toxicology STX is among the most potent members of this phycotoxin group. Murine intraperitoneal and oral LD50s for STX were recently determined to be approximately 8 and 260–960 mg/kg, respectively (Selwood et al., 2017). Other species, from pigeons to rats to monkeys, are similarly

STXs are known for their classical role in human PSPs, the onset of which is often within several minutes to a few hours after ingestion of various seafood types that accumulate the toxins (van der Merwe, 2014). The occurrence of human deaths from PSPs is relatively high in comparison to other marine toxins (Etheridge,

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2010). Poisonings of multiple people who dined on shellfish have been linked to harvests by individuals unaware of posted warnings of high risks of STXs (Selwood et al., 2017). Around 2000 human cases of STX toxicosis occur annually, and around 15% of affected individuals die. Children are believed to be more susceptible to STXs than adults (WHO, 2020d). Data from an episode of human PSP that affected 187 people who ate a soup made with clams revealed that the minimum lethal dose of STX equivalents for children weighing 25 kg was approximately 25 mg/kg body weight, and for adults it was 86–788 mg/kg. Half of the children exposed died, compared to only 7% of the adults. Symptoms and signs of STX poisoning in humans include paresthesias, often termed tingling or numbness, of the lips and mouth, which progresses to the face, neck, and extremities, as well as ataxia, nausea, vomiting, drowsiness, hypotension or hypertension, dysphagia, dysarthria, incoherent speech, and a feeling of floating. Acute deaths can occur from respiratory failure (de Carvalho et al., 1998.; Garcı´a et al., 2005; Hurley et al., 2014). There are no specific morphologic lesions associated with STX toxicosis, although cyanosis may be noted. The lungs of two fishermen who died 3–4 h after ingesting mussels and had STXs in their gastric contents were described as “crackling to the touch,” and they had gross evidence of pulmonary congestion and edema (Garcı´a et al., 2004). The “crackling” is consistent with emphysema, but whether it was chronic or had developed acutely in relation to severe respiratory effort shortly before death is unknown.

2.5. Diagnosis, Treatment, and Control A rapid diagnosis and astute intervention are essential to minimize death losses from STX poisonings. Evidence of consumption of seafood, especially shellfish, within a few hours prior to the onset of neurological signs is consistent with poisoning by STXs. Stomach contents, blood, urine, and organs should be analyzed for the presence of STXs and metabolites by immunoassays or derivatization or oxidation of the molecules for HPLC with fluorescence detection (Garcı´a et al., 2005; Etheridge, 2010; Turner et al., 2019).

There are no antidotes for STX poisoning. Activated charcoal is recommended to reduce absorption of STXs from the digestive tract. Humans sometimes die from respiratory paralysis or cardiac failure within 2–12 h of STX ingestion; thus, close monitoring and artificial respiration and cardiopulmonary resuscitation can be essential life-saving components of management (Garcı´a et al., 2004, 2005). Successfully treated patients often recover within 24 h (Hurley et al., 2014). Skiing or jet skiing through blooms of Dolichospermum or other cyanobacteria containing high concentrations of STXs and irrigating plants with water containing STXs creates risks of inhalation exposure (WHO, 2020d). Such activities should be avoided when freshwater, estuarine, or marine blooms of STX-producing organisms are present. Seafood monitoring programs have been established and are widely used to lessen risks of harvest and human ingestion of STXcontaminated bivalves (Etheridge, 2010). Mouse bioassays were used for decades for that purpose (Luckas et al., 2003). In recent years, however, many nations have adopted official analytical methods that measure concentrations of different STX congeners, as described by Turner et al. (2019). Using knowledge of the relative toxicity of STXs, many current human food safety laws prevent harvest and marketing when greater than 800 mg of STX equivalents per kilogram is present in shellfish tissue, although concentrations below that level may still be toxic (Terrazas et al., 2017). The increasing incidence of STXinduced mass fish kills and STX-associated risks to human health are spurring efforts to prevent HABs and to identify effective ways to detoxify shellfish and drinking water supplies (Etheridge, 2010). Enzymatic partial detoxification ot STX analogues is also being explored in efforts to develop new pharmaceutical drugs (Lukowski et al., 2019). Recently published guideline values, i.e., concentrations that should not be exceeded, for STXs have included 3 mg/L for drinking water in infants, 15 mg/L for adults, and 30 mg/L for recreational water (WHO, 2020d). Those concentrations were chosen based on abundant data from human cases of PSP. They were based, however, on short-term exposures only, and no long-term guideline values were reported. Fortunately, typical drinking water purification

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systems in developed countries seem to reduce STX concentrations to very low levels.

3. CYCLIC IMINES

OH H O

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3.1. Source/Occurrence

N

Cyclic imine (CI) phycotoxins include many structurally related compounds that have been named spirolides, pteriatoxins, pinnatoxins, prorocentrolides, spiro-prorocentrimines, symbioimines, gymnodimines, and portimine (Munday, 2008; European Food Safety Authority Panel on Contaminants in the Food Chain, 2010; Otera et al., 2011; Molgo´ et al., 2017) (Figures 5.3–5.6). Members of this group of phycotoxins have a macrocyclic ring, including from 14 to 17 carbon atoms, an essential cyclic imine (carbon–nitrogen double bond, usually as a spiroimine), and a spiroketal ring system (Gue´ret and Brimble, 2010; Molgo´ et al., 2017).

FIGURE 5.4 Structure of gymnodimine. Figure reproduced from Quilliam MA: Chemical methods for lipophilic shellfish toxins. In Hallegraeff GM, Anderson DM, Cembella AD, editors: Manual on Harmful Marine Microalgae. UNESCO, Paris, France, 2003, pp 211–246, in Toxicology and Diversity of Marine Toxins. In Gupta RC, editor: Veterinary Toxicology, Elsevier, 2012, Fig. 69.16, p. 927.

HN+

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COOH H

31

O

O

NH+

O

O

O

24

HO

O

OH

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O 2

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3

O HO

O

OH

10 13

R2 Spirolide

R1

R2

'2,3

MH+

√

692.5

A

H

CH3

B

H

CH3

C

CH3

CH3

D

CH3

CH3

desmethyl-C

CH3

H

desmethyl-D

CH3

H

694.5 √

706.5 708.5

√

692.5 694.5

FIGURE 5.3 Structures of the spirolides A–D and two desmethyl analogues from Alexandrium ostenfeldii. Figure reproduced from Sleno L, et al.: Structural study of spirolide marine toxins by mass spectrometry. Part I. Fragmentation pathways of 13-desmethyl spirolide C by collisioninduced dissociation and infrared multiphoton dissociation mass spectrometry, Anal Bioanal Chem 378:970, 2004, with permission.

FIGURE 5.5 Structure of pinnatoxin A. Figure reproduced from Quilliam MA: Chemical methods for lipophilic shellfish toxins. In Hallegraeff GM, Anderson DM, Cembella AD, editors: Manual on Harmful Marine Microalgae. UNESCO, Paris, France, 2003, pp 211–246, in Toxicology and Diversity of Marine Toxins. In Gupta RC, editor: Veterinary Toxicology, Elsevier, 2012, Fig. 69.17, p. 927.

CI toxins have often been found in marine ecosystems and marine aquaculture production facilities around the world. They are detected in seawater, sediments, clams, mussels, and oysters. Included in this group are compounds synthesized by dinoflagellates, as well as metabolites of such compounds that are generated by filter-feeding bivalves. The structures of the first recognized CI phycotoxins, pinnatoxin-A and gymnodimine, were identified after neurotoxicity was encountered in mouse bioassays of shellfish from the coasts of Japan and New Zealand (Selwood et al., 2010; Stivala et al., 2015).

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OH OH O OH N

OH

O OH O

O

definitive links between these toxins and human poisonings remain to be established (Stivala et al., 2015; Bacchiocchi et al., 2020). The adductor muscle, other edible tissues and viscera, and the digestive gland of shellfish have been found to contain the toxins (Stivala et al., 2015; Molgo´ et al., 2017). A number of the CI toxins are very slowly depurated after shellfish exposure to the toxigenic dinoflagellates has ceased.

O

HO OH

3.2. Toxicology

OH

FIGURE 5.6 Structure of prorocentrolide. Figure reproduced from Torigoe K, Murata M, Yasumoto T: Prorocentrolides, a toxic nitrogenous macrocycle from a marine dinoflagellate, Prorocentrum lima. J Am Chem Soc 110: 7876–7877, 1988, in Tubero A, Sosa S, Hungerford J: Toxicology and Diversity of Marine Toxins. In Gupta RC, editor: Veterinary Toxicology, Elsevier, 2012, Fig. 69.18, p. 927.

Many of the names of these toxins were derived from the genera of shellfish from which they were first isolated. The pinnatoxins were named based on their discovery in pen shellfish, which are bivalve mollusks of the genus Pinna. Pteriatoxins were first found in shellfish in the genus Pteria (Rundberget et al., 2011). Pinnatoxins are likely precursors of pteriatoxins, but whether the dinoflagellates directly produce the pteriatoxins or they are metabolites produced by shellfish is unknown (Stivala et al., 2015). The dinoflagellate, Vulcanodinium rugosum, is also a producer of pinnatoxins, portimine, and kabirimine (Stivala et al., 2015; Hermawan et al., 2019). The spirolides and gymnodimines have been associated with at least three species of dinoflagellate producers, Alexandrium ostenfeldii, Alexandrium peruvianum, and Karenia selliformes (Sleno et al., 2004). Spiroprorocentrimine was isolated from a laboratorycultured benthic species of a dinoflagellate of the genus Prorocentrum. Prorocentrolide was structurally identified after its isolation from Prorocentrum lima from coastal Japan (Torigoe et al., 1988). Symbioimine was isolated from marine dinoflagellates of the genus Symbiodinium. The potent oral toxicity and comparatively high stability of many of the CI toxins have prompted considerable research. Nevertheless,

Extensive studies on mechanisms of some of the CI phycotoxins and their derivatives and metabolites in laboratory rodents and in vitro have revealed antagonism at cholinergic receptors, and little to no activity at other receptors (Molgo´ et al., 2017). These lipophilic toxins act by blocking both nicotinic and muscarinic acetylcholine receptors in the central and peripheral nervous systems, including at neuromuscular junctions (Otera et al., 2011). Deaths in laboratory animals have been ascribed to respiratory paralysis (Amar et al., 2018). Portimine has also been linked to apoptosis-associated cytotoxicity (Hermawan et al., 2019). While kabirimine is a CI toxin, its effects on cholinergic receptors have not been reported but, interestingly, it has been found to have moderate activity against respiratory syncytial virus. Insufficient research has focused on absorption, distribution, metabolism, and excretion of CI phycotoxins in vertebrate animals (Farabegoli et al., 2018).

3.3. Clinical Signs and Pathology The acute oral and intraperitoneal toxicity of several of these toxins has been evaluated in rodent studies. A number of CI toxins have similar effects on mice. There is a cascade of neurologic abnormalities within a few minutes of intraperitoneal injection, followed either by death within 20 min or a rapid and complete recovery with no apparent sequelae (Otera et al., 2011). Rodents given spirolides can exhibit a “moderate hunched up appearance,” decreased activity, piloerection, hyperextension of the back, a stiff tail that arches toward the head, tremors, spasms, paralysis, and extension

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of the rear limbs. Mice also display abdominal breathing and dyspnea or respiratory arrest, tremors and contractions of the front legs, exophthalmia, lacrimation, urination, and death. Gymnodimines initially cause hyperactivity and jumping in mice, followed by paralysis, hind-leg extension, and a lack of response to external stimuli. Dyspnea and abdominal breathing progress to respiratory arrest and exophthalmia. Pinnatoxins cause hyperactivity, followed by decreased activity, abdominal breathing, and hind-leg extension. Respiration rate decreases and exophthalmia may be moderate to severe just before death. Gross lesions are not expected in animals that die from poisoning with CI phycotoxins. In a study of 13-desmethyl spirolide that was given intraperitoneally to rats at 2000 ug/kg and mice at 75, 260, or 2000 ug/kg, the brain was the only tissue found to be affected. The two species differed, however, in transcriptional and histopathologic markers of injury (Gill et al., 2003). The rats died within 2 min of dosing. The mice at the low, middle, and high doses died at 2, 4, and 8 min postdosing, respectively. In spite of the very short time frame, the rats reportedly had upregulated transcription of nicotinic and muscarinic receptors in the cerebellum and brain stem, but not in the cerebrum, and no histologic changes were noted. By contrast, in the mice, no changes in transcription were found, but they had widespread neuronal damage throughout the brain, and it was most severe in the hippocampus and brain stem. Pyramidal cells of the cornus ammonis 1 (CA1), CA2, and especially the CA3 of the dentate gyrus were affected in a dose-dependent manner, with changes that included hyperchromatic nuclei, pyknosis, karyorrhexis, ghost cells, and shrunken cytoplasm. Also, most notably in the mice at the high dose, the dendrites in the molecular layers of the hippocampus, especially those of the stratum lucidum and stratum radiatum of the CA1 region, were disrupted. Changes included discontinuity in staining of the dendritic fibers.

3.4. Human Exposure and Disease The risks associated with CI phycotoxins in humans are difficult to assess. Shellfish contaminated with these toxins have been encountered worldwide, including on both sides of the North

Atlantic, in multiple areas of the Mediterranean Sea, and on the coastlines of Japan, China, Australia, and New Zealand (Stivala et al., 2015). In some instances, the contaminated shellfish were not allowed to be sold for human consumption (Gue´ret and Brimble, 2010). However, an assessment by a food safety organization in 2010 suggested that most human exposures to CI phycotoxins from shellfish would fall well below those that have been associated with oral toxicity in mice, although more research is needed (European Food Safety Authority Panel on Contaminants in the Food Chain, 2010).

3.5. Diagnosis, Treatment, and Control Mouse bioassays have often been used to screen for neurotoxins, including CIs, in shellfish, but concerns regarding the humane treatment of animals have constrained their use. Increasingly, HPLC-MS/MS and, more recently, cholinergic receptor–based assays have been employed in assays for CIs (Quilliam, 2003; Molgo´ et al., 2017). However, analytical challenges in the HPLC-MS methods include the need for greater availability of certified CI toxin standards. By contrast, the receptor-based assays for these toxins are target-directed, they offer high sample throughput with detection of all known congeners of a given CI family, and they have value in identifying new CIs (Stivala et al., 2015). Unfortunately, very little research has examined the potential toxicity of CI phycotoxins to wild aquatic animals and ecosystems. Studies of wild vertebrates and laboratory animals that consume shellfish contaminated with CIs are needed to better assess risks to human health, wildlife health, and ecological sustainability.

4. DOMOIC ACID 4.1. Occurrence and Species Susceptibility Domoic acid (DA) is also discussed in Food and Toxicologic Pathology, Vol 3, Chap 2 as an example of a naturally occurring toxin in food. The several isomers of DA are structural analogs of the neuroexcitatory amino acids, glutamic acid and kainic acid (Figure 5.7) (Swanson and Sakai, 2009; Tubaro et al., 2012). DA is produced by diatoms of the genera Pseudo-nitzschia (Figure 5.8)

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CH3

CH3 COOH

H CH3

A

COOH

N

H2 C

COOH

COOH N

H

COOH

COOH H2 N

COOH

H

B

C

FIGURE 5.7 Structures of (A) domoic acid, (B) kainic acid, and (C) glutamic acid. Figure reproduced from Nijjar MS, Nijjar SS: Ecobiology, clinical symptoms, and mode of action of domoic acid, an amnesic shellfish toxin. In Botana LM, editor: Seafood and Freshwater Toxins: Pharmacology, Physiology and Detection, Dekker, New York, NY, United States of America, 2000, pp 325–358, in Tubero A, Sosa S, Hungerford J: Toxicology and Diversity of Marine Toxins. In Gupta RC, editor: Veterinary Toxicology, Elsevier, 2012, Fig. 69.6, p. 909.

FIGURE 5.8 Photomicrograph of Pseudo-nitzschia, showing the silica-impregnated cell walls. Image courtesy of Rozalind Jester. Figure reproduced from Beasley VR: Harmful algal blooms (phycotoxins). Reference Module in Earth Systems and Environmental Sciences, Elsevier, 2020. https://doi.org/10.1016/B978-0-12-409548-9.11275-8, Fig. 14, p. 15, with permission.

and Nitzschia, and it is also found in red algae (Bates et al., 2018). DA moves up aquatic food chains. Mussels, razor clams, other shellfish, including Dungeness crabs (Metacarcinus magister), and finfish (especially anchovies and sardines) can serve as vectors of DA to other species (Lefebvre and Robertson, 2010; Fire and Van Dolah, 2012). Low doses of DA from the red alga, Chondria armata, were used for centuries as an anthelmintic in Japan (Takemoto and Daigo, 1958). It was also employed as a natural insecticide (Addison and Stewart, 1989). The previous familiarity with DA and its experimental use in

exploring glutamate receptor activity enabled its structure to be rapidly confirmed after the first recognized poisonings of humans following ingestion of the toxin in cultured mussels from Cardigan Bay, Prince Edward Island, Canada (Iverson et al., 1989; Addison and Stewart, 1989; Perl et al., 1990; Iverson and Truelove, 1994). The syndrome became known as amnesic shellfish poisoning (ASP) due to the disorientation and recall problems of affected people (Iverson and Truelove, 1994; Lefebvre and Robertson, 2010). Blooms of Pseudo-nitzschia that present risks of DA toxicosis may not cause distinctive discolorations of the water (Grant et al., 2010). Upwellings of cold waters bring nutrients and the diatoms to the surface where sunlight and warming may stimulate photosynthesis, replication, and DA production (Lefebvre and Robertson, 2010). Increasingly frequent anomalous ocean warming events and nutrient pollution seem to result in more generations of Pseudonitzschia and increased risks of DA toxicosis (McCabe et al., 2016; McKibben et al., 2017). For example, in 2015, a record-breaking massive bloom of Pseudo-nitzschia along the west coast of the United States that was associated with elevated ocean temperatures resulted in widespread closures of fisheries resulting in significant economic loss (McCabe et al., 2016). Trophic transfer of DA from the diatoms to benthic invertebrates and planktivorous fish presents risks to aquatic predators and humans (Bejarano et al., 2008). DA has also been incriminated in death losses in fish and aquatic invertebrates, including sardines, sharks, and squid (Fire and Van Dolah, 2012).

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4. DOMOIC ACID

Marine wildlife with DA toxicosis have frequently been encountered along the Pacific coast of North America, from California to Alaska, and occasionally in the Gulf of Mexico (Bejarano et al., 2008; Fire and Van Dolah, 2012; Lefebvre et al., 2016). California sea lions (Zalophus californianus) are the most commonly affected species of marine mammals. Since 1998, when DA poisoning was first identified in sea lions following a bloom of Pseudo-nitzschia australis along the California coast, there has been an increased incidence of DA-producing diatom blooms with strandings of sea lions on the west coast (Scholin et al., 2000; Silvagni et al., 2005; Goldstein et al., 2008). Other marine species of the Pacific coast, including northern fur seals (Callorhinus ursinus), southern sea otters (Enhydra lutris), common dolphins (Delphinus sp.), harbor seals (Phoca vitulina), several baleen whale species, and fish-eating birds, have also been affected (Lefebvre and Robertson, 2010; Fire and Van Dolah, 2012; Lefebvre et al., 2010).

4.2. Toxicology DA is a heat-stable, water-soluble, excitatory neurotoxin that exerts effects through interactions with several ionotropic glutamate receptor subtypes at synaptic terminals, whose distributions tend to reflect regions of the brain that are most damaged (Nijjar and Nijjar, 2000; Colegrove et al., 2018). Both DA and the released glutamate activate postsynaptic N-methyl-D-aspartate (NMDA) receptor subunits, which promotes further glutamate release and activation of other postsynaptic glutamate receptors. Receptor activation by these ligands opens neuronal sodium-, potassium-, and calcium-selective ion-gated channels, resulting in osmotic tissue damage. Calcium influx also causes injury from reactive oxygen species as well as activation of phospholipases, endonucleases, and proteases such as calpain, damaging mitochondria, DNA, the cytoskeleton, and cell membranes. The result is neuronal death by either apoptosis or necrosis. Astrocytes and cardiomyocytes also express glutamate receptors, and thus they too are susceptible to DA effects (Pulido, 2008; Lefebvre and Robertson, 2010; Wang et al., 2018). Cynomolgus monkeys are much more sensitive to DA than rodents, and they may be a more appropriate model for human exposure.

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Vomiting in cynomolgus monkeys, an initial sign of DA toxicosis, may be noted after an oral dose of 1 mg/kg (Truelove et al., 1997). While cumulative injury at toxic doses seems likely, studies of nonhuman primates suggest that repeated oral dosing with DA at slightly below acutely toxic doses does not result in accumulations that exceed thresholds of toxicity.

4.3. Clinical Signs and Pathology Clinical signs of DA poisoning are quite varied, but most distinctive are those related to its effects on the brain. Experimental exposure of cynomolgus monkeys caused vomiting, mastication, and yawning (Tryphonas et al., 1990). In mice injected with DA, bizarre but characteristic behavior changes were noted, including scratching of the shoulders with the hind leg, inactivity, and seizures (Iverson et al., 1989). DA poisonings in association with a bloom of Pseudo-nitzschia australis off the coast of California in 1991 caused large death losses in brown pelicans and Brandt’s cormorants, as well as deaths of a few double-crested cormorants, pelagic cormorants, and western gulls (Work et al., 1993). The birds had eaten anchovies that had large numbers of P. australis in their stomachs as well as high concentrations of DA in their viscera and flesh (flesh refers to the whole fish minus the viscera, Work, personal communication, 2021). Signs in the pelicans included slow side-to-side head movements, partial extension of their wings, and continuous fine muscle tremors. Feathers of the pelicans were wet and ruffled. When they tried to fly, they did not retract their legs, ventrally flexed their neck, attempted to scratch their pouch, and soon landed awkwardly in the water. On land, signs were similar except pelicans vomited, rested on their hocks, and clenched their toes. Many of them became unresponsive to external stimuli, had torticollis, lay on their back or side, slowly paddled their feet, and died within 1 day of presentation for rehabilitation. Unlike brown pelicans, the Brandt’s cormorants did not exhibit prominent neurologic signs but were exceedingly passive when approached and handled. Elevated serum creatine kinase, blood urea nitrogen, and uric acid were noted in the affected birds. Most of the dead birds were in good condition. The only consistent

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gross lesions in affected birds were focal hemorrhages in the adductor, sartorius, gracilis and vastus medialis of the hindlimbs, and the biceps brachii of the forelimbs. Four of nine affected birds had microscopically evident focal necrosis of skeletal muscles, and one pelican had diffuse neuronal necrosis in the cerebrum accompanied by capillary endothelial cell hyperplasia. Southern sea otters along the coast of California tend to specialize on a subset of aquatic prey items, and those that focus on clams and crabs in areas with high populations of DAproducing diatoms have an increased risk of cardiomyopathy and lethal heart failure (Moriarty et al., 2021). Those prey species bioconcentrate and only slowly eliminate DA. Cardiomyopathy in the otters appears to be due to acute, subacute, and chronic DA exposure. DA-associated lesions in the central nervous system frequently coexist with cardiomyopathy and the most severely affected otters develop terminal dilative cardiomyopathy. Prime-age adult otters (4–8 years old) are often affected. The clinical signs and pathology of DA toxicosis have been extensively studied in sea lions affected in the wild (Scholin et al., 2000; Silvagni et al., 2005; Goldstein et al., 2008; Zabka et al., 2009; Gulland et al., 2012). Both acute and chronic syndromes have been identified. Seizures, head-weaving, ataxia, and coma have occurred in acutely affected animals. Scratching was also noted, reminiscent of the signs seen in DA-exposed mice and pelicans. Some of the sea lions with more chronic effects exhibited unusual or inappropriately aggressive behavior, intermittent seizures, and apparent blindness; however, they commonly seemed to be normal between neurologic events. Eosinophilia and low serum cortisol were also noted in clinically affected sea lions. Gross neurologic lesions of DA toxicity in California sea lions have included piriform lobe malacia in animals dying acutely, and unilateral or bilateral atrophy of the hippocampus in sea lions that died in the chronic stage of the disease (Figure 5.9). Gross cardiac lesions were less frequently observed and included myocardial pallor and hemorrhage, with myocardial fibrosis occurring with chronic toxicoses. Gravid animals had placental hemorrhage, abortion,

FIGURE 5.9 Domoic acid toxicosis in a California sea lion. Unilateral hippocampal atrophy (arrow) due to chronic domoic acid toxicosis (formalin fixed). Figure reproduced from Colegrove KM, Burek-Huntington KA, Roe W, Siebert U: Pinnipediae. In Terio K, McAloose D, St. Leger J, editors: Pathology of Wildlife and Zoo Animals, ed 1, Academic Press, New York, USA, 2018, pp 569–592. https://doi.org/10.1016/B978-0-12-805306-5.00 023-7, Fig. 23.2, p. 570, with permission.

and uterine torsion and rupture (Silvagni et al., 2005; Pulido, 2008; Goldstein et al., 2008). Histopathologically, the brain damage caused by DA has been relatively similar among affected species as reviewed by Pulido (2008). Brain lesions have been most evident in the limbic system and hippocampus (Figures 5.10–5.12), but also occurred in the olfactory bulb, the piriform and entorhinal cortices, the lateral septum, the subiculum, the arcuate nucleus, and several amygdaloid nuclei. Pyramidal neurons in cornus ammonis 3 (CA3), CA4, and the hilus of the dentate gyrus were the most severely affected regions of the hippocampus. The CA1 region was also affected, and the prominence of damage to the dentate gyrus seemed to be greater in sea lions than other species. The CA2 region tends to be less affected by DA in sea lions. Lesions noted with acute exposure included neuronal shrinkage and necrosis, vacuolization of the neuronal cytoplasm, edema, microvacuolation of the neuropil, and hydropic cytoplasmic swelling of resident astrocytes. Animals and humans surviving acute DA intoxication or those having potentially repeated exposures from food sources can show evidence of chronic histologic damage that is still present

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4. DOMOIC ACID

FIGURE 5.10 Acute domoic acid toxicosis in the brain of a California sea lion. There is extensive neuronal necrosis and neuropil vacuolation (see arrows for examples). Figure reproduced from Colegrove KM, BurekHuntington KA, Roe W, Siebert U: Pinnipediae. In Terio K, McAloose D, St. Leger J, editors: Pathology of Wildlife and Zoo Animals, ed 1, Academic Press, New York, USA, 2018, pp 569–592. https://doi.org/10.1016/B978-0-12805306-5.00023-7, Fig. 23.1, p. 570, with permission.

FIGURE 5.11 Normal hippocampus from a California sea lion. CA1–4, cornu ammonis sectors 1–4; DG, dentate gyrus. Figure reproduced from Colegrove KM, BurekHuntington KA, Roe W, Siebert U: Pinnipediae. In Terio K, McAloose D, St. Leger J, editors: Pathology of Wildlife and Zoo Animals, ed 1, Academic Press, New York, USA, 2018, pp 569–592. https://doi.org/10.1016/B978-0-12805306-5.00023-7, Fig. 23.3, p. 571, with permission.

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FIGURE 5.12 Chronic lesions of domoic acid toxicosis in the hippocampus of a California sea lion. There is parenchymal collapse, neuronal loss throughout the dentate gyrus (DG) and cornu ammonis (CAs), sectors 1–4, and gliosis. Figure reproduced from Colegrove KM, Burek-Huntington KA, Roe W, Siebert U: Pinnipediae. In Terio K, McAloose D, St. Leger J, editors: Pathology of wildlife and zoo animals, ed 1, Academic Press, New York, USA, 2018, pp 569–592. https://doi.org/10.1016/B978-0-12805306-5.00023-7, Fig. 23.4, p. 571, with permission.

months or even years after exposure (Strain and Tasker, 1991; Pulido, 2008). Lesions noted in sea lions with chronic DA toxicosis included hippocampal atrophy, neuronal loss in the hippocampus, dentate gyrus and adjacent temporal lobe cortex, gliosis within the hippocampus, and perivascular cuffing (Silvagni et al., 2005; Goldstein et al., 2008). There is also mounting evidence that chronic low-level exposure to DA may alter brain morphometry and chemistry resulting in cognitive deficits (Lefebvre et al., 2017; Petroff et al., 2019). Animals with DA toxicosis and clinical signs of spinal cord damage, such as paralysis or tremors, may have evidence of focal hemorrhage, neuronal swelling, and vacuolation at any segment, but predominantly in the ventral and intermediate gray matter (Silvagni et al., 2005; Pulido, 2008). Retinal lesions have been rarely noted in studies of DA with rats and cynomolgus monkeys. They consisted primarily of vacuolation and pyknosis of cells in the inner nuclear layer and outer plexiform layer. By contrast, sea lions with DA toxicosis developed vacuolation

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of cells in the ganglion layer (Iverson and Truelove, 1994; Pulido, 2008). Heart lesions noted in sea lions exposed to DA have been well described. They are most likely to occur in the interventricular septum and left ventricle, beginning at the base of the septum. Lesions have ranged from interstitial edema, myocyte vacuolation, and necrosis acutely to myocyte loss and replacement of myocytes by adipocytes and loose fibrous connective tissue in chronically affected animals (Figure 5.13) (Zabka et al., 2009). A spectrum of cardiovascular lesions has been identified in southern sea otters with DA toxicosis (Colegrove, personal observation). Cellular alterations have also been identified in cardiomyocytes of rats given DA (Vieira et al., 2016). The potential behavioral and neuropathologic consequences of exposure to DA in utero or early in life are just beginning to be evaluated (Dakshinamurti et al., 1993). There are concerns that developing animals may be far more sensitive to DA toxicosis than adults, such that current regulations on levels of DA in shellfish may not be low enough to protect developing human

FIGURE 5.13 Domoic acid cardiomyopathy in a California sea lion. Note the loss of myocytes and replacement with loose fibrous connective tissue and adipocytes, with reactive cardiomyocyte nuclei (arrows) surrounding those lesions. Figure reproduced from Colegrove KM, Burek-Huntington KA, Roe W, Siebert U: Pinnipediae. In Terio K, McAloose D, St. Leger J, editors: Pathology of Wildlife and Zoo Animals, ed 1, Academic Press, New York, USA, 2018, pp 569–592. https://doi.org/10. 1016/B978-0-12-805306-5.00023-7, Fig. 23.5, p. 571, with permission.

offspring (Costa et al., 2010). In rehabilitated sea lions, impacts during development have been postulated to produce neurologic consequences that may not manifest until years following the DA exposure and may result in sudden death (Simeone et al., 2019).

4.4. Human Exposure and Disease The first confirmed cases of DA poisoning were in humans in 1987 in Canada and were traced to ingestion of contaminated mussels (Perl et al., 1990). Since then, no confirmed human cases of ASP have been reported, presumably because risks of acute exposure have been lessened through testing shellfish and regulating harvests, based on toxin load (Lefebvre and Robertson, 2010). Nevertheless, chronic, low-level exposure is considered a risk, especially to recreational and tribal shellfish harvesters (Lefebvre et al., 2017). Clinical effects of DA toxicoses in humans include vomiting, diarrhea, abdominal cramps, cardiac arrhythmias, blood pressure fluctuations, headaches, confusion, ophthalmoplegia, hemiparesis, disorientation, agitation, seizures, and coma. The signature characteristic of human ASP, however, is permanent short-term memory loss, which occurs in approximately 25% of cases (Perl et al., 1990; Pulido, 2008). Older individuals have tended to experience more severe impacts of DA toxicosis. Of more than 200 DApoisoned humans, four died within 4 months of eating contaminated mussels. One male victim of the DA poisoning episode, who was in his mid-80s, had prolonged neurologic effects and died from pneumonia 3.25 years later (Cendes et al., 1995; Costa et al., 2010). Shortly after the DA exposure, he had nausea, vomiting, confusion, coma, generalized convulsions, and partial status epilepticus. Three months laster, he was no longer having seizures, but had severe residual memory deficit, and electroencephalograms (EEGs) revealed periodic epileptiform discharges. The EEG abnormalities had resolved at 8 months after the DA ingestion, but magnetic resonance imaging revealed bilateral atrophy of both hippocampi. Seizures and EEG abnormalities were noted again at 1-year after the exposure. Examination of the brain at postmortem revealed severe bilateral hippocampal sclerosis.

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4.5. Diagnosis, Treatment, and Control The presence of appropriate clinical signs and histopathologic findings with a history of exposure to a suspected source of DA are commonly used as presumptive evidence of a toxicosis. Signs and symptoms generally begin within hours or up to 2 days following exposure, and confirmation can be made in acute cases by finding DA in gastrointestinal contents, blood, feces, and/or urine. As the toxin is quickly metabolized, it is often not detected in cases of suspected chronic exposure. Treatment is supportive, with severe cases frequently requiring extended hospitalizations and antiseizure medication (Goldstein et al., 2008). Severely affected humans from the 1987 outbreak were elderly or had preexisting illness. Less severely affected individuals improved within 24 h, but recovery took up to 12 weeks (Perl et al., 1990). Monitoring for conditions conducive to Pseudonitzschia blooms and for high levels of the diatoms and DA concentrations in shellfish are preferred measures for prevention and control. Many countries have established regulatory limits of acceptable concentrations of DA in shellfish tissue, with levels in excess triggering closure of shellfish harvest areas (Lefebvre and Robertson, 2010). A standard method for DA quantification relies on HPLC with ultraviolet absorption detection, but more sensitive methods exist, including HPLC electrospray ionization time-offlight mass spectrometry (Quilliam et al., 1995;

Rossi et al., 2016). A recently published regulatory limit for DA in razor clams and other shellfish is 20 mg/kg of wet weight whole tissue, although up to 30 mg/kg is allowed in Dungeness crabs (Ferriss et al., 2017; FDA and EPA, 2021). The 20 mg/kg value is regarded as the equivalent of a human dose of around 0.1 mg/ kg and is believed to represent a relatively narrow margin of safety (Truelove et al., 1997).

5. BREVETOXINS 5.1. Source/Occurrence There are at least nine brevetoxins (BTXs) divided into two structural types, A and B (Figure 5.14). In addition, several BTX metabolites have been identified in shellfish, finfish, and other marine animals (Baden et al., 2005). The BTXs are lipophilic polyethers. They are produced primarily by marine dinoflagellates belonging to the genus Karenia. The genus is found worldwide, and toxic blooms of Karenia brevis (formerly Gymnodinium breve and Ptychodiscus brevis) account for most of the harmful red tides in the Gulf of Mexico, and occasionally on the Southeastern coast of the United States. Such blooms have been termed Florida red tides, and although they may persist for months, the factors that contribute to bloom behavior have not been clearly elucidated (Landsberg et al.,

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FIGURE 5.14 Structures of brevetoxins of types A and B. PbTx-1 is a synonym forbrevetoxin-1 (BTX-1). Examples of brevetoxin A subtypes include: brevetoxin-1 (PbTx-1) R ¼ eCH2C(¼CH2)CHO and brevetoxin-7 (PbTx7) R ¼ eCH2C(¼CH2)CH2OH. Examples of brevetoxin B subtypes include: brevetoxin-2 (PbTx-2) R ¼ eCH2C(¼CH2)CHO, brevetoxin3 (PbTx-3) R ¼ eCH2C(¼CH2)CH2OH, and brevetoxin-9 (PbTx-9) R ¼ eCH2CH(CH3) CH2OH. Created by Minutemen using BKchem 0.11.4 and Inkscape 0.44dOwn work, Public Domain, https://en.wikipedia.org/wiki/Breve toxin#/media/File:Brevetoxin_A.svg and https://en. wikipedia.org/wiki/Brevetoxin#/media/File:Brevetoxi n_B.svg, retrieved March 22, 2021.

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2009; Fleming et al., 2011; Fire and Van Dolah, 2012). In the Gulf of Mexico especially, BTXcontaining blooms have been associated with short-term, massive losses in fish populations that harmed commercial and recreational fishing industries. BTXs accumulate in filter-feeding mollusks, and consumption of contaminated shellfish is the major source of serious exposures to humans. One of the syndromes produced by BTXs in humans is called neurotoxic shellfish poisoning (NSP), and many cases have been documented along the coast of the Gulf of Mexico in the United States, especially in Florida (Watkins et al., 2008; Fleming et al., 2011). New Zealand has also had outbreaks of NSP that were linked to a mixture of BTXs (Morohashi et al., 1995). Direct inhalation of aerosols of contaminated water is another source of exposure for humans and marine mammals, as wind and surf can suspend intact K. brevis cells as well as lyse them, releasing BTXs (Landsberg et al., 2009; Fire and Van Dolah, 2012). Impacts after inhalation are likely to result from inhalation of both solubilized toxins and intact Karenia cells that gradually release them in the respiratory tract. Pulmonary system receptors are especially sensitive to BTXs (Baden et al., 2005). BTXs have often proven to be lethal to West Indian manatees (Trichechus manatus), especially along the southwestern coast of Florida. Manatees are endangered herbivorous marine mammals of the Sirenidae. The most common cause of lethal brevetoxicosis in manatees is the ingestion of K. brevis on sea grasses, which leads to respiratory paralysis and other neurologic effects. Manatees may also develop irritation of the upper airways after inhalation of contaminated aerosols (Bossart et al., 1998). BTXs have been associated with significant seabird and cetacean mortality events (Naar et al., 2007; Fire and Van Dolah, 2012; Fauquier et al., 2013a). Fish-eating birds and cetaceans can have high concentrations of the toxins in their stomach contents after eating planktivorous menhaden. Indirect effects from loss of prey species can also be a concern for cetaceans (Landsberg et al., 2009; Fire and Van Dolah, 2012).

5.2. Toxicology Toxicity varies among naturally occurring BTX analogues and mixtures (Baden et al., 2005).

Baden (1989) reported mouse intravenous, intraperitoneal, and oral LD50s for BTXs of 50, 500, and 500 mg/kg, respectively. A minimum lethal intraperitoneal dose for one of the most potent BTXs with the BTX A backbone, PbTx-1 (also termed BTX-1), was 50 mg/kg body weight (Ishida et al., 1995). BTXs act on nerve cell membranes by binding to site 5 on voltage-sensitive sodium channels, causing an influx of sodium ions (Baden, 1989). This causes spontaneous depolarization of the membranes and neuronal activation (Lombet et al., 1987; Fleming et al., 2011). Heart effects have been observed with high doses of BTXs. Studies of dogs and cats have revealed BTXinduced cardiac arrhythmias, bradycardia, and peripheral vasodilation (Baden, 1989). BTXdosed rats and guinea pigs had complete heart block. In the respiratory tract, the mechanisms of action are less studied, but there is evidence that BTXs can inhibit cathepsin catabolism, and cause DNA damage and apoptosis. Cathepsins are proteases that normally are intralysosomal. They are a trigger of apoptosis, in part via caspase activation and release of proapoptotic factors from mitochondria. Upper respiratory disease caused by inhalation of aerosolized BTXs may also be due, in part, to histamine release in the airways, and stimulation of axonal sodium channels that causes acetylcholine release at parasympathetic efferent nerves in respiratory smooth muscle cells (Franz and LeClaire, 1989). BTXs also suppress humoral immunity and lymphocyte proliferation, increasing susceptibility to infectious diseases (Fleming et al., 2011; Pierre et al., 2018).

5.3. Clinical Signs The clinical signs of brevetoxicosis are largely due to neurotoxicity, hemolysis, and, when inhaled, respiratory toxicity. Manatees poisoned by BTXs have been disoriented, exhibited difficulty maintaining a horizontal position in the water, and had labored breathing (Bossart et al., 1998; Fire and Van Dolah, 2012). In bottlenose dolphins, an increased rate of forceful exhalations or “chuffing” has been associated with red tide blooms (Fire et al., 2020b). Piscivorous birds believed to be poisoned by BTXs on the west coast of Florida have included double-crested cormorants (Phalacrocorax

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auritus), brown pelicans (Pelicanus occidentalis), great blue herons (Ardea herodias), and common loons (Gavia immer). Affected birds were disoriented, ataxic, unable to stand, and had seizures (Fauquier et al., 2013a). On the west coast of Florida in 2005 and 2006, during two K. brevis red tide events, a total of 318 sea turtles, including loggerhead (Caretta caretta), Kemp’s ridley (Lepidochelys kempii), green (Chelonia mydas), hawksbill (Eretmochelys imbricata), and leatherback (Dermochelys coriacea) turtles, became stranded, which was an increase from the 12-year average of 43 strandings (Fauquier et al., 2013b). A number of the sea turtles were found dead, and others died during rehabilitation. In total, 61 affected sea turtles were admitted for rehabilitation. Clinical signs included head bobbing, muscle twitching, jerky body movements, lethargy, unresponsiveness, paresis, and circling. The residues of BTXs in the plasma of live turtles and in feces, stomach contents and liver of those that died, the neurologic signs, and a lack of significant lesions in the sea turtles were believed to be consistent with BTX toxicosis. Fish poisoned with BTXs have been reported to violently twist and swim in a corkscrew pattern. Other signs included regurgitation, defecation, paralysis of pectoral fins, curvature of the caudal fin, loss of equilibrium, reduced activity, vasodilation, convulsions, and death from respiratory failure (Baden, 1989).

5.4. Gross and Histologic Findings The pathologic features of BTX poisoning are nonspecific, but patterns of changes consistent with the toxicosis after exposure via various routes of exposure have emerged. Some of the nonspecific lesions are secondary to neurotoxicity. Pathologic findings in West Indian manatees during a large die-off attributed to BTXs from a red tide were described by Bossart et al. (1998). Lesions of brevetoxicosis overlapped with those of other common causes of death in that species (Figure 5.15). For example, bloody discharge from the eyes and nares, and congested bloody organs, have been seen in manatees dying from BTX poisoning, but these may occur secondarily to acute shock from other causes such as watercraft trauma or acute

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hypothermia. Gross necropsy of BTX-poisoned manatees revealed reddening and edema of the nasopharyngeal, tracheal, and bronchial mucosae, with accumulation of thick red mucus in lumens. Some affected manatees had severe congestion and a red mottled appearance to the lungs, and lung margins that were pink to bright red. Copious amounts of blood and serosanguinous fluid were noted on cut section consistent with pulmonary edema. Histopathological findings in manatees with BTX toxicosis have included pulmonary congestion, hemorrhage, and edema that were compatible with nonspecific effects of acute agonal cardiovascular collapse or acute shock. Chronic-active catarrhal inflammation of the upper respiratory tract was commonly found, with infiltration of moderate numbers of submucosal lymphocytes and plasma cells, sparse neutrophils, and submucosal congestion and hemorrhage. The lesions often were most severe in the nasopharynx, suggesting respiratory irritation. Similar changes associated with respiratory mucosal irritation have been evident in humans and dolphins following inhalation of windborne BTXs (Bossart et al., 1998). Manatees with BTX poisoning have had hemosiderin deposition in multiple tissues, including the liver, spleen, and central nervous system, with siderophages and occasionally hemorrhage and congestion in the cerebrum, cerebellum, meninges, and spinal cord. These findings have been interpreted as evidence of chronic hemolysis, similar to hemosiderosis noted in fish and birds following BTX exposure that was accompanied by chronic hemolytic anemia or a consumption coagulopathy. Nonsuppurative leptomeningitis, primarily affecting cerebellar meninges, was noted. In the brain, both lymphocytes and microglial cells exhibited positive immunohistochemical staining for BTXs (Bossart et al., 1998). Rats do not appear to be an optimal model species for inhalation toxicity of BTXs by humans. A study using rats found that they tolerated inhalation exposure that was 2–3 orders of magnitude higher than those associated with respiratory effects in humans on beaches during red tide events (Benson et al., 2004). BTX-3 is a major component of K. brevis extract. The rats were exposed via nose-only inhalation to an aerosol containing purified BTX-3 at 532 mg/m3

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FIGURE 5.15 Gross necropsy and histologic findings from a West Indian manatee believed to have died from brevetoxicosis. (A) Foamy nasal discharge. (B) Congested primary bronchus, filled with bloody foam and mucus. (C) Photomicrograph of nasal mucosa revealing submucosal thickening, congestion, and leukocyte infiltrate (20). Such findings can be found in manatees dying from a variety of causes besides brevetoxicosis. The diagnosis of brevetoxicosis needs to be confirmed with additional findings such as exposure to a bloom of Karenia and brevetoxins in ingesta and tissues. Photographs (A) and (B) courtesy of the Florida Fish and Wildlife Conservation Commission; photograph (C) courtesy of David Rotstein. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 3, Academic Press, 2013, Fig. 38.10, p. 1165, with permission.

for 0.5 or 2 h/day on 5 consecutive days. The authors estimated that the delivered doses were 8.3 or 33 mg/kg/day, respectively. Although some of the high-dose rats were transiently lethargic and lost body weight, none of the treated rats developed clinical signs consistent with neurologic or respiratory toxicity. Necropsy and histopathologic findings were unremarkable. Nevertheless, some immunologic responses were observed. In both the low- and high-dose rats, there were >70% reductions in plaque-forming splenic lymphocytes in response to administered sheep red blood cells. A recent review on the effects of both BTXs and ciguatoxins addressed respiratory and

immunologic toxicity in greater detail, documenting findings in medaka fish embryos, loggerhead sea turtles, mice, rats, sheep, manatees, dolphins, and humans (Pierre et al., 2018). The authors of that report indicated a need for studies of neuroimmunologic mechanisms of BTXs.

5.5. Human Exposure and Disease A retrospective analysis of an outbreak of human brevetoxicosis from a bloom of K. brevis that traveled in the Gulf Stream to the coast of North Carolina in 1987 found that 65% of individuals who ingested 12 or more BTX-

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contaminated oysters developed symptoms of NSP (Watkins et al., 2008). The risk of poisoning increased with the number of oysters consumed. Finfish may also contain BTXs, but at much lower concentrations, and their consumption is generally not restricted during red tides. Concentrations of BTXs are higher in fish viscera than muscle, and humans who eat fish fillets appear to be at low risk of acute poisoning (Naar et al., 2007). In humans with NSP, gastrointestinal signs may include vomiting, diarrhea, and abdominal cramps. Neurological signs and symptoms include ataxia, vertigo, weakness, and especially paresthesia, manifested by numbness or a tingling sensation of the face, lips, and extremities, as well as a reversal of hot and cold sensations. To our knowledge, no human deaths from NSP have been reported. However, the long-term risk of exposure to BTXs over time is unknown. In addition, BTXs may persist in shellfish for months after a harmful bloom of K. brevis. Inhalation of sea spray aerosols by humans during red tides containing BTXs has been associated with acute ocular and throat irritation, nasal congestion, wheezing, coughing, chest tightness, bronchoconstriction, asthma, dizziness, and tunnel vision (Kirkpatrick et al., 2004; Fleming et al., 2011). Even a relatively shortterm exposure to BTX-containing aerosols can aggravate signs of asthma lasting for days (Kirkpatrick et al., 2011).

5.6. Diagnosis, Treatment, and Control The mouse bioassay is used in screening for the presence of BTXs in shellfish for regulatory purposes. Immunoassays, such as radioimmunoassays (RIAs) and ELISAs, have been developed to screen blood and urine for BTX metabolites, and can also be used with samples of seawater, shellfish, and tissues. HPLC-MS can be used to detect specific BTXs and their metabolites (Abraham et al., 2008, 2018). A presumptive diagnosis of NSP in humans is generally dependent upon a clinical history of consumption of mollusks 3–4 h before the onset of respiratory, gastrointestinal, and neurological symptoms and signs. Noting such effects in multiple individuals that have had the same exposure is also supportive of the diagnosis (Fleming

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et al., 2011). Identifying the toxins in samples of shellfish that were consumed or in gut contents of the affected species supports diagnoses of BTX poisoning (Fire and Van Dolah, 2012). In birds that died or were euthanized after exposure to a dense bloom of K. brevis, the highest concentrations of BTXs found were in bile, stomach contents, and liver (Fauquier et al., 2013a). Sequential sampling of blood and feces from live seabirds during rehabilitation indicated that BTX was cleared within 5–10 days after admission for rehabilitation. BTXpoisoned birds generally had no significant gross abnormalities when examined at necropsy. In the report of Fauquier et al. (2013b) on the effects of the K. brevis bloom along the west coast of Florida in 2005 and 2006, BTXs were found in 52 of 56 live stranded sea turtles, and 42 of 43 dead stranded sea turtles. BTX concentrations were highest in feces, stomach contents, and liver, and the toxins were cleared from the blood of survivors within 5–80 days after admission for rehabilitation (Fauquier et al., 2013b). The residues of BTXs and the lack of significant lesions in most of the turtles that died were regarded as consistent with BTX toxicosis. The clearance of BTXs from the blood of sea turtles was much slower than in mice and rats. Treatment of human patients for BTX poisoning is largely supportive. Most BTXpoisoned human patients recover within a few days (Watkins et al., 2008). Activated charcoal has been given to bind BTXs in people who were admitted within 4 h of ingestion of contaminated shellfish. Sedation and pain mitigation have also been employed. However, data are lacking on the effectiveness of interventions for human BTX toxicoses. Brevanal, another metabolite of K. brevis, has been effective in antagonizing toxic effects of BTXs in laboratory studies of both cells and fish. It is under consideration for treatment of BTX poisoning, and has been suggested as a potential future treatment for BTX-poisoned manatees. Brevenal is also being examined as a potential drug candidate to reduce pulmonary inflammation in human asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (Keeler et al., 2019). A recent report indicated that intravenous lipid emulsion therapy administered to sea turtles presented to rehabilitation facilities for

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brevetoxicosis accelerated removal of BTXs from plasma, reduced recovery times, and improved overall survival (Perrault et al., 2021). Control of exposure to BTXs is accomplished by avoiding contact with contaminated waters and seafood. Closures of beaches can decrease potential respiratory exposures to beachgoers (Fleming et al., 2011). The presence of K. brevis in algal blooms is monitored, and densities of the organism that exceed acceptable cut-off points can result in closures of commercial shellfish harvesting (Plakas and Dickey, 2010).

6. CIGUATOXINS 6.1. Source/Occurrence Human ciguatera fish poisoning (CFP) results from consumption of tropical and subtropical coral reef fish contaminated with ciguatoxins (CTXs) (Poli et al., 1997). CTXs are lipid-soluble cyclic polyethers that are structurally similar to brevetoxins. Around 50 congeners occur, and they differ in toxicity, but the most common and potent is CTX-1 (Figure 5.16). Pacific CTXs are generally more toxic than those of the Indian Ocean or the Caribbean Sea (Pasinszki et al., 2020). Cases of CFP are likely to be vastly underreported (Friedman et al., 2008). Nevertheless, with estimates that more than 25,000 people experience CFP each year, the syndrome is believed to be the most common marine biotoxin food poisoning around the world today (Lewis and Ruff, 1993; Pasinszki et al., 2020). Producers of CTXs include Gambierdiscus toxicus and other members of that genus, as well as Fukuyoa spp. and possibly several other benthic dinoflagellates of the genera Ostreopsis,

Prorocentrum, Amphidinium, and Coolia. The toxins are concentrated in coral reef food chains, and high levels are most commonly found in predatory fish, including species such as barracuda (Sphyraena spp.), grouper (Epinephelus and Mycteroperca spp.), red snapper (e.g., Lutjanus spp.), and amberjack (Seriola spp), some of which are top selling in fish markets (Barton et al., 1995; Dickey and Plakas, 2010; Friedman et al., 2017; Gaiani et al., 2020). CFP outbreaks are common in the tropical Caribbean and subtropical regions of the North Atlantic and the Pacific (Gillespie et al., 1986; Barton et al., 1995). Residents of Pacific atoll islands who often rely on fish as a major component of their diets are especially at risk (Lewis and Ruff, 1993). Being on the windward side of tropical islands, earthquakes, severe tropical storms, tsunamis, explosions, construction, and shipping activities have been implicated in triggering movement of the dinoflagellates from residence beneath the sand so that they become available in food chains leading to predatory fish. Damage to coral reefs, nutrient pollution, and coastal development have been associated with increases in cases of CFP (Lewis and Ruff, 1993; Litaker et al., 2010). CTXs have been detected in tissues from a high percentage of stranded Hawaiian monk seals (Neomonachus schauinslandi) that died due to various causes, as well as from blood samples collected during live seal health assessments. Relatively high levels of the toxins have been found in some seals that were alive years later. Though these findings suggest that some level of toxin exposure may be tolerated by these endangered tropical pinnipeds, more studies are needed to better assess potential health effects in such species, including after sequential CTX exposures (Bottein et al., 2011). CH3

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FIGURE 5.16 Structure of CTX-1B, one of the most toxic ciguatoxins; a cause of ciguatera fish poisoning. Created by Minutemen using BKchem 0.11.4 and Inkscape 0.44dOwn work, Public Domain, https://commons.wikimedia.org/w/ index.php?curid¼1167399, retrieved March 19, 2021.

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6.2. Toxicology CTXs are metabolized in herbivorous, omnivorous, and carnivorous fishes, forming oxidized and more potent derivatives that accumulate to toxic levels in edible fish flesh (Pasinszki et al., 2020). CTXs are agonists on receptors of voltage-gated sodium channels of neuromuscular junctions, sensory neuron membranes, and other excitable cells (Legrand et al., 1982; Swift and Swift, 1993; Strachan et al., 1999). Like brevetoxins, CTXs act on site 5 of the alpha subunit of sodium channel receptors to increase excitability and prolong refractory periods of sensory and motor nerves. The result of the high-affinity interaction of CTXs with such sodium channels is the opposite of the effects of tetrodotoxin or saxitoxin, which block sodium channels (Cameron et al., 1991a,b; Dechraoui et al., 1999). In vitro, CTX induces contraction of the ileum and exerts a positive inotropic effect on guinea pig cardiac muscle (Lewis and Hoy, 1993).

6.3. Maitotoxins Maitotoxins (MTXs) have been implicated along with ciguatoxins (CTXs) in some reports on CFP. MTXs are structurally similar to both brevetoxins (BTXs) and CTXs, but MTXs are much larger molecules (Pisapia et al., 2017). All of these toxins are ladder-shaped cyclic polyethers, but MTX has 32 fused ether rings, 28 hydroxyl groups, 21 methyl groups, 2 sulfates, and 98 chiral centers. Although water-soluble MTXs may coexist with CTXs in epibenthic Gambierdiscus spp. and Fukuyoa spp., and MTXs can be isolated from herbivorous fish, the risk of human poisoning from them is not well established. The bulk of MTXs in fish is likely to be in their digestive tracts, which are not routinely eaten by humans. Moreover, while MTXs can be extraordinarily toxic when given intraperitoneally, they are much less toxic after ingestion. At sufficient doses, MTXs cause rapid influx of calcium into cells via a voltage-independent mechanism in the plasma membrane. This is believed to account for depolarization of neurons; smooth muscle contraction; secretion of dopamine, norepinephrine, gamma aminobutyric acid (GABA), and insulin; increases in inflammatory mediators; and development of

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cytolytic pores. Studies are needed to identify potential contributions of MTXs to CFP or other toxic syndromes in exposed humans (Swift and Swift, 1993; Caillaud et al., 2010; Munday et al., 2017). Also, since fish-eating wildlife typically ingest whole fish, investigating the exposures and susceptibilities of piscivores to MTXs alone and in combinations with CTXs should be a priority.

6.4. Clinical Signs and Pathology Signs of toxicosis in mice following oral or intraperitoneal exposure to CTX include hypothermia, diarrhea, lacrimation, hypersalivation, penile erection, dyspnea, cyanosis, convulsions, and death (Ito et al., 1996). In cats that consumed contaminated fish, clinical signs included vomiting, diarrhea, inappetence, hypersalivation, ataxia, and partial tetraparesis (Clark and Whitwell, 1968). Gross lesions in experimental mice dosed with CTX included large amounts of mucus in the upper colon, similar to that seen with cholera toxin. However, histologic evidence of damage to the intestinal mucosa was not a consistent finding. Ultrastructural lesions included swelling of nerve fibers and synapses in the small intestinal muscle layers and a loss of synaptic vesicles (Terao et al., 1991). In the heart, histologic findings included focal necrosis and swelling of cardiac myocytes and effusion into cardiac interstitial spaces. Capillaries in the heart had swollen endothelial cells, narrowing of capillary lumina, and intracapillary platelet accumulation. Repeated intraperitoneal or oral administration of CTX caused bilateral ventricular hypertrophy as well as diffuse interstitial fibrosis of the interventricular septum and ventricles (Terao et al., 1992). Mice had marked degeneration of the adrenal medulla and those with severe dyspnea had severe pulmonary edema and congestion (Terao et al., 1991).

6.5. Human Exposure and Disease CFP has been known for centuries in tropical and subtropical regions, but with their popularity as tourism destinations and their exports of fish, CFP has become a worldwide health

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concern (Hashmi et al., 1989; Dickey and Plakas, 2010; Solin˜o and Costa, 2020). In addition, Gambierdiscus spp. was recently found in both the eastern Atlantic and the Mediterranean Sea. CTXs have been detected in more than 400 species of fish. Although CTXs can cause abnormal behavior or be lethal in fish, toxincontaining fish may also appear healthy, and have normal taste and smell. The variability in toxin accumulation in fish and the lack of understanding of the factors affecting the occurrence and toxigenicity of Gambierdiscus blooms make predicting risks of CTX poisoning extremely difficult (Litaker et al., 2010; Solin˜o and Costa, 2020). The clinical manifestations of CFP have been best described in man and include a combination of gastrointestinal, cardiovascular, and neurologic effects (Morris et al., 1982; Friedman et al., 2008). Symptoms may vary somewhat based on geography due to the presence of different congeners in local environments and fish species (Friedman et al., 2017). Repeated exposures may result in more severe and prolonged effects, likely due to the fat-soluble CTXs accumulating over time (Dickey and Plakas, 2010). The first symptoms and signs of CFP may appear within 10–30 min postingestion and typically include gastrointestinal upset that subsides within 2 days (Farstad and Chow, 2001). The condition has been described as “usually very debilitating with acute symptoms persisting up to 3–4 weeks after onset” (Cameron et al., 1993). Common effects that support a diagnosis of CFP include paresthesias marked by onset of prickling and numbness of the oral and perioral regions, which sometimes extend to the hands and feet. Chronic dysesthesia, characterized by a reversal of hot and cold sensations, is also consistent with CFP. It has been suggested that CTX-induced discharge of A-delta and Cpolymodal nociceptors may be involved in the abnormalities in temperature sensation. Other early effects include nausea, vomiting, watery diarrhea, abdominal cramps, myalgia, arthralgia, dizziness, and, on occasion, hypersalivation (Fasano et al., 1991). Chills and sweating may also be present. Exhaustion, weakness and fatigue, paresthesia, and pruritis can persist for weeks to months, and in some patients symptoms of chronic fatigue have lasted for years

(Friedman et al., 2007; Dickey and Plakas, 2010). Bradycardia with hypotension also occurs, and may be accompanied by respiratory distress; however, coma and death from CTX poisoning are rare (Friedman et al., 2017). Nerve biopsies from human patients have shown Schwann cell swelling with compression of axons and myelin vesicular degeneration (Friedman et al., 2017).

6.6. Diagnosis, Treatment, and Control A provisional diagnosis of CFP is usually made based on the combination of appropriate clinical signs and symptoms, and a history of recent consumption of predatory fish. Symptoms appearing in multiple individuals who recently consumed the same fish, and exclusion of other diseases that cause similar symptoms, including neurotoxic shellfish poisoning, are supportive of a diagnosis of CFP. Confirmation of the CTXs as the cause of toxicosis depends upon identifying the toxins in the fish consumed (Hoffman et al., 1983; Friedman et al., 2017; Pasinszki et al., 2020). The mouse bioassay has been used classically for regulatory testing and confirmation of the presence of CTXs in fishery products. A carefully obtained fish extract can also be tested in a bioassay that involves injecting mosquitoes. Methods that offer more specific diagnostic information with regard to the presence of CTXs in food samples and specimens from suspected CFP patients include cell-based assays, receptor binding assays, immunoassays, and HPLC-MS/MS. CTXs are potent and an estimated minimum lethal dose of CTX for people is 0.02 mg/kg body weight. Concentrations of CTX >1 mg/kg of fish flesh pose risks to human health. The European Food Safety Authority and US Food and Drug Administration have set a recommended safety limit for human consumption of Pacific CTX-1 equivalents of 0.01 mg/kg of fish tissue (Pasinszki et al., 2020). Because several CTXs are yet to be structurally characterized and analytical standards for many such compounds are unavailable, their roles remain to be adequately understood. Treatment for CTX poisoning is usually only supportive, including controlling fluid and electrolyte balance and maintaining vital cardiorespiratory functions (Friedman et al., 2017).

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Although contradictory reports exist, some success in reducing the severity of neurologic symptoms in confirmed cases has been reported from the use of intravenous infusions of mannitol (Hashmi et al., 1989; Cameron et al., 1993; Lewis et al., 1993; Friedman et al., 2017). Other aspects of treatment for CFP were reviewed by Farstad and Chow (2001).

7. OKADAIC ACID AND DINOPHYSISTOXINS 7.1. Source/Occurrence Diarrheic (alternatively diarrhetic or diarrheal) shellfish poisoning (DSP) is the common name of a human toxicosis stemming from this group of toxins. Okadaic acid (OA) (Figure 5.17) and structurally related dinophysistoxins (DTXs), including DTX-1, which is 35-methylokadaic acid, are lipophilic polyethers that are produced by several species of two genera of dinoflagellates, Prorocentrum and Dinophysis, and by bacteria in the Roseobacter clade that associate with those algae (Lafay et al., 1995; Sugiyama et al., 2007; Tong et al., 2015). DSP cases have been documented in Japan, Europe, North and South America, Thailand, Australia, and New Zealand (Tubaro et al., 2012). In one incident of OA poisoning in Italy, over 300 people were poisoned (Tubaro et al., 2012). OA was originally isolated from sea sponges of the genus Halichondria, including Halichondria okadai, but symbiotic Prorocentrum lima were the likely producers of the toxin (Baden and Trainer, 1993). Prorocentrum and Dinophysis occur in tropical to temperate waters, and human poisonings from eating contaminated bivalve shellfish have been reported from many parts O

H

HO OH

of the world. Fortunately, lethal poisonings by these toxins are rare in humans, and hospitalization is generally not necessary. Unfortunately, much less is known about the effects of these toxins in other species. Additional studies are needed to better understand the potential impacts of OA and DTXs in marine mammals (Broadwater et al., 2018). Dinophysis and Prorocentrum are among the phycotoxin-producing genera that are commonly encountered in the western Gulf of Mexico. Off the coast of Texas in early 2008, blooms of Dinophysis, Prorocentrum, and, to a limited degree, Pseudo-nitzchia were present when more than 100 bottlenose dolphins (Tursiops truncatus) stranded and died. The dolphins had low levels of OA in their digestive tract contents, and near the end of the die-off, low residues of DA were also found in some of them. The findings confirmed exposures, but causation of the deaths could not be conclusively established (Fire et al., 2011). When apparently healthy South American sea lions (Otaria byronia) and Peruvian fur seals (Arctocephalus australis) were examined on the coast of Peru during a haul-out in 2012, they had low levels of OA in their feces (Fire et al., 2011, 2017). The latter findings suggest that some exposures to OA may be well tolerated by certain pinnipeds.

7.2. Toxicology In a variety of test systems, OA and DTXs have been shown to have cytotoxic, mutagenic, carcinogenic, tumor-promoting, neurotoxic, immunotoxic, hepatotoxic, nephrotoxic, cardiotoxic, and embryotoxic effects (Fujiki et al., 1988, 1991; Valdiglesias et al., 2013; Abal et al., 2018). In laboratory rodents, OA and different DTXs have oral

O

O O H OH

H

H

OH O

O H

O

H

OH

H

O

FIGURE 5.17 Structure of okadaic acid, which causes diarrheic (alternatively diarrhetic or diarrheal) shellfish poisoning. Author, Charlesy. This image of a simple structural formula is ineligible for copyright and therefore is in the public domain. It consists entirely of information that is common property and contains no original authorship, https://commons. wikimedia.org/wiki/File:Okadaic_acid.svg, retrieved March 22, 2021.

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and intraperitoneal LD50s ranging from around 200 to 2300 mg/kg (Aune et al., 2007; Valdiglesias et al., 2013; Abal et al., 2018). A recent comparison in orally dosed mice indicated that DTX-1 was most toxic, OA was intermediate, and DTX-2 was least toxic (Abal et al., 2018). OA, DTX-1, DTX-2, DTX-3, and several other DTX analogues are potent inhibitors of serine/ threonine protein phosphatase types 1 (PP1) and 2A (PP2A) (Perez et al., 2008; Valdiglesias et al., 2013). OA is a more potent inhibitor of PP2A than PP1, with IC50 values ranging from 0.07 to 0.2 nM for PP2A, and from 3.4 to 19 nM for PP1. While other mechanisms of action may also be involved in the cytotoxicity, genotoxicity, and mitotic arrest attributable to these toxins, the inhibition of protein phosphatases is usually regarded as a highly important initiator that culminates in major effects including oxidative stress, caspase activation, decreased mitochondrial membrane potential, loss of cytochrome c from mitochondria to the cytosol, inhibition of protein synthesis, and cytoskeletal disruption (Vale and Botana, 2008; Feng et al., 2018). In contrast to the many neurotoxic phycotoxins, the acute clinical manifestations of OA and DTX poisoning are largely related to the gastrointestinal tract. Following ingestion, the inhibition of PP1 and PP2A results in increased phosphorylation of myosin and other proteins, which accounts for smooth muscle contraction and damage to gastrointestinal epithelial cells. The outcome is abdominal cramping and intraluminal fluid accumulation in the digestive tract.

7.3. Clinical Signs and Pathology Terao et al. (1986) described the effects that followed intraperitoneal dosing of suckling Balb/c mice with DTX-1. Gross lesions were limited to the small intestine, and included the accumulation of mucus, with distension of the proximal small intestine. Histopathologic findings included edema of the lamina propria of villi and, at higher doses, vacuolation of mucosal epithelial cells. The villous and submucosal vessels were congested, especially at higher doses. Transmission electron microscopy (TEM) revealed three dose-dependent sequential levels, or stages, of degenerative change to the

intestine. The first stage involved edema and extravasation of serum into the lamina propria, resulting in some separation of the overlying epithelial cells. In the second stage, epithelial absorptive cells contained dilated cisternae of the Golgi apparatus that were filled with flocculent material, and vesicles within the apical portions of the cells. At the higher doses, there was also degeneration of epithelial cells and loss of microvilli. The third stage was characterized by congestion of villous and submucosal vessels, and detachment of many mucosal epithelial cells from the basement membrane with focal erosions on the mucosal surface. Abal et al. (2018) completed an oral toxicity study in mice comparing OA, DTX-1, and DTX-2 to controls. The toxin-treated mice experienced a rapid onset, but short duration of diarrhea. They also showed signs including piloerection, ocular squinting, cyanosis, and reductions in food and water intake, responsiveness (termed apathy), and body weight. The stomach and intestines of the affected mice were distended and contained gas and liquid ingesta. TEM of the gastrointestinal mucosae in the more severely affected mice revealed mitochondrial swelling, shortened microvilli, swelling of rough endoplasmic reticulum, and increased phagophores containing damaged organelles. Myocardial cells of OA-treated mice had disruption of the outer mitochondrial membrane. Both OA- and DTX-1-treated mice had dilated rough endoplasmic reticulum in the gut epithelium and cells of the spleen, kidney, and brain. OA and DTX-1 may also cause hepatic damage, with subcapsular hemorrhage, degeneration of hepatic sinusoidal endothelial cells, and dissociation of ribosomes from endoplasmic reticulum as well as autophagic vacuoles within midzonal hepatocytes. In the skin of topically treated mice, OA and DTX-1: (i) inhibited protein phosphatases, (ii) increased phosphorylation of nuclear and cytosolic proteins, and (iii) were effective tumor promoters (Fujiki et al., 1988, 1991).

7.4. Human Exposure and Disease OA and DTXs accumulate in the hepatopancreas of shellfish. Mussels are the most common

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source that leads to clinical toxicoses, although other filter-feeding species, including clams and scallops, and crustaceans that prey on shellfish can also present hazards to human consumers (Torgersen et al., 2005). The onset of clinical signs of DSP in humans generally ranges from 30 min to 12 h after ingestion, and symptoms usually resolve within 3 days (Munday, 2013). Acute poisoning is primarily manifested by nausea, vomiting, abdominal pain, and severe diarrhea that is often watery. Around 10% of affected individuals develop fever. The syndrome can readily be confused with bacterial gastroenteritis. Cancers of the digestive tract, liver, and pancreas have been associated with previous diarrheic shellfish poisoning in humans (Valdiglesias et al., 2013).

7.5. Diagnosis, Treatment, and Control OA and DTXs can be quantified in shellfish, urine, feces, and blood with LC-MS/MS (Abal et al., 2018; Sakaguchi et al., 2021). OA is widely distributed in the body, but concentrations tend to be high in the liver and digestive tract contents. The toxin can often be found in the feces for several days following oral exposures (Valdiglesias et al., 2013). Unfortunately, phycotoxin analyses are infrequently used for diseases caused by this class of toxins. Instead, the diagnosis is usually based primarily on the onset of clinical signs within a few hours of ingestion of filter-feeding bivalve mollusks, such as mussels. This leaves doubt in regard to the actual frequency of OA and DTX poisoning as well as a risk that other phycotoxins that may account for the problems will remain unrecognized. There is no specific antidote for poisoning by OA or DTXs. Treatment is symptomatic, e.g., oral rehydration, and complete clinical recovery usually occurs within 3 days. OA equivalents have been developed, based on knowledge of its toxicity and that of DTX analogues. To protect consumers, food safety agencies have chosen a maximum level of 160 mg of OA equivalents per kg of edible shellfish tissue for human consumption (Abal et al., 2018). With scallops, removing the hepatopancreas and eating only the adductor muscle can greatly reduce risks of poisoning.

8. AZASPIRACID TOXINS 8.1. Source/Occurrence Azaspiracids (AZAs) are lipophilic polyether toxins that were first identified in 1995 after eight individuals of the Netherlands who had consumed mussels, Mytilus edulis, originating from Killary Harbour, Ireland, were poisoned (Twiner et al., 2008) (Figure 5.18). Since then, AZAs have been found in shellfish samples from several places around the world, including several European countries, northwest coastal Africa, eastern Canada, and the east and west coasts of South America. AZAs are produced by small dinoflagellates of the genera Azadinium and Amphidoma (Dai et al., 2019). Bivalves take up AZAs when filter feeding on those dinoflagellates as well as when they ingest Protoperidinium crassipes, another dinoflagellate that is believed to accumulate the toxins through predation on Azadinium spp. Ingestions of mussels have accounted for the vast majority of human AZA poisonings to date; however, other bivalves, such as oysters, clams, scallops, and cockles, as well as crabs (Cancer pagurus) can also contain AZAs (Furey et al., 2010). Characteristics that distinguish AZA phycotoxins from other toxic polyethers produced by marine dinoflagellates include the presence of a cyclic amine, known as an aza group, and a trispiro assembly. Also, the first AZA discovered, AZA1 contained an end carboxylic acid. Thus, the name reflected three features: the aza, spiro,

O O

HO O

H H OH

O

H

H H

NH

O HO O H

O O

O

H

FIGURE 5.18 The structure of azaspiracid-1. Author, Charlesy. This image of a simple structural formula is ineligible for copyright and therefore is in the public domain. It consists entirely of information that is common property and contains no original authorship, Own work, https:// commons.wikimedia.org/wiki/File:Azaspiracid-1.svg, retrieved March 22, 2021.

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and acid groups (Twiner et al., 2008). Several additional AZAs have been described, e.g., AZA2, AZA3, AZA4, AZA5, and AZA6, which differ primarily in the numbers and locations of methyl, carboxyl, and hydroxyl groups. Overall, at least 62 AZA analogs have also been found to date, but often AZA1, AZA2, and AZA3 account for most of the overall AZA profile in contaminated shellfish (Furey et al., 2010; Dai et al., 2019; Boente-Juncal et al., 2020). Some of the analogues are products of metabolism of parent AZA compounds in shellfish, and others result from structural artifacts produced during extraction.

8.2. Toxicology The minimum acutely lethal intraperitoneal dose of AZA1 in mice has been reported to be as low as 150–200 mg/kg (Twiner et al., 2008; Furey et al., 2010). By that route of administration, AZA2 and AZA3 were slightly more toxic; while AZA4 and AZA5 were significantly less toxic. The lethal intragastric dose of an extract containing concentrated AZA1 was about 1.5–3 times higher than the lethal intraperitoneal dose (Ito et al., 2002a; Furey et al., 2010). Although the signs and symptoms of AZA poisoning in humans are very similar to those of diarrheic shellfish poisoning, the toxins are structurally distinct from OA (Twiner et al., 2008). The molecular target and mechanism of AZA toxicosis have not been conclusively demonstrated, but they differ from OA, as AZAs do not inhibit protein phosphatases 1 or 2A. AZA1 is cytotoxic to a range of cultured cell types, including neurons. It increased cyclic AMP, stimulated a mitogen-activated protein kinase pathway, and increased caspase activity in cultured neuronal cells (Furey et al., 2010). A recent study with human embryonic kidney (HEK293) cells revealed that AZA1 reduced proliferation, and both AZA1 and AZA2 inhibited sodium currents in voltage-gated ion channels, which are also important in brain and spinal cord (Boente-Juncal et al., 2020). Both AZAs and glutaric acid can be found in the same shellfish, and a synergistic effect between AZA1 and glutaric acid in altering sodium flux was revealed in other studies of cultured HEK293 cells (Chevallier et al., 2015). The

clinical importance of impacts of AZAs on the sodium channels of intact vertebrates requires further study.

8.3. Clinical Signs and Pathology Although reports of AZA toxicoses in humans relate primarily to gastrointestinal signs, studies of mice have revealed damage not only in the digestive tract, but also other organs, including liver, lung, pancreas, thymus, and spleen. AZA is also a tumor initiator in mice (Ito et al., 2000, 2002a; Aasen et al., 2010). Despite the development of intestinal lesions, diarrhea has not been characteristic of acute AZA toxicosis in mice. Manifestations of acute toxicosis following intraperitoneal injection included progressive paralysis with dyspnea, convulsions, and, at high doses, death within 20–90 min (Ito et al., 2000; Furey et al., 2010). Unlike the effects of AZA in intraperitoneally dosed mice, the onset of clinical signs in intragastrically dosed mice was delayed, and even a highly lethal dose of 900 mg/kg caused no behavioral changes during the first 4 h (Ito et al., 2002a; Furey et al., 2010). Nevertheless, light microscopic lesions were noted in mice as soon as 1 h after intragastric dosing with an extract rich in AZA1, and by 24 h, damage was found in several organs (Ito et al., 2000). Small intestinal lesions included congestion and mild fluid accumulation. Light and scanning electron microscopy revealed atrophy of the small intestine with necrosis in the lamina propria of villi, epithelial vacuolization and sporadic degeneration, and eroded, shortened, and sometimes fused villi (Figure 5.19). The large intestine had widespread degeneration of mucosal epithelial cells. The stomach was largely spared. The mice also had liver enlargement and pallor due to an increase in lipid (Figure 5.20). Mice given AZA at 300 mg/kg had fatty change in the liver by 1 h postdosing. The liver was pale by 4 h. At 24 h after dosing at 500 mg/kg, liver weight had increased by 38%. Other changes included hepatocellular degeneration and single cell necrosis, severe splenic atrophy, and lymphoid necrosis in the spleen, Peyer’s patches, and thymus. Both B and T lymphocytes were affected. The time to recovery from lesions in mice that survived intragastrically administered AZA1 at doses from 250 to 450 mg/kg on one or two

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FIGURE 5.19 Scanning electron micrograph (A) and H and E light microscopy (B) of section of the upper small intestine from a mouse given azaspiracid intragastrically at 300 mg/kg 4 h previously. Epithelial cells (E) covering intestinal villi are sporadically degenerated (arrows). Vacuolar degeneration (circle) is widespread and atrophy of the lamina propria (LP) has caused spaces to form between the epithelial cells and lamina propria. Figure reproduced from Ito I, Satake M, Ofuji K, Kurita N, McMahon T, James K, Yasumoto T: Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland, Toxicon 38:917–930, 2000, Fig. 2, p. 922, with permission.

occasionsdwith 2 days between dosingsdvaried depending on the organ or tissue involved (Ito et al., 2002a). Mice died at doses as low as 250 mg/kg. Mucosal erosions and shortened villi were noted in the stomach and small intestine. Lung lesions included alveolar edema, hemorrhage, and interstitial pneumonia. The liver had fatty change, polynuclear giant cells surrounding hematoxylin-stained debris, and small granulomas around central veins. The recovery times were: lymphoid tissues, 10 days; liver, 20 days; lung, 56 days; and small intestine and stomach, more than 12 weeks, but the recovery of the gastric mucosa was impaired by bacterial infection. Intragastric dosing of ICR mice with AZA1 at 1, 5, 20, or 50 mg/kg twice weekly for up to 145 days revealed dose-dependent toxicity (Ito et al., 2002a). The highest dose resulted in greatly reduced activity, severe damage to the intestinal mucosa, and marked losses in body weight as well as heart, liver, kidney, spleen, and thymus

weights. All of the mice in that group were euthanized before the end of the study. The small intestine was gas-filled, presumably due to insufficient peristalsis. Villi were shortened, and the intestinal wall was edematous, which may have caused malabsorption and the observed weight loss. The affected mice developed interstitial pneumonia and sometimes congestion, with increases in lung weight. Lung, stomach, and intestinal lesions were also prominent in the mice dosed at 20 mg/kg. The gastric mucosa was hyperplastic in most of the mice of the 20 mg/kg/dose group. At 50 mg/kg, the spleen and thymus were severely atrophied with marked reductions in lymphocytes. Lymphoid depletion was less severe at 20 mg/ kg. Lung tumors were observed in 1 of 10 mice in the 50 mg/kg/dose group at about 100 days into the study, and in 3 of 10 in the 20 mg/kg/ dose group at 2–3 months after the dosing period ended. No consistent lesions were noted at 1 or 5 mg/kg. Immunohistochemical staining of the

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rather AZA35 and six novel AZAs. Effects on the mussels were mainly in the digestive gland, and included thinning of tubules, reduction in neutral lipid deposits, and an accumulation of lipofuscin. Hemocytes had inhibited phagocytosis and damaged lysosomal membranes. There was also a biphasic change among the populations of different hemocytes, which suggested the possibility of AZA-associated immunosuppression in the mussels.

8.4. Human Exposure and Disease FIGURE 5.20 Sudan III–stained section of liver from mouse given azaspiracid orally at 500 mg/kg 24 h previously. Reddish intracytoplasmic vesicles are evidence of fat accumulation and were distributed in the peripheral regions of the hepatocytes throughout the liver. Figure reproduced from Ito I, Satake M, Ofuji K, Kurita N, McMahon T, James K, Yasumoto T: Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland, Toxicon 38:917–930, 2000, Fig. 7, p. 927, with permission.

tumor cells suggested a possible neurogenic origin. There is a need for additional studies of the potential for AZAs to cause lung and other cancers since the study of Ito et al. (2002a) examined small numbers of mice/group, and there was no clear dose–response relationship. Also, in other research, control ICR mice have had a high incidence of spontaneous lung tumors (Giknis and Clifford, 2005). Embryos of Japanese medaka (Oryzias latipes) that were injected with AZA1 had delayed development, bradycardia, reduced hatching, and increased death losses (Colman et al., 2005). Future studies should explore the possibility of similar impacts in embryos of finfish that have fed on contaminated shellfish. In a laboratory trial, mussels (Mytilus galloprovincialis) were allowed to bioaccumulate AZAs from Azadinium dexteroporum that had originated from the Mediterranean Sea. They were exposed for a period of 3 weeks followed by 3-week recovery period. The mussels retained a subset of the ingested AZAs (Giuliani et al., 2019). Unlike the better-known producers of AZAs, A. dexteroporum did not produce AZA1–2, but

To our knowledge, naturally occurring AZA poisonings have been documented in humans, but not in other species of animals. Effects may include severe diarrhea, nausea, vomiting, and stomach cramps (Twiner et al., 2008; Dai et al., 2019). Clinical signs in poisoned humans have resolved within 2–5 days.

8.5. Diagnosis, Treatment, and Control LC-MS/MS is an effective method for determining the presence and concentrations of AZAs in Azadinium extracts (Dai et al., 2019). Resin extractions from large volumes of water have found AZA1 and AZA2, with a predominance of AZA1 (Twiner et al., 2008). Shellfish from known areas of contamination in Europe have been tested for AZAs by mouse bioassays, and several analogs can be identified through targeted LC-MS/MS. The toxins are often concentrated in the hepatopancreas, which is the organ generally used for mouse bioassays. In mussels, however, AZAs have been found to migrate, resulting in higher concentrations in other tissues. Analytical standards are available for AZA1– 3, but a lack of analytical standards for other analogs presents challenges to surveillance and diagnostic programs. A regulatory limit of 160 mg AZA/kg whole shellfish flesh was set by the European Union to protect human consumers because the preferred, i.e., safer, lower limit of 80 mg/kg could not be detected by mouse bioassays (Furey et al., 2010). Concentrations of AZA are sometimes far higher than the 160 mg AZA/kg action level. Depuration appears to be very slow, so shellfish can be

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contaminated for many months (Twiner et al., 2008). It seems likely that some cases of AZA poisoning are misdiagnosed as diarrheic shellfish poisoning, such as when people develop gastrointestinal signs of disease after eating shellfish, especially mussels, and analytical confirmation is not pursued. Shellfish that are negative for diarrheic shellfish toxins, such as OA, and that may have been cleared for human consumption following mouse bioassays for diarrheic shellfish poisoning might still contain AZAs. Precooked and frozen mussels can also result in AZA poisonings. Future studies of other laboratory mammals are warranted to determine whether they react to AZAs in ways more similar to humans. Research should also examine AZA transfer and toxicity to marine molluscivores, including other invertebrates, fish, birds, and marine mammals. The potential for interactions of AZAs with other marine phycotoxins, including those that also interfere with sodium channels, should be assessed in cultured cells, laboratory animals, and representative aquatic animals that live in phycotoxin-contaminated food webs.

9. CYLINDROSPERMOPSINS 9.1. Source/Occurrence Cylindrospermopsin (CYN) is a sulfated tricyclic alkaloid that combines a guanidine group and a hydroxymethyl uracil. Its analogs include deoxycylindrospermopsin (7-deoxy-CYN) and 7-epicylindrospermopsin (7-epi-CYN) (Buratti et al., 2017) (Figure 5.21). CYN has been studied most, and it is highly water soluble, environmentally stable, cytotoxic, hepatotoxic, and nephrotoxic (Norris et al., 1999; Banker et al., 2000; Lewis, 2000; Li et al., 2001; Humpage and Falconer, 2003; Kinnear, 2010). The first-reports of poisoning implicating CYN described how, in 1979, water in Solomon Dam, Palm Island, Queensland, Australia, that contained a bloom of Cylindrospermopsis raciborskii (synonym Raphidiopsis raciborskii) was associated with an outbreak of severe hepatoenteritis affecting 148 Indigenous people, most of whom required hospitalization and intensive care. Initially, that syndrome was known as “Palm

H N

O OH O = S= O O

O NH

H H

H OH

N

NH † NH

FIGURE 5.21 Structure of cylindrospermopsin, a hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria of several genera. Created by Cacycle with ChemDraw and IrfanView, Own work, Public Domain, https://commons.wikimedia.org/wiki/File:Cylindrosp ermopsin.png, downloaded March 22, 2021.

Island mystery disease” (Blyth, 1980; Hawkins et al., 1985; Ohtani et al., 1992; Griffiths and Saker, 2003; de la Cruz et al., 2013). In the mid1990s, eutrophic lakes in Florida were also found to contain C. raciborskii that produced CYNs (Chapman and Schelske, 1997). In recent decades, strains of C. raciborskii have become globally invasive. They have been found in tropical, subtropical, and temperate areas of New Zealand, Asia, Africa, Europe, South America, and North Americadincluding Mexico, the United States, and Canada. A number of the specimens of C. raciborskii found, however, were not examined for CYNs, or they were assayed, but did not contain detectable concentrations (Kinnear, 2010; de la Cruz et al., 2013; Antunes et al., 2015; Yang et al., 2017). In addition to C. raciborskii, biosynthesis of CYNs has been linked to Chrysosporum ovalisporum (formerly, Aphanizomenon ovalisporum), Chrysosporum bergii (formerly Anabaena bergii), Dolichospermum flos-aquae (formerly Anabaena flos-aquae), Moorea wollei (formerly, Lyngbya wollei), Umezakia natans, and Raphidiopsis curvata (Li et al., 2001; Cronberg et al., 2003; Preussel et al., 2006; Seifert et al., 2007; Sarma, 2013). CYNs are possible contaminants of drinking water, and blooms of C. raciborskii are often present below the surface, potentially near the intakes of water treatment plants (Carson, 2000; US EPA, 2015a). Also, concentrations of CYNs are commonly higher in aqueous solution than in a suspension of the remaining intact cells. As such, simple filtration may have limited

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efficacy in protecting drinking water supplies, and most water treatment plants are not designed to remove these toxins. In open surface waters, CYN concentrations are generally well below 10 mg/L, but occasionally, they range up to 800 mg/L (WHO, 2020b). CYN concentrations of finished drinking water are usually less than 0.7, but the toxin has been found at up to 97 mg/L. CYNs in eutrophic freshwater can accumulate in the viscera and to a lesser extent the muscle of shellfish, including bivalves, snails, prawns and crayfish, as well as finfish. CYN is also found in food plants irrigated with contaminated water (de la Cruz et al., 2013; US EPA, 2015a). Because growing children and other young animals drink and eat more than adults, they may have proportionately higher levels of exposure to CYN.

9.2. Toxicology Cattle have died in less than 7 days of their initial exposure to a drinking water source that contained CYN at concentrations >1 mg/L, which comprised a dose of about 50 mg/kg/ day (Shaw et al., 2002). Those authors described two incidents of CYN toxicosis, one involving deaths of 10 cattle and the other that involved 45. Mice that were evaluated for the same report were less sensitive, as drinking water that contained CYN at as high as 5 mg/L for up to 90 days did not cause apparent toxicity. A range of studies suggests that an oral LD50 of CYN in mice is approximately 2–6 mg/kg (US EPA, 2015a). Mice that were dosed orally for 30 days with CYN at the lower doses of 75– 300 mg/kg/d had several alterations in hepatic gene expression, including: (i) increased Bcl2associated X protein (BAX), which participates in the metabolic pathway that leads to apoptosis; (ii) reductions in fatty acid–binding protein 4 (FAB4), which is involved in uptake, transport, and metabolism of fatty acids; (iii) increased 60S ribosomal protein L6 (RPL6), which is involved in hepatocellular regeneration; and (iv) decreased protein C (PROC), which inactivates factors Va and VIIIa, thereby interfering with coagulation (Chernoff et al., 2018). Lesions and renal effects in these mice are discussed below.

The mechanisms of CYN toxicity are complex and they vary as a function of dose. CYN accumulates in liver over time, binds to DNA, causes DNA fragmentation, and inhibits protein synthesis (Terao et al., 1994; US EPA, 2015b). Inhibition of protein synthesis in vivo and in vitro has been attributed to the parent toxin (Buratti et al., 2017). Phase I and Phase II metabolism are important in the toxicity of CYN. Cytochrome P450 enzymes are not required for CYN to inhibit protein synthesis, but they are important in generating toxic metabolites of CYN (Froscio et al., 2003). In studies using primary hepatocytes, inhibition of cytochrome P450 enzymes decreased CYN cytotoxicity (Runnegar et al., 1995b). Lower concentrations of CYN inhibited hepatocyte protein synthesis, while higher concentrations acted in concert with P450 to trigger other mechanisms of hepatocytotoxicity resulting in cell death (Froscio et al., 2003). Although supported by indirect evidence in vitro and in vivo, CYN biotransformation to one or more potent cytotoxic compound(s) remains to be clarified, and metabolites believed to account for CYN-induced genotoxicity are yet to be identified (Shaw et al., 2000; Norris et al., 2002). In human hepatoma HepG2 cells, CYN itself induced upregulation of Phase I enzymes (CYP1A1, CYP1B1, ALDH1A2, and CES2) as well as Phase II enzymes (UGT1A6, UGT1A1,  NAT1, and GSTM3) (Straser et al., 2013). Induction of CYP 450 enzymes caused HepG2 cells to become more vulnerable to CYN toxicity; however, Phase I metabolites of CYN could not be detected in those cells or in fractions of liver tissue (Liebel et al., 2015; Kittler et al., 2016). Other studies of CYN toxicity found metabolic disturbances, including oxidative damage, and inhibition of glutathione (GSH) synthesis (Runnegar et al., 1994, 1995b), which would interfere with conjugation of reactive metabolites by glutathione-s-transferases (GSTs). CYNassociated oxidative injury might therefore be expected to contribute to cytotoxicity in several organs, including liver, kidneys, and heart. However, as with metabolism that produces bioactive metabolites, the roles of oxidative stress and GSH in CYN toxicity are only partially understood, and toxic effects have

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been found to be dose dependent (US EPA, 2015a; Buratti et al., 2017). Indeed, the study of  Straser et al. (2013), which focused on toxicogenomics in human hepatoma, HepG2, cells that were exposed to a “noncytotoxic but genotoxic concentration of CYN” of 0.5 mg/mL for 12 and 24 h, indicated that oxidative stress was minimal, and that, while catalase (CAT) and thioredoxin reductase (TXNRD1) were upregulated, other relevant genes were unaffected. Nevertheless, CYN induced the FOS and JUN gene families in concert with marked deregulation of the P53 tumor suppressor pathway and upregulation of NF-kB signaling. There was also strong upregulation of the growth arrest and DNA damage inducible genes, GADD45A and GADD45B, as well as cyclin-dependent kinase inhibitors, CDKN1A and CDKN2B, checkpoint kinase 1 (CHEK1), and genes that assist in DNA repair (XPC and ERCC4). The findings were consistent with cell cycle arrest, nucleotide excision, and double-strand break repair. A more recent study that examined the effects of CYN on HL1-hT1 cells, which express characteristics of adult human liver stem cells, indicated that a “subcytotoxic concentration” of CYN (1 mM; 0.415 mg/mL) that did not cause oxidative stress, ER stress, or DNA damage, nevertheless, increased transcription coding for stress-related transcription factor ATF3 and increased the phosphorylation of both ERK1/2 and p38 kinase, consistent with increased mitogen-activated protein kinase (MAPK) activity (Raska et al., 2019). Additional information on mechanisms of CYN toxicity is available in a number of summary documents (US EPA, 2015a,b; de la Cruz et al., 2013; WHO, 2020b).

9.3. Clinical Signs and Pathology As noted above, cattle have been reported to be susceptible to lethal poisoning by CYN (Shaw et al., 2002). The affected cattle had pale mottled livers and distension of their gall bladders. Histopathologic examination revealed hepatocyte degeneration and necrosis as well as nephrosis and multifocal degeneration of cardiac myocytes. At least four cattle died from a herd following exposure to water from a dam containing C. raciborskii and CYN. Necropsy of a calf from that herd that rapidly

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deteriorated over 24 hours revealed epicardial and small intestinal serosal hemorrhages, and a pale, swollen liver with bile duct hyperplasia (Saker et al., 1999). Histopathology showed severe hepatocyte degeneration and necrosis with only isolated areas of intact hepatocytes remaining, and widespread hepatic fibrosis. Widespread hepatic fibrosis would not be expected in this time frame if a single acute exposure was involved, but the time frame of exposure in this case was not reported. Prolonged monitoring of potential or confirmed exposures to CYN, and ruling out other hepatoxic substances and pathogens, would be helpful to better understand the potential range of effects of CYN on cattle, other livestock, and wildlife. Mice that were dosed intraperitoneally with extracts of cultured C. raciborskii, from the Solomon Dam reservoir mentioned above, developed hepatomegaly and grossly evident lobular congestion and hemorrhage, as well as congestion of the lungs, kidneys, and adrenals (Hawkins et al., 1985). Some of the mice had a pale liver, white foci on the hepatic margins, and/or focal pulmonary hemorrhages. Histopathology revealed hepatocellular coagulative necrosis that ranged from centrilobular to massivedaffecting all hepatocytes. Eosinophilic material and necrotic cells were evident in central veins, and thrombi were noted in the portal veins and lungs. Surviving mice developed hepatocellular lipidosis. The kidneys had variable degrees of epithelial necrosis, which was usually mild and affected proximal tubules. There was scattered degeneration and necrosis in the adrenals, and congestion and edema in the small intestine. Seawright et al. (1999) characterized the clinical signs and lesions of mice exposed orally to C. raciborskii containing CYN. Mice consuming these cells had reduced activity, anorexia, loss of body weight, and a rough hair coat due to piloerection. Although they were anorexic, their stomachs were full of the normal diet and algae, indicating a marked delay in gastric emptying. The liver was the primary target organ, appearing mottled, pale, and swollen. The mice had ulcers in the esophageal portion of the gastric mucosa and fresh blood in the stomach and intestine. The kidneys were pale and slightly swollen, and there was atrophy of the thymus and spleen. Histopathologic findings included

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hepatocellular lipid accumulation that gave the cytoplasm a foamy appearance. The lower doses of C. raciborskii were principally associated with periacinar hepatocyte fatty infiltration. At higher doses, there was more generalized lipid accumulation, as well as periacinar coagulative necrosis and inflammatory cell infiltrates that included macrophages. Acute tubular necrosis was present in the kidneys, cortical lympholysis in the thymus, and lymphophagocytosis in the spleen. The authors suggested that the damage to extrahepatic organs, including kidneys, thymus, and spleen, may have resulted from tissue anoxia and stress from dehydration and being moribund. Subacute exposure of mice to CYN in their drinking water was associated with increased plasma cholesterol and elevated hematocrit, as well as acanthocytes in the peripheral blood, which may be related to liver injury (US EPA, 2015b). Terao et al. (1994) dosed mice intraperitoneally with purified CYN, from U. natans, at 200 mg/kg body weight, followed by euthanasia over the following 100 h for gross, histopathologic, and ultrastructural examinations. Hepatomegaly progressively increased and, by 72 h, the liver was yellow. Histopathologic findings included hepatocytes that were pale and enlarged, as well as centrilobular necrosis with hemorrhage in both centrilobular and midzonal areas. Ultrastructural lesions in the liver progressed over time. Earlier hepatocellular changes were more common in centrilobular regions and included: detachment of ribosomes from the endoplasmic reticulum; formation of dense, rounded, and small nucleoli; proliferation of the smooth endoplasmic reticulum; Golgi bodies that occasionally formed membrane whorls; fat droplets displacing organelles; and autophagic vacuoles in hepatocytes near central veins. The bile canaliculi were dilated and contained flocculent material. The authors described four phases: (i) ribosomal and nucleolar changes associated with inhibited protein synthesis, (ii) agranular membrane proliferation, (iii) accumulation of fat droplets, and (iv) cell death. By 100 h, all of the hepatocytes “were destroyed.” The kidneys were pale on gross examination, and at the ultrastructural level, there was proliferation of the smooth endoplasmic reticulum in proximal convoluted tubular epithelial cells as well as fat droplets

beneath the brush border. Single cell necrosis was noted in proximal and distal convoluted tubules. The thymus had severe cortical lympholysis, and there was occasional single cell necrosis in the heart. Scattered hepatocellular apoptosis, necrosis, and hepatic inflammatory cell infiltrates were observed in mice treated intraperitoneally with CYN at 830 mg/kg (Figures 5.22 and 5.23; Haschek unpublished data). Ultrastructural changes in the liver were similar to those described by Terao et al. (1994). In the kidneys, single cell death was localized to the pars recta of the proximal tubules. Rogers et al. (2007) dosed pregnant mice intraperitoneally with purified CYN from 8 to 128 mg/kg on gestation days 8–12 and encountered significant lethality in the dams that were given >32 mg/kg. Surviving dams were euthanized. Litter sizes, fetal weights, and the incidence of anomalies were not altered by CYN exposure. In a second trial, the toxin was similarly administered at 50 mg/kg on gestation days 8–12 or 13–17. Both groups had fewer pups than controls, but only the pups of the late-dosed dams had reduced body weights

FIGURE 5.22 Section of liver from a mouse dosed intraperitoneally with cylindrospermopsin at 830 mg/kg. There is scattered hepatocellular apoptosis (arrowhead), karyorrhexis (arrow), and phagocytized apoptotic bodies (oval). H and E stain, 400. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 3, Academic Press, 2013, Fig. 38.16, p. 1172, with permission.

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FIGURE 5.23 Section of liver from a mouse dosed intraperitoneally with cylindrospermopsin at 830 mg/ kg. The hepatic parenchyma shows evidence of hepatocellular necrosis with nuclear debris, which was most severe within centrilobular regions (arrow) and a few foci of neutrophilic infiltrates. Binucleated hepatocytes are occasionally seen (arrowhead). H and E stain, 400. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 3, Academic Press, 2013, Fig. 38.17, p. 1172, with permission.

and more deaths. Also, the surviving offspring had hemorrhage in the digestive tract and hematomas in the tail. Although they had been crossfostered by control females until weaning, the males of that group were stunted, and they remained so, even at 15 months of age. Chernoff et al. (2011) treated pregnant mice with purified CYN intraperitoneally at 50 mg/ kg for 5 days, either early or late in gestation. Toxin treatment caused lesions of varying severity among the dams, but those treated during gestation days 8–12 had earlier and more marked effects than the ones treated during gestation days 13–17. Manifestations of CYN toxicosis included reduced maternal weight gain and activity, hemorrhage in and around the eyes, and bleeding from the vagina, distal tail, and gastrointestinal tract. Serum enzyme markers of hepatic disease as well as markers of renal dysfunction (blood urea nitrogen and creatinine) increased, while the serum concentrations of glucose and albumin decreased. Several mice died before the end of the study or became moribund and were

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euthanized. Histopathology of the liver of the dams revealed centrilobular hepatocellular necrosis and apoptosis, and inflammatory cell infiltrates that generally affected less than 10% of the hepatic parenchyma. Approximately 25% of the dams treated during gestation days 8–12 developed chronic interstitial nephritis or mild tubular epithelial necrosis. Oral dosing of mice with purified CYN at 75, 150, or 300 mg/kg/day for 90 days caused no overt signs of toxicity, but males had increased liver and kidney weights (Chernoff et al., 2018). Females had more severe hepatic lesions, but less notable changes in serum chemistry values than males. Both sexes had enlarged hepatocytes and hepatic cord disruption, but only the females were affected at all three doses. Males at the highest dose had increased serum alanine aminotransferase activity, and at the two highest doses, they had reduced serum cholesterol. The males also had dilated renal tubules with enlarged and basophilic epithelial cells. CYN is mutagenic and can cause whole chromosome loss (Falconer and Humpage, 2001). Mice that were dosed orally up to three times with CYN in an extract from C. raciborskii were compared to controls. A subset of the mice of each group were then given food twice weekly that contained O-tetradecanoylphorbol 13-acetate, a potent tumor promoter. Tumors were noted in mice with and without the tumor promoter. Of 53 CYN-exposed mice, 5 developed neoplastic changes, most often in the liver, whereas none were found in 27 control mice. It seems reasonable to suspect that CYN is a potential carcinogen in other species, and additional research on the carcinogenic potential for CYN would be of value. High-level cutaneous exposures of mice to CYN caused primary irritation as well as a hypersensitivity reaction (Stewart et al., 2006). Gutie´rrez-Praena et al. (2012) dosed tilapia via gavage or intraperitoneally with purified CYN at a single sublethal dose of 200 mg/kg, and noted histological damage in several organs that increased in severity over the 5-day observation period. Lesions were most severe in the liver and kidneys. Fish treated intraperitoneally tended to have more pronounced changes than those treated via gavage, except in the gastrointestinal tract. They developed hepatocellular

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steatosis with large lipid droplets, substantial glycogen deposits, increased nuclear size, and loss of normal parenchymal architecture. Cells in pancreatic regions appeared degenerated, with cytoplasmic vacuoles and scarce granularity, and occasional cells were necrotic. Ultrastructural studies revealed tumefaction of mitochondria. The kidneys had membranous glomerulopathy with hyalinization and dilation of Bowman’s capsule, atrophy of glomeruli, capillary hyperemia, and reduced width of the proximal and distal convoluted tubules. Cardiac changes associated with CYN treatment included edema and myofibrolysis. Gastrointestinal changes included catarrhal enteritis that progressed to necrosis of enterocytes, calciform cells, loss of microvilli, edema, hyperemia, and microhemorrhages. The gills had swollen lamellae with erosions, microhemorrhages, hyperemia, edema, and inflammatory foci.

9.4. Human Exposure and Disease A few days prior to the outbreak on Palm Island, Australia, the major drinking water source, Solomon Dam reservoir, was treated with unreported levels of copper sulfate to control a dense algal bloom. By lysing cyanobacterial cells, copper sulfate increases free cyanotoxins in the water. Only households connected to the reservoir were affected by the illness. Retrospective epidemiological and ecological assessments have indicated that the predominant cyanobacterial species in the reservoir, C. raciborskii, was the likely source of the illness (Griffiths and Saker, 2003; Hawkins et al., 1985). Nevertheless, one consultant has asserted that the copper sulfate had been dumped near the water intake for the treatment plant, such that it was a more likely cause of the poisonings (Prociv, 2004). In our view, the possibility of combined copper and CYN toxicosis should not be dismissed. The total number of people exposed in that incident was not reported. However, of the 148 cases, 138 were children, with a mean age of 8.4 years, range 2–16 years, and 41% were male and 59% female. The ages and sexes of the adults were not reported. The clinical signs and symptoms included fever, headache, vomiting, profuse bloody diarrhea, and hepatomegaly. There was also renal damage as indicated by dilute urine with losses of electrolytes, proteins, ketones and carbohydrates, dehydration, hypovolemia, and shock. The findings

from Palm Island may suggest greater susceptibility of children than adults, and/or that the children drank more water. The principal ongoing concerns regarding human exposures to CYN relate to ingestion of drinking water and swallowing surface water during recreation (US EPA, 2015a). Humans and other species can also be exposed from eating contaminated plants and animals, including irrigated vegetables and wild or aquaculture-derived invertebrates, fish, and amphibians.

9.5. Diagnosis, Treatment, and Control Analysis for CYNs can be accomplished with LC with UV absorbance detection, LC with photodiode array detection, and LC-MS/MS. The current US EPA method relies on the latter technology (WHO, 2020b). While quantitative criteria remain to be established, a presumptive diagnosis of CYN toxicosis relies on identifying compatible lesions, ruling out other etiologies, and evidence of exposure, with toxin analysis of source materials generally relying on ELISA, HPLC-diode array detection, or HPLC-MS. In one case involving cattle, the water had a CYN concentration of approximately 1.5 mg/L (Saker et al., 1999). In two other incidents cattle that died had CYN at 570 or 5700 mg/L in their rumen contents (Shaw et al., 2002). In the first of these, the water had CYN at 1050 mg/L and liver CYN concentrations (apparently wet weight) ranged from 7.4 to 51 mg/kg, while kidney CYN ranged from 9.4 to 29 mg/kg. The effectiveness of treatment to counteract CYN toxicoses in exposed humans and other animals warrants additional study. Many of the individuals who were apparently affected by CYN on Palm Island, Australia, required intravenous therapy to correct electrolyte imbalances and, in some cases, therapy for acidosis, and hypovolemic shock (Blyth, 1980; Griffiths and Saker, 2003; de la Cruz et al., 2013). Across the world, the values for CYN used as legal limits or recommended maximal concentrations in drinking water have ranged from 1 to 15 mg/L (Buratti et al., 2017). Recently the US EPA identified a reference concentration of CYN at 15 mg/L to protect human health related to recreational exposures from water bodies (US EPA, 2019). WHO (2020b) set provisional

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guideline values, meaning concentrations that should not be exceeded, for CYN of 0.7 mg/L for life-time drinking water, 3 mg/L for shortterm drinking water, and 6 mg/L for recreational water. These guideline values were based on nephrotoxicity, which was regarded as the most sensitive effect of CYNs. In that regard, WHO relied on two reports. The first was that of Humpage and Falconer (2003), which found an increase in relative kidney weight in mice given CYN at >60 mg/kg bw/day and a noobserved-adverse-effect level (NOAEL) of 30 mg/kg bw/day. Humpage and Falconer (2003) indicated, however, that in terms of histopathologic effects, the liver was slightly more sensitive than the kidneys: minor histopathologic changes were noted in the liver at as low as 120 mg/kg bw/day, while the lowest dose that was associated with damage to renal proximal tubules and a decrease in urine specific gravity was 240 mg/kg bw/day. The second source of information relied upon by WHO in deriving their provisional guideline values was the report of Chernoff et al. (2018) that described effects on mice that received CYN at 75–300 mg/ kg bw/day for up to 90 days. In that study, toxic effects were noted at all doses, including increases in liver and kidney to body weight ratios, and lesions in both organs. The lowest dose caused toxic effects; thus, they did not derive an NOAEL. In males given 75 mg/ kg bw/day, the mean increase in liver/body weight ratio was 20%, while the mean increase in kidney/body weight ratio was approximately 40%. WHO (2020b) also indicated that 7-deoxyCYN and 7-epi-CYN may be of similar potency to CYN, so that they should be included in calculations of total CYN exposures. Preventing cyanobacterial blooms through limitations on dissolved phosphorus is highly important in avoiding high CYN concentrations (WHO, 2020b). The large extracellular concentration of CYN presents a unique challenge to drinking water managers. Nevertheless, removal of cyanobacterial cells has value, and CYN can be reduced by prefiltration while keeping the water pH above 6 to avoid lysis. Dissolved CYN can be inactivated or removed by ultraviolet light treatment, biodegradation, activated carbon filtration, nanofiltration, reverse osmosis, and oxidation by interaction with chlorine or ozone, but not chloramine or

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chlorine dioxide (US EPA, 2015b; WHO, 2020b). Additional information on drinking water management to reduce risks of CYN toxicoses is provided by WHO (2020b). Field studies should examine whether CYN is a cause of human cutaneous irritation, which sometimes follows exposure to cyanobacterial blooms. The potential for Cylindrospermposisand CYN-associated skin irritation and hypersensitivity reactions were revealed by a mouse bioassay. Depending on the findings of additional research on dermotoxicity, revised guidelines to limit or prevent recreation-related topical exposure to water containing these cyanobacteria and this phycotoxin may be warranted (Stewart et al., 2006).

10. MICROCYSTINS AND NODULARINS 10.1. Source/Occurrence The most commonly recognized poisonings caused by cyanotoxins are the acute hepatotoxicoses that follow oral exposures to microcystins (MCs) and nodularins (NDs). The structurally similar MCs and NDs are monocyclic heptapeptides and pentapeptides, respectively (Carmichael et al., 1988a,b; Rinehart et al., 1988; Carmichael, 1997) (Figure 5.24). Most of the variations in the MCs are in their two additional L-amino acids (e.g., Gago-Martinez, 2007). One of the most common, most potent, and most studied of these is MC-LR, where LR indicates that the variable amino acids are leucine and arginine. The number of identified MCs has expanded in recent years to around 250, whereas the NDs total only 10 (Spoof and Catherine, 2017). Across the world, Microcystis is the genus most often involved in poisonings by MCs, and, among the three identified toxigenic species, Microcystis aeruginosa, Microcystis viridis, and Microcystis wesenbergii, the most intensively studied has been M. aeruginosa (Figure 5.25). Other genera that produce MCs include Dolichospermum (formerly Anabaena), Nostoc, Planktothrix, and Oscillatoria (Duy et al., 2000; Vergalli et al., 2020). Some toxigenic species of Planktothrix were previously in the genus Oscillatoria (Kurmayer et al., 2016). Early studies of Nodularia revealed hepatotoxicity in intraperitoneally dosed mice that was quite

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O

CO2H N

HN O

OCH3

NH O

O H N

HN

O CO2H

N H

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O NH

B

H N O

NH2

A

NH

O

H 2N

O

OH N

N H NH H N

O

O NH

O

O

OH

N H

FIGURE 5.24 Structures of the cyclic heptapeptide and pentapeptide cyanotoxins, microcystin-LR (A) and nodularin-R (B), respectively. Both are potent hepatotoxins. Author, Charlesy, Own work, Public domain, https://en.wikipedia.org/wiki/Microcystin-LR#/media/File:Mi crocystin-LR.svg and Author, Ed (Edgar 181), Own work, Public Domain, https://en.wikipedia.org/wiki/Nodularin#/me dia/File:Nodularin_R.svg.

similar to that produced by hepatotoxic Microcystis (Runnegar et al., 1988). Strains of Nodularia spumigena and benthic Nostoc produce nodularin-R (ND-R). The mechanism of action and potency of ND-R are very similar to those of MC-LR (Jokela et al., 2017). MC poisonings have been documented in many cases involving dogs and are occasionally seen in cats as well (Backer et al., 2013). The effects in dogs are similar to those of hepatotoxic mushrooms, necessitating diagnostic assessments. Because dogs often have access to stagnant water bodies and they live in close contact with humans, they can serve as sentinels for risks of microcystin toxicoses to people and other species (Backer et al., 2013; Hilborn and Beasley, 2015; Foss et al., 2019). Other mammals, including cattle, sheep, pigs, and multiple species of wild mammals, as well as birds have developed hepatotoxicoses from ingesting Microcystis and MCs, and MCs have

FIGURE 5.25 Typical compact colonies of Microcystis spp. held together by a clear organic matrix that surrounds most cells. Most of the individual cells had an average diameter of 3.0–5.5 mM. Courtesy of Birgit Puschner. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 3, Academic Press, 2013, Fig. 38.18, p. 1173, with permission.

also been associated with netpen liver disease in salmon (Jackson et al., 1984; Galey et al., 1987; Andersen et al., 1993; Oberholster et al., 2009; Rankin et al., 2013; Classen et al., 2017). The producer of the MCs that cause netpen liver disease is unknown. Human and animal dietary supplements have repeatedly been found to be contaminated with MCs, although reports of toxicoses from such products are limited (Roy-Lachapelle et al., 2017). An 11-year-old pug that was given a cyanobacterial supplement intended for dogs that contained a mixture of MCs for 3.5 weeks developed a poor appetite, lethargy, polyuria, polydipsia, and apparent discomfort as well as elevated serum liver enzymes and a coagulopathy (Bautista et al., 2015). After withdrawal of the contaminated supplement, hospitalization, and ample supportive care, the dog gradually recovered. Another recent case report linked MC contamination of a cyanobacterial supplement given to an 8-year-old gelding with the onset of anorexia, mild colic, icterus, and an elevated blood ammonia concentration (Mittelman et al., 2016). The horse became obtunded, consistent with hepatoencephalopathy, and died.

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The deaths of 21 southern sea otters off the coast of California were linked to their ingestion of MC-contaminated marine bivalves (Miller et al., 2010). The shellfish had filtered highly toxigenic Microcystis from Monterey Bay on the coast of California, near the mouth of a river that received outflow from a lake that had a dense bloom of the cyanobacteria. In contrast to the many freshwater sources of MCs, Nodularia produces blooms in brackish water, which likely limits animal and human poisonings. A possible exception was the likely role of NDs from Nodularia in the first report of cyanotoxin-poisoned animals (Francis, 1878). In that outbreak, cattle, sheep, horses, pigs, and dogs died after drinking from a bloom in Lake Alexandrina near the mouth of the Murray River in coastal South Australia. Dogs have also been poisoned by ND when they drank from water in coastal areas of South Africa and Finland (Harding et al., 1995; Simola et al., 2012). Stewart et al. (2012) reviewed concentrations of ND that have been found in tissues of a wide range of marine vertebrates and invertebrates. In addition, they described an artificial lake in southeastern Queensland, Australia, that, in 2009, had a dense bloom of N. spumigena and a large ND-induced die-off in planktivorous mullet (Mugil cephalus). That report raised concern regarding ND transfer to fish-eating humans and other animals.

10.2. Toxicology When given intraperitoneally or intravascularly, MCs achieve much higher concentrations in the body than after equivalent oral doses (Nishiwaki et al., 1994). Intraperitoneal LD50 doses of MCs in mice have ranged from 36 mg/ kg to >1 mg/kg (US EPA, 2015c; Stotts et al., 1993). Toxicity equivalency factors (TEFs) based on intraperitoneal LD50 doses have been proposed for different analogs, using MC-LR as the index compound (TEF ¼ 1) (US EPA, 2015c). Among three of the most commonly encountered MCs, MC-LR was most toxic, MCYR was intermediate, and MC-RR was least toxic. One oral LD50 reported for MC-LR in mice was 10,900 mg/kg (Yoshida et al., 1997). To determine LD50s, clinical signs, and lesions of MC-LR, Fawell et al. (1999a) dosed mice one time either intraperitoneally or via gavage.

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They derived approximate intraperitoneal and oral LD50s of 50–158 mg/kg and 5000 mg/kg, respectively, which indicated that MC-LR was 30–100 times less toxic by the oral route than intraperitoneally. The orally dosed mice that died had reduced activity and piloerection. The intraperitoneally dosed mice that died were prostrate, and they developed convulsions and slowed respiration. In mice that died after dosing by either route, the liver was distended and dark in color, and the kidneys, spleen, and adrenals were pale. Histopathologic examination of the mice that died after dosing by either route of administration revealed hepatic centrilobular hemorrhage. In a study that examined the effects of MC-LR on swine, Beasley et al. (2000) noted that a single intravascular dose at 25 mg/kg was toxic but sublethal, while 75 mg/kg was consistently lethal. By contrast, low oral doses of MC-LR may be well tolerated by swine, even when given on a daily basis for a period of weeks. For example, dosing of pigs via oral gavage with MC-LR at either 2.0 or 8.0 mg/kg/day for 35 days produced no signs or gross lesions of liver damage, no toxin-related changes in plasma albumin, total protein or liver enzyme activities, and no changes in the blood or liver metabolomes or in the liver lipidome (Welten et al., 2020). MCs and NDs target the liver largely because abundant bile acid transporters, also termed organic anion transporting polypeptides (OATPs), carry them from the ileal lumen into the epithelium, and from the hepatic portal blood into hepatocytes (Dahlem et al., 1989; Runnegar et al., 1995a; Stotts et al., 1997; Greer et al., 2018). The intracellular mechanism of toxicity of MCs and NDs is binding to and inhibition of cellular serine/threonine protein phosphatases, including types 1, 2A, and several others, culminating in excessive phosphorylation of essential proteins (Runnegar et al., 1993; Honkanen et al., 1995; Solter et al., 1998; Fontanillo and Kho¨n, 2018). Effects include collapse of cytoskeletal structures, oxidative injury, inhibition of gluconeogenesis, enhanced glycolysis, apoptosis, and necrosis (Wickstrom et al., 1995; Khan et al., 1996; Liu and Sun, 2015). In severe toxicoses, hepatocytes round up and lose cell-to-cell adhesion with loss of the normal hepatic architecture; destruction of sinusoids and intrahepatic

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hemorrhage are often severe (Hooser et al., 1989; Beasley et al., 2000; Meng et al., 2011) (Figure 5.26). Different species of animals vary in their distributions of OATPs in the body, and different OATPs vary in their capacity to transport different MCs. Renal and brain cells have similar OATPs, which confer some susceptibility of MCand presumably ND-exposed animals and humans to nephrotoxicity and neurotoxicity (Fischer et al., 2005; Dias et al., 2009; Feurstein et al., 2009; Foss et al., 2019). Xu et al. (2020) and Hu et al. (2016) published reviews of the literature on the nephrotoxicity and neurotoxicity of MCs, respectively. Mice dosed with tritiated-dihydro-MC-LR had low but detectable concentrations of the toxin in the brain after intraperitoneal or oral administration (Meriluoto et al., 1990; Nishiwaki et al., 1994). Studies of primary brain cells of mice that examined relative inhibition of intracellular protein phosphatases suggested that MC-LR is less readily transported by OATPs into neurons than MCLF (Feurstein et al., 2009, 2010). Zhang et al. (2018) integrated findings from human neuroblastoma cells and intact rats, and suggested

that MC-LR-induced inhibition of PP2A and possibly other protein phosphatases resulted in marked hyperphosphorylation of tau, axonal degeneration, and neuronal death. Additional research on the neurotoxicity of MCs and NDs is needed to deduce whether neuronal toxin concentrations and effects documented by in vitro studies are similar to those seen in humans and other vertebrate animals after ingestions of drinking water or surface water. MCs are conjugated to GSH and eliminated via the bile and urine (Foss et al., 2019). Unfortunately, high-level exposures to MCs may deplete GSH, interfering with the further detoxification and elimination of the MCs, and reducing the normal protection provided by GSH against oxidative injury. A recent report suggested that additional harm from oral exposure to MCs may arise through changes in the gut microbiome (Lee et al., 2020). Mice provided water over a period of 30 weeks that contained either purified MC-LR at a concentration of 10 mg/L or an equivalent concentration of the toxin from a M. aeruginosa lysate had distinct increases in intestinal Firmicutes and reductions in Bacteroidetesdchanges that repeatedly have been associated with increased susceptibility to obesity. The mice of both MC-LR-treated groups also had reductions in the ratio of Bacilli to Clostridiadchanges that may predispose to gut inflammation. In addition, the two MC-treated groups experienced reductions in gut microbial diversity, which might contribute to poor nutrient absorption.

10.3. Clinical Signs and Pathology

FIGURE 5.26 Section of liver from a calf dosed intraruminally with Microcystis aeruginosa containing microcystin-LR collected from a dairy farm in southern Wisconsin where several cattle had died after ingesting the material. Shown are intrahepatic hemorrhage and disassociation of hepatic cords. Photograph courtesy of Beasley Laboratory Group. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 3, Academic Press, 2013, Fig. 38.22, p. 1175, with permission.

The predominant clinical signs, clinical pathology changes, and lesions seen in acutely poisoned mammals after natural or experimental exposure to MCs and NDs are related to hepatotoxicity. Acute fatalities can occur within hours to several days of exposure, and can result from intrahepatic hemorrhage and shock, potentially severe hypoglycemia, and, in some cases, terminal hyperkalemia (Hooser et al., 1989; Takahashi et al., 1995; Beasley et al., 2000; Rankin et al., 2013). Manifestations include elevations of serum liver enzyme activities, bilirubin, bile acids, urea nitrogen, and creatinine, as well as mucous membrane pallor, tachycardia, and

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tachypnea likely from acidosis (Galey et al., 1987; Hooser et al., 1989; Beasley et al., 2000; Luo et al., 2014). Additional clinical signs in mammals include anorexia, abdominal pain, vomiting, diarrhea that can be bloody, lethargy, ataxia, recumbency, dehydration, mental derangement, coma, and death (Galey et al., 1987; Rankin et al., 2013). In cattle and sheep, other manifestations of liver failure, such as hepatogenous photosensitivity, may follow sublethal toxicoses (Carmichael and Schwartz, 1984; Carbis et al., 1995). Oral exposures to Microcystis and intraperitoneal exposure to MC-LR have also been implicated in tumor promotion (Falconer et al., 1988; Falconer, 1991; Falconer et al., 1994; Nishiwaki-Matsushima et al., 1992; US EPA, 2015c). Gross lesions of acutely lethal MC or ND poisoning may include icterus, serous fluid in the abdominal cavity, a large, dark, swollen liver with an accentuated lobular pattern, miliary foci of intrahepatic hemorrhage, and swollen kidneys (Francis, 1878; Hooser et al., 1989; Simola et al., 2012). Some affected animals develop edema in and around the gall bladder. Hemorrhagic enteritis and pulmonary edema also may be seen. Necropsy of the southern sea otters that died off the coast of California from feeding on MCcontaminated shellfish showed icterus with hepatic swelling and hemorrhage (Miller et al., 2010). Histopathology revealed parenchymal hemorrhage, hepatocellular swelling with intracytoplasmic vacuolation, apoptosis, and necrosis. Histopathologic examinations of liver following experimental exposures of laboratory rodents and a calf to a single acutely lethal dose of MCs, as well as a field exposure of a dog to ND, revealed dissociation of hepatocytes, and marked intrahepatic hemorrhage (Galey et al., 1987; Hooser et al., 1989; Simola et al., 2012). Centrilobular apoptosis and necrosis of hepatocytes may extend to periportal regions. Dislodged hepatocytes and fragments thereof may be seen in central veins, and pulmonary and renal vessels (Hooser et al., 1989, 1990) (Figure 5.27). Also, renal tubular epithelial damage has been observed in MC-dosed rodents and carp, as well as in a dog with ND toxicosis

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FIGURE 5.27 Dislodged hepatocytes in pulmonary vessels of a rat experimentally treated with microcystinLR. Courtesy of Stephen Hooser. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 2, Academic Press, 2002, Fig. 14, p. 640, with permission.

(Hooser et al., 1989; Fischer and Dietrich, 2000; Simola et al., 2012). A gelding that developed severe liver failure and clinical encephalopathy after being given a dietary supplement containing MCs had a small, flaccid, soft, friable, orange-tan to green-brown liver (Mittelman et al., 2016). Histopathology revealed severe periportal to massive necrosis, sinusoidal dilation and hemorrhage, and relative sparing of centrilobular zones. Additional changes included multifocal hepatocyte necrosis, and megalocytosis as well as polykaryosis, portal-to-portal bridging fibrosis, and mild infiltrations of lymphocytes and macrophages. In the brain, Alzheimer type II cells were present in the medulla, cerebellum, mesencephalon, diencephalon, and cerebral cortex. Sublethal lesions of MC-LR differ from those of acutely lethal MC toxicosis. Also, a sublethal exposure may reduce the lethality of a subsequent potentially lethal dose (Lovell et al., 1989). Three days after mice were given what was usually a sublethal (i.e., an LD23) dose of MC-LR, they were given a second dose at

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40 mg/kg, which had been determined to be a minimum LD100 dose in earlier dosing: the previous smaller dose of MC-LR increased survival. Histopathology studies of the surviving mice revealed hepatocyte regeneration in the damaged liver. Guzman and Solter (2002) studied mice that were given either a single dose or multiple once-daily doses of MC-LR at 45 mg/kg intraperitoneally. At only 2 h after the single dose, centrilobular and midzonal hepatocytes had changes consistent with glycogen depletion as well as hypertrophy with mild karyomegaly and increased eosinophilic staining. The affected areas had taken up MC-LR as indicated by immunohistochemical staining. At 4 h postdosing, affected hepatocytes had darkly eosinophilic cytoplasm in areas that also stained for cytokeratin, indicating cytoskeletal collapse (Figure 5.28). Staining for MC-LR was prominent in the nucleus and cytoplasm of centrilobular and midzonal areas, but periportal regions were unaffected (Figure 5.29). Western blotting of an extract of hepatocyte nuclei confirmed the binding of MC-LR to PP2A. At 24 h after the single dose of MC-LR, some of the hepatocytes in the hypertrophic areas had undergone apoptosis. Staining for MC-LR was maintained in mice dosed repeatedly. After two daily doses, centrilobular hepatocyte nuclei were apoptotic or had fragmented into small pyknotic bodies. Mice dosed for 4 or 7 days had hepatic enlargement and pallor with an accentuated reticular pattern. Histopathology revealed marked hepatocytomegaly and karyomegaly, with moderate parenchymal disarray involving the entire hepatic lobule. None of the mice in this study of sublethal MC-LR toxicosis had the typical swollen and severely hemorrhagic liver that is seen in mice given an acutely lethal dose of MC-LR. Male mice dosed by oral gavage for 5 days/ week with MC-LR at 1000 ug/kg had severe liver lesions, including hepatocellular dissociation, as well as necrosis and apoptosis in centrilobular areas, with cellular infiltrates, at 4 and 8 weeks (Elmore et al., 2017). Marked hepatocytomegaly, karyomegaly, multinucleation, and abnormal mitotic figures were also observed (Figure 5.30). These changes were largely resolved at 13 weeks, indicating that these lesions had regressed in spite of continued

FIGURE 5.28 H and E-stained section of liver from a mouse given microcystin-LR intraperitoneally at 45 mg/kg 4 h earlier. There are eccentric areas of eosinophilic cytoplasmic condensation (arrow). Additional experiments showed those areas to be positive for cytokeratin, suggesting cytoskeletal damage. Bar ¼ 10 mm. Figure reproduced from Guzman RE, Solter PF: Characterization of sublethal microcystin-LR exposure in mice, Vet Pathol 39:17–26, 2002, Fig. 4a, p. 20, with permission.

dosing. Lesion severity correlated with changes in liver weight as well as serum transaminase activity. Primary lesions were not found in other body systems, including the male reproductive organs. Fawell et al. (1999a) gave repeated doses of MC-LR via oral gavage to pregnant mice and found that almost one-third of the dams died at a daily dose of 2000 mg/kg. The dams were more sensitive than their offspring. The noobserved-adverse-effect level (NOAEL) for liver damage in the dams was 40 mg/kg, whereas the NOAEL for developmental toxicity in their offspring was 600 mg/kg. The authors suggested that the reduced fetal weight and delayed skeletal ossification at the highest doses were likely due to maternal toxicity.

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FIGURE 5.29 Immunohistochemically stained liver from a mouse treated with microcystin-LR intraperitoneally at 45 mg/kg 4 h earlier. Both cytoplasmic and nuclear staining for microcystin-LR demarcate centrilobular to midzonal regions from periportal regions. The hepatocytes within these regions are hypertrophied and there is karyomegaly. C ¼ central vein. Bar ¼ 50 mm. Figure reproduced from Guzman RE, Solter PF: Characterization of sublethal microcystin-LR exposure in mice, Vet Pathol 39:17–26, 2002, Fig. 6, p. 20, with permission.

In another part of their study, Fawell et al. (1999a) dosed mice daily with MC-LR via gavage for 13 weeks. At the highest dose of 1000 mg/kg/ day, the male mice lost body weight, the females had increases in packed cell volume and hemoglobin, and both males and females had increased serum alkaline phosphatase and transaminase activities. Liver lesions noted at 200 and especially 1000 mg/kg/day included chronic inflammation, hemosiderin deposits, and hepatocyte degeneration. Mice given MC-LR at 200 mg/ kg/day had relatively minor lesions, and only a few were affected. Studies that examined other time frames and endpoints have indicated that mice can be sensitive to lower oral doses of MC-LR. An example involved the administration of MC-LR by oral gavage at a relatively low dose of 40 or 200 mg/kg every other day for 90 days (He et al., 2017). The treated mice developed a syndrome consistent with nonalcoholic steatohepatitis (NASH) as determined by an integrated analysis of proteomic, metabolic, histological, and cytokine profiles. MC-LR significantly inhibited fatty acid b-oxidation and hepatic lipoprotein secretion, and it caused hepatic inflammation. Another example is the

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report of Cao et al. (2019), which found that chronic oral exposure to low doses of MC-LR can damage the small intestine. Six months of oral exposure of mice to MC-LR in drinking water at 1, 30, 60, 90, or 120 mg/L produced lesions in the jejunum. Mild lesions were evident even with MC-LR at 1 mg/L, which the WHO has chosen as the upper limit for drinking water. The higher concentrations caused more severe lesions in the jejunum, including increased goblet cells, and a disorganized mucosa with lymphocyte infiltration. Studies to further explore small intestinal sensitivity to MC-LR are warranted.

10.4. Human Exposure and Disease MC contamination of dialysis solutions poses an extreme hazard to patients undergoing treatment for renal insufficiency. Jochimsen et al. (1998) described impacts on humans after an accidental contamination of dialysis fluid with MCs. Of the 131 patients who were exposed to the MCcontaining solution, 116 became ill, 100 had acute liver failure, and 52 died (Jochimsen et al., 1998; Carmichael et al., 2001; Azevedo et al., 2002; Yuan et al., 2006). Manifestations included headache, eye pain, night blindness, nausea, vomiting, weakness, and liver failure. Histopathology showed “disruption of liver plates and cell deformity, extensive necrosis, apoptosis, severe cholestasis, cytoplasmic vacuolization, mixedleukocyte infiltration, and occasional multinucleated hepatocytes.” A subsequent incident in a different region of Brazil involved sublethal exposures to microcystins via dialysate solutions (Soares et al., 2006; Hilborn et al., 2007; WHO, 2020c). Serum MC-LR equivalents were measured, and of 44 who were exposed to the contaminated solutions, 13 had detectable MC concentrations in their blood samples. These individuals were monitored over a period of 8 weeks and several of them had changes consistent with liver injury, including elevations in the serum activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transpeptidase. Near shore waters may contain MCs in mg/L concentrations due to the accumulation of cyanobacterial scums. Such concentrations can present a high risk of human poisoning from accidental ingestion of even small volumes of water

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FIGURE 5.30 Liver from male mice of the control (A), or microcystin-LR-treated groups (B–D). (B) After 4 weeks of MC-LR treatment via gavage, there is hepatocyte dissociation, multinucleation, karyocytomegaly, and loss of hepatocytes. Infiltration by mononuclear cells and a few neutrophils is also present. (C) After 8 weeks of MC-LR treatment, hepatocyte apoptosis, apoptotic bodies, and karyocytomegaly are prominent. (D) After 13 weeks of MC-LR treatment, there is almost complete regeneration; however, cell proliferation continues with some abnormal mitoses, mild cellular infiltrates, and hepatocellular cytoplasmic alteration. Figure reproduced from Elmore SA, Aeffner F, Bangari DS, Crabbs TA, Fossey S, Gad SC, Haschek WM, Hoane JS, Janardhan K, Kovi RC, Pearse G, Wanket LM, Quist EM: Proceedings of the 2017 National Toxicology Program Satellite Symposium, Toxicol Pathol, 45:799–833, 2017. https://doi.org/10.1177/0192623317733924, Fig. 9, p. 824.

(WHO, 2020c). In early 2015 in Uruguay, repeated recreational exposure of a 20-monthold child to water containing Microcystis blooms and MCs at up to 8200 mg/L was associated with diarrhea, vomiting, elevated serum liver enzymes and bilirubin, and liver failure severe enough to necessitate a liver transplant 20 days after admission. Histopathology of the removed liver revealed marked hepatocellular damage, hemorrhage, and nodular regeneration. Analysis of a methanolic extract of the liver found MC-LR and especially D-Leu-MC-LR.

Inhalation of aerosols from and immersion in cyanobacterial blooms from such activities as water skiing and riding jet skis may also result in toxicologically significant MC exposures. In early 2007, at a recreational lake in Argentina, a young man who was riding a jet ski accidentally ended up in a bay containing a bloom that he described as “green paint” (Giannuzzi et al., 2011). He was immersed for 2 h and then swam to the shore dragging his jet ski. The bloom consisted of Microcystis wesenbergii and M. aeruginosa, and a sample of the water

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contained MC-LR at 48.6 mg/L. Four hours later, he developed nausea, vomiting, weakness, abdominal pain, and fever. Three days later, he experienced not only renal failure as indicated by increased serum creatinine and urea nitrogen, but also dyspnea, respiratory distress, and hypoxemia sufficient to warrant being hospitalized, placed on a ventilator for 3 days, and given an antibiotic. On that third day after admission, he had evidence of hepatotoxicity including marked elevations in serum alanine aminotransferase, aspartate aminotransferase, and gamma glutamyl transferase. Cholestasis was not present as indicated by normal serum bilirubin and alkaline phosphatase. Other aspects of therapy were not described, but the man made a complete recovery by 20 days after admission. Humans exposed to waters containing these toxins may also develop skin and eye irritation, hay-fever like symptoms, dizziness, fatigue, and gastroenteritis, but it is unknown whether these signs and symptoms are from hepatotoxins, neurotoxins, or endotoxins in cyanobacteria, or, alternatively, whether some such effects are due to allergies. Coastal and inland water contamination with MCs, toxic impacts in fish, and contamination of fish, shrimp, prawns, mussels, and snails in the human diet were recently reviewed by Preece et al. (2017) and discussed to some degree in WHO (2020c). High intake of seafoods containing elevated MC concentrations would appear to present a low risk to human health. The liverand viscera of finfish, whole fish, and whole mussels typically have higher MC concentrations than fish muscle, which generally has concentrations below 100 mg/kg fresh weight. In Lake Erie, the muscle tissues of yellow perch tended to have low MC concentrations with a mean of 12 mg/kg wet weight, while white perch had a mean of 38 mg/kg wet weight, and walleye were higher yet, with a mean of 84 mg/kg and a maximum concentration of 303 mg/kg (Wituszynski et al., 2017). The overall findings suggested that eating fish occasionally would not result in intakes that exceed WHO guidelines for maximal MC intake. However, frequently eating large servings of white perch or walleye from waters with high MC concentrations could result in exposures that exceed the recommended maximum tolerable daily intake.

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Most human exposures to MCs and NDs result from ingestion of contaminated drinking water. An increase in serum liver enzyme activities in communities or households who rely on surface water sources should trigger water sampling with examinations for cyanobacterial producers and assays for cyanotoxins (Falconer et al., 1983). MCs were found in multiple samples of drinking water from the area near the Three Gorges Reservoir in China. Children, ages 7– 15, who relied on that water had daily MC intakes greater than the tolerable daily intake proposed by the WHO. They had detectable MC in their blood as well as elevated serum activities of aspartate aminotransferase and alkaline phosphatase (Li et al., 2011). The review by Xu et al. (2020) cited a number of studies that correlated MC exposures in human populations to increases in blood urea nitrogen, serum creatinine, and chronic kidney disease. The authors concluded, “The evidence suggests that drinking water and aquatic product intake of MCs may be one of the critical risk factors for renal function damage.” Detection of MCs in surface-derived drinking water has also been associated with increases in the incidence of human primary liver cancer in areas of China (Yu, 1989; Ueno et al., 1996). In 2014, Toledo, Ohio’s public water provider, which relies upon the microcystin-impacted western part of Lake Erie, issued a “do not drink or boil” advisory to roughly 500,000 residents because the finished water had a total MC concentration of 2.5 mg/L. Such findings motivated a study in Ohio, which utilized satellite multispectral remote sensing data with in situ monitoring and census data to explore relationships between potential exposures to cyanotoxins and hepatocellular carcinoma in the human population (Gorham et al., 2020). The researchers found a 14% higher hepatocellular carcinoma rate in people of the state who were believed to be served by a water utility that relied on surface waters impacted by cyanobacterial blooms than in those whose water providers were likely to be using surface waters without such blooms. In addition, the incidence of hepatocellular carcinoma in the former group was 17% higher than in other Ohio residents whose water providers were thought to rely on groundwater. The report by WHO (2020c) provides additional information on studies that

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examined potential risks of liver cancer associated with MCs, including from ingestion of MCs in both water and seafood.

10.5. Diagnosis, Treatment, and Control Monitoring for MCs and NDs in water or cyanobacterial lysates has historically relied on mouse bioassays for lethality with development of characteristic gross and microscopic lesions. Other methods for testing water for potential MCs and NDs include cyanobacterial identifications and counts, as well as toxin biosensors, immunoassays such as ELISAs, analyses using HPLC-MS/MS (which only detect free MCs or NDs), and oxidative cleavage of the unique ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10phenyl-4,6-decadienoic acid) side chain of MCs and NDs followed by analysis for MMPB (2methyl-3-methoxy-4-phenylbutyric acid) with LC-quadrupole/time-of-flight MS in tandem mode (LC-QTOF-MS/MS) to quantify free and bound forms of a broad array of these toxins (Backer et al., 2010; Foss et al., 2019; Anaraki et al., 2020). Confirmation of cyanobacterial identity and genetic capability to produce these toxins may also be bolstered by rapid and inexpensive PCR assays (Carmichael and Li, 2006; Massey et al., 2020; Yuan et al., 2020). Diagnoses depend upon a history of exposure to a source of MCs or NDs. If ingestion has been recent, colonies of cyanobacteria, such as Microcystis, may still be identifiable in vomitus or gastrointestinal contents (Foss et al., 2019). Appropriate clinical signs and clinical pathology changes, and lesions consistent with those caused by MCs and NDs, are typically present in acute poisoning cases. Confirmation may rely upon immunoassay or HPLC-MS/MS or LC-MS/MS detection of MCs or NDs in vomitus, feces, urine, digestive tract contents, and liver (Beasley et al., 1989; Schmidt et al., 2014; Foss et al., 2019; Massey et al., 2020). Although they are research rather than diagnostic tools, immunohistochemistry methods may be employed to localize: (i) the toxins in liver; (ii) inhibition of liver protein phosphatases; and (iii) increased phosphorylation of nuclear p53 (Guzman et al., 2003; Yuan et al., 2006).

In addition to rapidly bathing cyanobacteria off the hair coat of grooming animals, such as dogs, so that they do not lick the material, absorption from the digestive tract should be reduced with oral or intragastric administration of cholestyramine or, when it is not available, activated charcoal (AC) (Dahlem et al., 1989; Rankin et al., 2013). The initial dose of cholestyramine or AC may be accompanied by a sorbitol cathartic to hasten gut motility, and thus enable both toxin binding and evacuation. Repeated administration of cholestyramine or AC may be of value, with care to prevent constipation. Intravenous fluids may be indicated to counteract dehydration and perfuse vital organs, and hypoglycemia and/or hyperkalemia may warrant therapeutic correction. Plasma may be indicated as a source of clotting proteins, and a blood transfusion may be needed to compensate for blood loss into the liver. Animal poison control centers may also recommend rifampin or cyclosporin-A to counteract liver uptake and inflammation, silybins to help restore liver function (Jayaraj et al., 2007), and sometimes Bvitamins and vitamin K as supportive care. Studies of in vitro or in vivo exposures to MCs have demonstrated that toxicity could be reduced by inhibition of hepatocellular uptake using bile acid transport inhibitors (e.g., antamanide, sulfobromophthalein, and rifampin) and bile salts (e.g., taurocholate) (US EPA, 2015e). Livestock that may be poisoned with MCs should be removed from the source and provided clean water without causing avoidable stress. Feeding AC and supportive care to counteract hypovolemia and electrolyte imbalances as well as antioxidants, such as vitamin E and selenium, have been recommended (Varga and Puschner, 2012). Animals that begin to exhibit photosensitization should be housed indoors when possible. Avoiding intake of cyanobacterial bloom material by water utilities and appropriate water treatment to remove or neutralize the toxins are important in risk management for human populations. MCs can be inactivated or removed from contaminated drinking water using methods similar to those discussed for cylindrospermopsin. Guidelines for MCs in drinking and recreational water supplies, i.e., concentrations that

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should be avoided, have been reported by Chorus and Bartram (1999), WHO (2003, 2020c) and US EPA (2015c). The most recent of these (WHO, 2020c) listed 1 mg/L for life-time drinking water, 12 mg/L for short-term drinking water, and 24 mg/L for recreational water. Because of their higher water consumption per unit of body weight, WHO (2020c) indicated that bottle-fed infants and small children should be provided with an alternative water source whenever MC concentrations exceed 3 mg/L. Activated charcoal (AC) can bind free MCs in drinking water facilities, but there are differences in binding depending on the kind of AC, the particular MCs present, and the pH and natural organic matter of the water. A recent report confirmed that chlorination of water can help detoxify MCs, but the duration of time in contact with chlorine influences the extent of detoxification, and MCdisinfection by-products may present toxic risks to consumers of treated drinking water as indicated by in vitro testing that showed persistent inhibition of protein phosphatase 1 (Zong et al., 2015). Aquatic pH and salinity may also alter the effectiveness of chlorination in detoxification of MCs. Additional research is needed to characterize the structures and concentrations of the MCdisinfection by-products, their transport via OATPs, their inhibition of protein phosphatase 2A, their various effects on primary hepatocytes and other cells, and their acute and chronic effects on intact animals. Management of water bodies to reduce contamination by MCs and NDs may include reducing nutrient inputs and binding nutrients, among other approaches. For example, researchers who had studied treated and control enclosures within a hypertrophic urban pond that had high Microcystis densities and high concentrations of a mixture of MCs recommended management including the removal of contaminated sediment via dredging, use of a binder containing lanthanum-modified clay to reduce dissolved phosphorus concentrations, and marked reductions in public feeding of fish and ducks (Lu¨rling and Faasen, 2012). While copper in different forms can kill cyanobacteria, the associated lysis releases cyanotoxins to the water column, potentially resulting in an increased risk of poisoning, and copper itself is toxic. Among domestic animals, sheep are especially susceptible to copper toxicity.

11. ANATOXINS 11.1. Source/Occurrence The anatoxins (ANTXs) have caused periodic poisonings of multiple species of wildlife, livestock, birds, carnivores, and fish in many nations around the world. The path to the isolation and characterization of ANTX-a began in the early 1960s in studies of toxic strains from blooms of what was long known as Anabaena flos-aquae (Lyngb.) de Bre´b that had poisoned cattle at Burton Lake in Saskatchewan, Canada. A. flos-aquae was recently renamed Dolichospermum flos-aquae. The toxic effects of ANTX-a in dosed mice were rapid (25 min), and therefore, until its structure was identified, it was termed Anabaena veryfast-death-factor (Gorham, 1964). The “a” in ANTX-a was based on its being the first toxin from Anabaena to be structurally identified (Carmichael and Gorham, 1978). [Please note that the structurally unrelated toxin, ANTX-a(s), was recently renamed guanitoxin, as discussed in a separate section below (Fiore et al., 2020).] ANTX-a is a bicyclic alkaloid (Figure 5.31), and producers may also biosynthesize similarly acting but more or less potent analogs, including homoANTX-a, dihydro-ANTX-a, and dihydrohomoANTX-a (WHO, 2020a; Puddick et al., 2021). Failure to test for such analogs may account for discrepancies between concentrations found in water and toxic effects seen in exposed animals, particularly when analogs of higher toxicity than ANTX-a are present at high concentrations. Colas et al. (2021) provided

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FIGURE 5.31 Structure of anatoxin-a. Created by Cacycle, Own work, Public Domain, https://commons. wikimedia.org/wiki/File:Anatoxin-a.png, downloaded March 22, 2021.

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insights on the synthesis, and pelagic, benthic mat, and biofilm concentrations of ANTX-a, as well as its environmental abiotic and biotic degradation. ANTXs are produced by members of several cyanobacterial genera, including Dolichospermum (formerly Anabaena), Aphanizomenon, Arthrospira (formerly Spirulina), Blennothrix, Chrysosporum, Cuspidothrix, Cylindrospermum, Kamptonema, Oscillatoria, Phormidium, Planktothrix, Cylindrospermopsis (Raphidiopsis), Tychonema, Microcoleus, and Hydrocoleum, which lives in marine ecosystems (Bruno et al., 2017; Beasley, 2020; Bire´ et al., 2020; Puddick et al., 2021; Colas et al., 2021). As such, they are encountered in surface blooms, crusts when such blooms evaporate, in the water column, in biofilms formed by epiphytic and benthic cyanobacteria, and in filter-feeding organisms. Multiple episodes of ANTX-a toxicosis have involved deaths in cattle, dogs, lesser flamingos, and waterfowl (Devlin et al., 1977; Puschner et al., 2008; Beasley, 2020; Colas et al., 2021). A lack of access to uncontaminated water has often led to ANTX-a-induced deaths in terrestrial animals. Poisonings may also occur from accidental swallowing of water or via voluntary consumption of dried algal crusts (Bruno et al., 2017). Pigs and dogs may be extremely sensitive to oral exposure, and the latter sometimes seek out ANTX-a-containing benthic bloom materials and dried crusts (Codd et al., 1992). Reported lethal poisonings by ANTX-a have also involved large numbers of bats and carp (Colas et al., 2021). In May 2017, after swimming in or drinking from a mesotrophic lake in Berlin, Germany, at least 12 dogs developed weakness in their hind and front limbs, collapse, dyspnea, and cyanosis (Fastner et al., 2018). Several of the dogs died despite treatment. There were no floating blooms, but dense growths of floating water moss (Fontinalis antipyretica) carried large amounts of cyanobacteria of the genus Tychonema. The investigation team found Tychonema as well as a high concentration of ANTXa (8700 mg/L) in stomach contents, while assays for other cyanotoxins and pesticides were all negative. The lake was undergoing management to achieve reoligotrophization, i.e., efforts to reduce nutrient concentrations, which, in concert with local weather conditions, may have resulted in a period of “Goldilocks” nutrient concentrations, i.e., “just right,” for the

cyanotoxin producer. A similar incident involved the deaths of three dogs at a lake in Bavaria in southern Germany in August 2019 (Bauer et al., 2020). At that location, there was a red-brown biofilm of Tychonema on plants in the water, and there were mats of Tychonema floating on the water and resting on the banks. LC-MS/MS analysis of stomach contents of the affected dogs revealed ANTX-a and dihydroANTX-a at 563 and 1207 mg/L, respectively.

11.2. Toxicology James et al. (2007) reviewed the toxicology of ANTX-a. It is a potent postsynaptic depolarizing neuromuscular blocking agent (Carmichael et al., 1975; WHO, 2020a). However, it acts not only at neuromuscular junctions but also in the central nervous system and at autonomic ganglia (Sire´n and Feuerstein, 1990). ANTXa has a higher affinity for nicotinic acetylcholine receptors than either acetylcholine or nicotine. ANTX-a is rapidly absorbed from the digestive tract (Carmichael et al., 1975). Its lethality is often due to respiratory paralysis, although cardiotoxicity may also play a role. Reported oral LD50 values for ANTX-a in mice have ranged from 6 to 14 mg/kg (US EPA, 2015d; WHO 2020a). A maximum tolerated repeated dose was 2.5 mg/kg/day (US EPA, 2015d). Reported oral LD50s for medaka fish were 5.75 and 11.5 mg/kg (Colas et al., 2021). ANTX-a, homoANTX-a, dihydro-ANTXa, and dihydro-homoANTX-a are all highly toxic, but various ANTX-a degradation products are generally considered to be less potent (Bruno et al., 2017). Several other derivatives of ANTX-a are also found in producing organisms, and analogues have been synthesized as potential drug candidates. More comprehensive discussions of ANTXs are provided by Colas et al. (2021) and the US EPA report from 2015 (US EPA, 2015d).

11.3. Clinical Signs and Pathology ANTX-a poisoning can be lethal within minutes to a few hours, depending upon the toxin concentration(s) in the bloom, the amount ingested, the species of animal, and the amount of food in its stomach. Clinical signs of ANTXa poisoning follow a progression of muscle fasciculations, decreased movement, abdominal

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breathing, cyanosis, convulsions, and death. In smaller laboratory animals, death is often preceded by leaping movements, while in field cases, larger animals collapse and die. Opisthotonos with a rigid “s-shaped” neck has been observed in ANTX-a-poisoned birds. Clinical signs of toxicosis following intraperitoneal exposure of mice to ANTX-a can occur within 2 min, and death from respiratory arrest may occur within 15 min. Animals poisoned after ingestion or stomach tube administration of bloom materials may experience prolonged toxic effects, potentially lasting more than 24 h (Carmichael et al., 1975, 1977). However, intravascularly dosed anesthetized rats that were artificially ventilated largely recovered from ANTX-a-induced impairment of nerve transmission within 2 h (Valentine et al., 1991). Also, mice that survived intravenous doses of ANTX-a that were lethal to other mice recovered from visible effects within minutes (Fawell et al., 1999b). ANTX-a toxicosis is not associated with consistent lesions other than the possibility of cyanosis (Carmichael and Schwartz, 1984).

11.4. Human Exposure and Disease Concentrations of ANTX-a in drinking water and recreational waters were reviewed by the US EPA (2015d) and WHO (2020a). ANTX-a is often present in surface and drinking water, and concentrations are highly variable. Although ANTX-a concentrations in open water are rarely greater than tens of mg/L, samples of bloom-contaminated lake water in the United States have had up to approximately 2000 mg/ L. ANTX-a is largely contained within cyanobacterial cells and it dissipates and rapidly degrades after lysis. Repeated occupational exposure to ANTXs in cells and in solution via the inhalation of aerosols of irrigation water contaminated by cyanobacterial blooms may be an underappreciated risk (WHO, 2020a). ANTX-a concentrations in drinking water have ranged up to 8.5 mg/L. Extracts from Arthrospira (formerly Spirulina) and Aphanizomenon flos-aquae have been sold as human food supplements, and a minority of specimens of cyanobacterial food supplements have been found to contain ANTX-a. To our

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knowledge, the highest reported concentration of ANTX-a in supplements was 33 mg/kg (Rella´n et al., 2009; WHO 2020a). Colas et al. (2021) recently reviewed the available literature on human exposures to ANTX-a. Human poisonings from ANTX-a are not well documented, but toxic exposures to ANTXs are still a concern (Testai et al., 2016; WHO, 2020a). Human poisonings from recreational exposures may be uncommon because of the objectional appearance and unpleasant odor of contaminated blooms. Finfish can be poisoned by ANTX-a, but they do not appear to bioaccumulate the toxin to present an important hazard to humans or piscivorous species (Colas et al., 2021). However, other aquatic species, even in marine environments, might contain sufficient ANTX-a to be of concern when they enter the human diet. Between 2011 and 2018, 26 people in France, ranging in age from 20 to 80, were reported to have been poisoned after eating filter-feeding sea figs (Microcosmus spp.), which are also termed sea squirts, sea lemons, or sea potatoes. Some consumers regard them as a delicacy (Bire´ et al., 2020). Affected individuals had symptoms and signs including blurred or double vision, ataxia, dizziness, weakness, headache, muscle cramps, paresthesias, nausea, vomiting, and diarrhea. Food leftovers from four of those cases were analyzed for multiple phycotoxins. Regulated lipophilic marine phycotoxins were not found, but specimens from 2011, 2012, and 2018 contained ANTX-a at from 194 to 1240 mg/kg as revealed by HILIC-MS. It is not clear that the reported concentrations of ANTX-a in the sea figs were sufficient to result in those apparent human poisonings, but even higher concentrations of ANTX-a and analogues of the toxin in the sea figs could have been missed. Ingestion of giant clams (Tridacna maxima) contaminated by Hydrocoleum and homoANTX-a has also been implicated in human poisonings, but concentrations of the toxin were not determined (Me´jean et al., 2010). To better understand risks to human as well as aquatic wildlife health, future monitoring of filter-feeding marine organisms for cyanobacterial producers of phycotoxins and ANTXs is needed.

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11.5. Diagnosis, Treatment, and Control The diagnosis of poisoning by ANTXs relies upon finding the producing organisms and quantifying the toxin(s) in source water and digestive tract contents, appropriate clinical signs, normal cholinesterase values, and ruling out other etiologies. Approaches used to detect both ANTX-a and homoANTX-a include receptor-based assays, ELISA kits, derivatization for HPLC with fluorescence detection, HPLCMS-MS, and GC-MS (Furey et al., 2005; Vasas et al., 2004; Colas et al., 2021). ANTX-a is sensitive to sunlight and high pH, and the compounds created by photochemical or oxygen-mediated degradation, such as dihydroxy-ANTX-a, tend to be much less toxic than the parent compound. When they are included in assays, the oxidative derivatives can serve as additional indicators of ANTX-a exposure. Treatment for poisoning by ANTXs has not been examined in depth. Two calves given bloom material containing ANTX-a by stomach tube survived as long as artificial respiration was provided, but they did not recover the ability to breathe on their own even at the end of the trials, one of which lasted until 28.5 h postdosing (Carmichael et al., 1977). The lack of recovery may have been a result of ongoing absorption of the toxin(s) from the digestive tract. That observation and the rapid recovery (largely within 2 h) of nerve function in anesthetized rats after intravenous dosing with ANTX-a suggests that sustained life-saving artificial respiration should be used along with measures to limit absorption from the digestive tract, such as lavage and administration of activated charcoal with a cathartic (Valentine et al., 1991). However, some rats given ANTX-a and provided artificial respiration nevertheless died. This suggests that, in addition to respiratory paralysis, effects on the heart may also contribute to lethality. Other researchers who investigated ANTX-a toxicity have documented reductions in heart rate as well as increased secretions of catecholamines and increased blood pressure (Colas et al., 2021). There is no antidote for poisoning by ANTX-a, and animals often die before emergency care can be provided (Carmichael et al., 1977). Accordingly, avoiding bloom exposures and early detection of ANTXs in

source materials are essential components of control and prevention of poisonings. Colas et al. (2021) summarized formal recommendations regarding concentrations of ANTXa and/or other ANTXs in drinking water, recreational water, or foods. California issued guideline values that included a limit of 5 mg of ANTX-a/kg of fish flesh. The State of Washington issued a recreational guidance level of 1 mg/L. Ohio and Oregon set drinking water standards of 20 and 3 mg/L, respectively. Quebec chose 3.7 mg/L as its temporary acceptable maximum threshold for drinking water. New Zealand set a provisional maximum drinking water standard of 6 mg/L for ANTX-a and 2 mg/L for homoanatoxin-a. Recently, WHO (2020a) reported provisional health-based reference values for exposed adults for ANTX-a of 30 mg/L for acute or short-term exposure in drinking water and 60 mg/L for recreational water. Because of a lack of studies, no reference values were set in regard to chronic human exposure to ANTX-a. Recognizing the higher water intake of children, WHO (2020a) indicated that bottle-fed and small children should be given water from an alternative source whenever ANTX-a concentrations exceed 6 mg/L.

12. GUANITOXIN [FORMERLY ANATOXIN-A(S)] 12.1. Source/Occurrence Until recently, guanitoxin was termed anatoxin-a-sub s [anatoxin-a(s)] (Fiore et al., 2020). The choice of the initial name was intended to be temporary because the structure was unknown and the “sub s” designation was included only to denote the excessive salivation that it produced. The name change to guanitoxin was necessary to avoid ongoing confusion with ANTX-a. Guanitoxin is the phosphate ester of a cyclic N-hydroxyguanidine; thus, it is structurally unrelated to ANTX-a and is not a nicotinic agonist (Figure 5.32). Moreover, in contrast to the relatively stable ANTX-a, guanitoxin is unstable and readily hydrolyzed, which likely influences its capacity to poison different kinds of animals, and makes diagnoses a unique challenge (Cook et al., 1989a).

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12. GUANITOXIN [FORMERLY ANATOXIN-A(S)]

N HN H2N +

N –

O

CH3

CH3 O P O

O

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been documented in several parts of North America and Europe (Patocka et al., 2011). Additional surveillance for guanitoxin is an important need, simply because Dolichospermum is globally distributed and guanitoxin is rapidly acting, extraordinarily potent, and readily lethal.

CH3 12.2. Toxicology

FIGURE 5.32 Structure of guanitoxin [formerly anatoxin-a(s)], the only known naturally occurring organophosphorus cholinesterase inhibitor. Author Leyo. This image of a simple structural formula is ineligible for copyright and therefore is in the public domain. It consists entirely of information that is common property and contains no original authorship, https://commons.wikimedia.org/wiki/ File:Anatoxin-a(S).svg, downloaded March 22, 2021.

Guanitoxin is the only known naturally occurring organophosphorus cholinesterase inhibitor. Covalent binding of guanitoxin to an essential serine residue of acetylcholinesterase is believed to account for its irreversible inhibition of the enzyme (Mahmood and Carmichael, 1986, 1987; Hyde and Carmichael, 1991; Vale´rio et al., 2010; Patocka et al., 2011). Guanitoxin is stabilized in acid, which predisposes monogastrics to acute toxicosis, and pigs, dogs, and water birds have been affected in field cases. By contrast, at higher pH, guanitoxin tends to be hydrolyzed (Matsunaga et al., 1989; Fernandes et al., 2021). Hydrolysis in the upper digestive tract might explain why guanitoxin has not been associated with poisoning of orally exposed functional ruminants (Cook et al., 1989a).

12.3. Clinical Signs and Pathology

FIGURE 5.33 Spherical vegetative cells of Dolichospermum spiroides (formerly Anabaena spiroides) arranged in typical filaments and a large spore. A heterocyte (arrow), also termed heterocyst, is a specialized cell type that fixes nitrogen to support survival and growth of Dolichospermum spp. when dissolved nitrogen is low. Courtesy of Beasley Laboratory Group. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 2, Academic Press, 2002, Fig. 10, p. 638, with permission.

Guanitoxin is produced by planktonic species of Dolichospermum (formerly Anabaena) (Figure 5.33). Poisoning of animals by guanitoxin was first reported in Saskatchewan, Canada, but lethal poisonings of mammals and bird kills have

Cook and colleagues (1989a,b,c, 1990, 1991) published a series of papers on guanitoxin exposures of a variety of mammalian species and Muscovy ducks. They observed consistent peripheral inhibition of cholinesterase, digestive tract upset, bradycardia, and terminal respiratory paralysis. The polar toxin apparently lacks the capacity to cross the blood–brain or blood– retinal barriers. As a consequence, cholinesterase inhibition in the periphery is thought to account for the entire clinical syndrome. In mice and rats, guanitoxin poisoning often causes excessive salivation, urination, and defecation with marked diarrhea, and lacrimation. Poisoned rats may produce profuse red-colored tears, termed chromodacryorrhea (Figure 5.34). When mice are given doses of guanitoxin sufficient to cause death within a few minutes, they may fail to exhibit excessive salivation and lacrimation (Henriksen et al., 1997).

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FIGURE 5.34 Lacrimation with chromodacryorrhea due to cholinergic stimulation in a rat dosed intraperitoneally with guanitoxin. Courtesy of Beasley Laboratory Group. Figure reproduced from Solter PF, Beasley VR: Phycotoxins. In Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, ed 3, Academic Press, 2013, Fig. 38.27, p. 1178, with permission.

Anesthetized rats dosed with guanitoxin had marked reductions in mean arterial blood pressure, heart rate, respiratory rate, tidal volume, and minute volume (Cook et al., 1990). Increases in phrenic nerve activity but absence of normal responses of the diaphragm were consistent with (i) inability of the toxin to reach the brain and (ii) peripheral depolarizing blockade in the muscles of respiration. Abnormally high acetylcholine at neuromuscular junctions presumably induces the fasciculations, tremors, and respiratory paralysis. Excessive acetylcholine at muscarinic receptors likely accounts for the hypersalivation and bradycardia. A buildup of acetylcholine at muscarinic receptors of the digestive tract and nicotinic receptors in autonomic ganglia would plausibly cause the defecation and diarrhea. Ducks and grebes in Denmark died after ingesting water contaminated with a bloom containing guanitoxin (Henriksen et al., 1997). In

Illinois, Muscovy ducks were poisoned from a pond contaminated by a bloom of Dolichospermum with a toxin that inhibited peripheral but not central cholinesterase (Cook et al., 1989a). The ducks became ataxic, were in sternal and lateral recumbency, and had wing paralysis. Five of the 15 ducks died. Muscovy ducks that were orally dosed with the bloom material displayed profuse salivation as well as regurgitation, watery excreta, polydipsia, tremors, openmouth breathing, intermittent tonic seizures, and leg and wing paralysis. Stimulation from being picked up increased the neuromuscular abnormalities. The ducks died after two or more daily doses with the bloom-contaminated water. Thirteen of 108 pigs suddenly died after drinking from a different farm pond in Illinois that contained a cholinesterase-inhibiting bloom of Dolichospermum (Cook et al., 1989a). Clinical signs in two surviving pigs included excessive salivation, dyspnea, ataxia, and recumbency. Pigs that were dosed intragastrically with the bloom from the field case involving Muscovy ducks developed hypersalivation and mucoid nasal discharge within 30 min. Other signs included dilation of the anal sphincter, bruxism, fasciculations and tremors, vomiting, defecation, urination, coughing, prolonged capillary refill time, cold ears, dyspnea, and cyanosis. Death ensued within 90 min of dosing. Postmortem findings associated with guanitoxin poisonings may reflect exposures to cyanobacteria, e.g., bloom material containing cells of Dolichospermum on the surface of the animal and in the digestive tract, as well as evidence of diarrhea and sometimes excessive saliva in the esophagus. No remarkable histologic abnormalities are expected in cases of guanitoxin poisoning.

12.4. Human Exposure and Disease To our knowledge, there are no published reports of guanitoxin poisoning in humans. As with other cyanobacteria, the appearance and odor associated with water containing cyanobacterial bloom material likely result in avoidance of significant exposures. It is also likely that animal poisonings may reflect the toxic risks associated with the water. The removal of cyanobacterial cells via filtration and the instability of

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guanitoxin are likely to reduce risks related to exposures via municipal drinking water and well water.

12.5. Diagnosis and Treatment Antemortem diagnosis of guanitoxin poisoning relies on evidence of ingestion of Dolichospermum (many will call it Anabaena), compatible clinical signs, inhibition of blood cholinesterase, and, when possible, laboratory analysis for the toxin. Vertebrates examined postmortem will have inhibited peripheral tissue cholinesterase and normal brain cholinesterase. Bloom material should be refrigerated and rapidly examined by an expert capable of morphologic identification of the cyanobacteria. Acidified and frozen bloom material should be submitted to a capable laboratory for toxin identification. Fast atom bombardment-mass spectrometry and nuclear magnetic resonance data have been effective in identifying guanitoxin (Onodera et al., 1997a). A more recently reported method relied upon LC-MS/MS (Do¨rr et al., 2010). Unfortunately, however, instability of the toxin and a lack of available analytical standards for the parent toxin and its hydrolysis products make it difficult for laboratories to routinely provide such analyses (Fernandes et al., 2021). Biosensors that rely on cholinesterase inhibition have been developed for use in monitoring for guanitoxin in water (Devic et al., 2002). Because organophosphorus and carbamate insecticides may be present in the environment and they also inhibit cholinesterases, a biosensor method was developed using a mutant cholinesterase to help distinguish between guanitoxin and those insecticides. An important therapeutic challenge is that animals can die within minutes of ingestion of cyanobacteria containing guanitoxin, as occurred in dogs that swam in a waterbloom containing Dolichospermum and, upon exiting the water, licked the toxigenic cells from their fur (Mahmood et al., 1988). Management of guanitoxin poisoning therefore includes removal of cyanobacteria from hair, feathers, and skin to prevent ingestion during grooming, urgent detoxification of the digestive tract, administration of activated charcoal, and atropine or preferably another antimuscarinic

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agent, such as glycopyrrolate that, like the toxin, cannot cross the blood–brain barrier. Artificial respiration should be used as needed. While these are all rational components of therapy, studies on the effectiveness of such interventions are needed.

13. LYNGBYATOXINS AND APLYSIATOXINS 13.1. Source/Occurrence Benthic mat-forming filamentous cyanobacteria of the order Oscillatoriales, including the tropical to subtropical, largely estuarine and marine Moorea producens (previously Lyngbya majuscula) and the freshwater and more cosmopolitan Microseira wollei (previously Lyngbya wollei), produce many bioactive compounds (Osborne et al., 2001; Engene et al., 2012; McGregor and Sendall, 2015). Among the important cyanotoxins from these species are lyngbyatoxin A, which is structurally identical to teleocidin A1, and the functionally related aplysiatoxin and debromoaplysiatoxin (Osborne et al., 2001; Jiang et al., 2014b) (Figure 5.35). Aplysiatoxins were first identified in the sea hare, Stylocheilus longicauda (previously Aplysia longicauda), a species that grazes heavily on cyanobacteria including M. producens (Osborne et al., 2001). Sea hares, which are also called sea slugs, are large marine gastropods, some of which lack an external shell. Sea hares periodically wash up on beaches in large numbers. Accidental contact with sea hare extract resulted in skin irritation in humans. Sea hares sequester not only aplysiatoxins, but also many other bioactive compounds from cyanobacteria (Pennings et al., 1996). A common adverse effect of lyngbyatoxins, aplysiatoxin, and debromoaplysiatoxin in humans is skin irritation, termed “swimmer’s itch.” In addition to M. producens and M. wollei, filamentous cyanobacteria in the genera Schizothrix and Oscillatoria may also produce aplysiatoxins and cause “swimmer’s itch.” An unrelated cause of “swimmer’s itch” is the cutaneous penetration by cercariae of various trematodes, such as species that complete their life cycle in wildlife.

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5. PHYCOTOXINS

H N

N

OH

O N H

A

O

O OH O O

O

O

Br

O OH

OH

B

O

O OH O O

O

O

O

C

OH

OH

FIGURE 5.35 Structures of the skin irritant cyanotoxins, lyngbyatoxin A (A), aplysiatoxin (B), and debromoaplysiatoxin (C). The author of all three images is Charlesy. These images of simple structural formulae are ineligible for copyright and therefore are in the public domain because they consist entirely of information that is common property and contain no original authorship, https://commons. wikimedia.org/wiki/File:Lyngbyatoxin_A.svg, https://en.wikip edia.org/wiki/Aplysiatoxin#/media/File:Aplysiatoxin.svg, and https://en.wikipedia.org/wiki/Debromoaplysiatoxin#/media/Fil e:Debromoaplysiatoxin.svg, downloaded March 22, 2021.

The first reports of human dermatitis from exposure to M. producens were related to an outbreak of contact dermatitis affecting 125 people on a beach of Oahu, Hawaii, in 1958 (Grauer and Arnold, 1961; Moore, 1977; Serdula et al., 1982; Osborne et al., 2001). Skin damage was subsequently documented in Japan and in Australia, with the latter associated with large blooms in Moreton Bay, near Brisbane, Queensland (Hashimoto et al., 1976; WHO, 1984; Yasumoto and Murata, 1993; Dennison et al., 1999). Also, on the coasts of Australia, human ocular and respiratory irritation were related to exposures to M. producens from water and aerosols, and from cleaning fish nets and crab pots (Osborne et al., 2001). One human death on the

coast of Maui, Hawaii, United States, was linked to respiratory exposure to sea spray contaminated with M. producens (Osborne et al., 2001). To our knowledge, the first detection of these toxins in the continental United States was from a study of Florida’s inland waters; specimens containing M. producens or M. wollei had low concentrations of debromoaplysiatoxin (2–4 mg/kg dry wt). No illness was associated with those findings (Williams et al., 2006). More recently, however, a dog developed marked skin lesions after swimming in a lake in northern California. The water contained not only an array of cyanobacteria, some tentatively identified as Lyngbya, but also debromoaplysiatoxin at 3.8 mg/L (Puschner et al., 2017). Oral exposure to these toxins is also an important concern. A report from 1904 indicated that several horses died following ingestion of M. producens on the shore of the “Gulf of Manor” (presumably the Gulf of Mannar), in southeastern India (Palmer, 1959). Humans in Hawaii were poisoned after they ingested aplysiatoxin and debromoaplysiatoxin in an edible seaweed, the red alga Graciliaria coronopifolia, which was thought to be contaminated by epiphytic cyanobacteria (Nagai et al., 1996). Finfish may also be a dietary source of these toxins, but research is needed on the potential for such relay toxicoses (Osborne et al., 2001). Poisonings from these toxins have been strongly suspected in people and dogs after consumption of sea turtle meat in locations around the world (Osborne et al., 2001).

13.2. Toxicology Lyngbyatoxins are indole alkaloid terpenoids, and aplysiatoxins are bislactone terpenoids. Lyngbyatoxin A and the best known aplysiatoxins are potent inflammatory agents and tumor promoters (Osborne et al., 2001). Lyngbyatoxin A had an LDmin of 0.3 mg/kg in mice (route of administration not stated), and aplysiatoxin and debromoaplysiatoxins had an LDmin of 0.3 mg/kg in intraperitoneally dosed mice (Moore, 1977; Gorham and Carmichael, 1979). These toxins bind to phorbol ester receptors and activate protein kinase C, resulting in excessive phosphorylation of regulatory proteins, which likely accounts for many of their clinical and pathologic effects (Moore et al., 1986; Fujiki et al., 1981, 1990; Arthur et al., 2008). Some of

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them have been found to exert antiviral or antileukemic effects, and/or to block potassium channels (Zhang et al., 2020). There are at least 45 aplysiatoxins, and several are being examined as potential drug candidates (Nagai et al., 2019; Zhang et al., 2020).

13.3. Clinical Signs and Pathology After ingestion of these toxins, animals may develop oral and intestinal irritation, edema, and cellular damage. Lyngbyatoxin given intraperitoneally to mice caused death from small intestinal hemorrhage (Ito et al., 2002b). Epithelial cells of the stomach became “disconnected.” The mice had macroscopically visible mucusfilled “lines” on the mucosal surface and occasional gastric erosions. The epithelial cells of the small intestinal villi underwent degeneration or loss, exposing the lamina propria. The large intestine became edematous with loss of epithelial cells resulting in ulceration. In the lung, leukocytes were present within alveoli, alveolar septa, and in the arteries, and the alveolar septa appeared to be edematous. Administration of lyngbyatoxin A to mice by gastric tube caused gastrointestinal lesions that were similar to those that followed intraperitoneal administration of the toxin, although the signs of poisoning were milder and the lethal dosage was much higher (Ito et al., 2002b). Gastrointestinal injury and pulmonary edema were less severe, but they were still evident at 5 weeks after dosing. There were increased numbers of mitotic figures in the liver for up to 2 weeks after dosing. A dog that was believed to be poisoned by debromoaplysiatoxin in California had severe pruritis, erythema, and excoriations on its chest and neck, and urticaria in the inguinal areas and ventral abdomen, as well as vomiting, diarrhea, and dehydration. It recovered after treatment with dexamethasone, diphenhydramine, cephalexin, metronidazole, dolasetron, and intravenous fluids (Puschner et al., 2017). Green sea turtles (Chelonia mydas) are commonly impaired by fibropapillomatosis, a multifactorial disease in which chelonid herpesvirus 5 plays an important role. Exposures to elevated sea surface temperatures, eutrophication linked to HABs, ingestion of Moorea producens and lyngbyatoxin-A, and dissolved lyngbyatoxin-A,

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as well as dinoflagellates (Prorocentrum spp.) and brevetoxins have also been correlated with finding fibropapillomas in this species (Dujon et al., 2021; Arthur et al., 2008). It is thought that lyngbyatoxin-A may act as a tumor promoter in some of the affected sea turtles. Additional research is needed on potential involvement of lyngyatoxin-A, other phycotoxins, and anthropogenic contaminants in fibropapillomatosis of sea turtles.

13.4. Human Exposure and Disease The usual history of dermotoxicity with these cyanotoxins involves turbulence that suspends M. producens in the water, exposure inside the bathing suit, and continued wearing of the garment after leaving the water (Osborne et al., 2001, 2007). A single exposure through direct contact with skin, eyes, or the respiratory tract or ingestion has often resulted in clinical signs (Serdula et al., 1982). Acute dermatitis may become evident between 4 and 20 h after exposure to water containing these toxins, and impacts may last 2 to 12 days (Osborne et al., 2001; Werner et al., 2012). Symptoms include itching, burning, and pain. Often, lesions are found on the genitals, perineum, and perianal area, but they may also occur on other exposed

FIGURE 5.36 Vesicles and papules on the abdomen of a 13-year-old girl. The lesions shown were present 1 day after swimming in rough waters off the coast of Oahu, Hawaii, and are consistent with Moorea producens– induced contact dermatitis. Figure reproduced from Werner KA, Marquart L, Norton SA: Lyngbya dermatitis (toxic seaweed dermatitis), Int J Dermatol 51:59–62, 2012, Fig. 1, p. 60, with permission.

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skin, the lips, and eyes. Skin lesions include rash, vesicle formation, and deep desquamation (Figure 5.36). Patch testing of 33 volunteers produced lesions in every case, including escharotic dermatitis (Osborne et al., 2001). Histopathology of human skin lesions has revealed an acute, vesicular dermatitis (Figure 5.37) that may be accompanied by infiltrates of lymphocytes, eosinophils, and some neutrophils (Osborne et al., 2001). Although tumor promotion is a potentially important impact of lyngbyatoxin exposure, to our knowledge, lyngbyatoxins have not been directly associated with human cancers. In addition to toxic effects from direct contact with skin and eyes or inhalation, relay toxicoses to humans who eat contaminated aquatic animals may be important. Lyngbyatoxins have been implicated in outbreaks of human poisoning after ingestion of sea turtle meat. Effects included abdominal pain, diarrhea, nausea, and vomiting, as well as inflammation in the mouth, esophagus and stomach, oral ulcers, and foul breath (Yasumoto, 1998; Ito et al., 2002b; Jiang et al., 2014b). Headaches, dizziness, tachycardia, and fever were also reported. Green sea turtles (Chelonia mydas) have repeatedly been involved in human toxicoses. As juveniles, green turtles are largely

omnivorous, but adults mainly feed on sea grasses, algae, and invertebrates (Carrio´n-Cortez et al., 2010; Perrault et al., 2020). In 2010, after a community feast on an island of the Federated States of Micronesia where the main dish was a stew made from hawksbill turtle (Eretmochelys imbricata), 4 children and 2 adults died and 95 others became ill (Pavlin et al., 2015). Hawksbill turtles are largely carnivorous, but they also eat sea grasses and algae. Sore throat, mouth pain, and abnormal thirst were present in most of the individuals who ate the stew. Several infants nursing from mothers who ate the stew also became ill. Many of the affected individuals had pale yellow exudative lesions on the tongue or in the oropharynx. Six dogs deemed likely to have eaten from the turtle also died in the same time frame. The reef surrounding the island was in poor condition, with many dead corals as well as large amounts of cyanobacteria believed to be M. producens. Although the responsible toxin was not identified in that large outbreak because of the cyanobacteria noted on the reef, the time frame, the clinical signs, and the character of the observed lesions, the authors suspected poisoning by lyngbyatoxin A. Ito et al. (2002b) described several incidents of human poisonings after eating sea turtles that involved effects that were compatible with those of lyngbyatoxin A or aplysiatoxin poisoning. Some of those incidents involved hundreds of people and included significant numbers of deaths. When poisoning from eating sea turtles, termed chelonitoxism, is encountered, analyses should include a focus on lyngbyatoxins, aplysiatoxins, and other phycotoxins. Moreover, because all species of sea turtles are endangered, human consumption of these species or their eggs should cease. For more on chelonitoxism and chelonitoxins, see Chapter 8, Animal Toxins.

FIGURE 5.37 Cutaneous punch biopsy of a blistering rash on the abdomen of a 13-year-old girl 1 day after swimming in rough waters off the coast of Oahu, Hawaii. The section is of the base of an intraepidermal blister that had superficial desquamation of the blister roof. The clinical history, distribution of the rash, and the histopathology are consistent with Moorea-induced contact dermatitis. Figure reproduced from Werner KA, Marquart L, Norton SA: Lyngbya dermatitis (toxic seaweed dermatitis), Int J Dermatol 51:59–62, 2012, Fig. 4, p. 60, with permission.

13.5. Diagnosis, Treatment, and Control Presumptive diagnoses of poisoning by M. producens toxins are usually based on characteristic clinical signs and lesions in individuals after recent contact with the filamentous cyanobacteria. Symptoms and signs can develop within hours of contact and usually begin with itching or burning of the skin in concert with development of erythematous blisters. Such lesions generally resolve within 2 weeks.

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14. b-METHYLAMINOALANINE

Exposed areas should be thoroughly washed as soon after exposure as possible. Other treatment is symptomatic. In view of the potential for severe irritation and tumor promotion, people and pets should avoid exposure to water contaminated by Moorea, Microseira, Schizothrix, and Oscillatoria. Thus, swimming or wading in areas where these cyanobacteria are present should be prohibited. Direct contact with bloom material washed up onto the beach should be prevented. Horses and other species of animals should not be allowed to ingest the bloom material. Where Moorea or these other cyanobacteria have washed onto beaches, it should be immediately cleared by local councils. In these circumstances it is important to take precautions to minimize contact with the bloom material during collection, transit, and disposal operations.

14. b-METHYLAMINOALANINE 14.1. Introduction The cyanotoxin, ß-N-methylamino-L-alanine (BMAA), is a nonproteinogenic diamino acid (Figure 5.38). BMAA was originally isolated from seeds of the tropical cycad, Cycas micronesica (formerly C. circinalis), and it has since been implicated in a neurologic disease that occurred with a high incidence on Guam. It received much attention in the medical, scientific, and public health arenas when it was proposed as a critical factor in the genesis of several other neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), as well as Parkinson’s and Alzheimer’s diseases,

O H 3C

N H

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which occur worldwide. However, questions have been raised as to whether existing data actually support a hypothesis of BMAA as a cause of neurodegenerative disease (Duncan et al., 1988; Meneely et al., 2016; Chernoff et al., 2017; Spencer et al., 2018). The literature regarding BMAA is confusing, largely because older analytical techniques were inaccurate, overestimating BMAA concentrations in environmental and tissue samples and possibly misidentifying BMAA producers (Chernoff et al., 2017; Bishop and Murch, 2020). For example, recent studies using direct detection of BMAA with liquid chromatography and tandem mass spectrometry, as described below, have shown that most cyanobacteria do not produce BMAA or produce very low quantities (Kru¨ger et al., 2010), as opposed to older studies that reported BMAA in most cyanobacterial species (Cox et al., 2005).

14.2. Sources/Occurrences/Exposures BMAA is produced by both prokaryotic (cyanobacteria) and eukaryotic (diatoms and dinoflagellates) microorganisms that occur worldwide (Cox et al., 2005; Jiang and Ilag, 2014: Jiang et al., 2014a). BMAA has been detected in a variety of environments where cyanobacteria are found, both aquatic (oceans, lakes, and desert springs) and terrestrial. BMAA, found in C. micronesica, is produced by Nostoc sp., a symbiotic filamentous cyanobacterial species associated with roots of the plant. Like other phycotoxins, BMAA production in various cyanobacterial species depends upon nutrient availability and other environmental conditions.

14.3. Toxicology

OH NH2

FIGURE 5.38 Structure of ß-N-methylamino-Lalanine (BMAA). Created by Yikrazuul with Inkscape, Own work, Public Domain, https://commons.wikimedia.org/ wiki/File:3-Methylamino-L-alanine.svg, downloaded March 22, 2021.

BMAA is water soluble and can also be protein-associated, leading to bioaccumulation up the food chain (Jonasson et al., 2010). BMAA has been reported to accumulate in seafood and experimentally in plants exposed to contaminated water or soil. Shark flesh and especially shark fins can contain high concentrations of BMAA. Most recently, BMAA has been reported in the brains of dolphins (Davis et al., 2019).

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14.4. Animal Studies Several studies in rats and monkeys have investigated the pharmacokinetics and transport mechanism(s) of BMAA. Both intravenous (iv) and oral administration of BMAA to rats were followed by rapid clearance from plasma (Duncan et al., 1991). Plasma concentrations after oral administration were approximately 80% of those achieved after iv dosing. Following either single dosing or continuous infusion for 2 weeks, BMAA was found to cross the blood–brain barrier (BBB), but to a limited extent without any indication of selective brain deposition or retention (Duncan et al., 1991). Oral administration to macaques (Macaca fascicularis) resulted in a similar pattern of bioavailability (Duncan et al., 1992). BMAA was shown to be transported across the BBB of rats by a large neutral amino acid carrier which generally functions as an amino acid exchanger rather than as a facilitator of accumulation (Smith et al., 1992). Because of interest in the potential of BMAA to induce neurodegenerative disease, many in vivo studies have been performed, as reviewed by Karamyan and Speth (2008). However, the high doses of BMAA used in these studies are viewed as being excessive and not relevant to potential human exposure. Only a few studies from that review are mentioned here. Acute neurotoxic effects were reported following intraperitoneal (ip) administration of BMAA to chicks, rats, and mice. Most studies in adult rodents found functional disturbances or neurodegenerative changes relevant to motor dysfunction. In rats, transient changes consistent with cerebellar dysfunction were observed starting from a single ip dose of the L-isomer (with the D-isomer being inactive) at 900 mg/kg, while 500 mg/kg/day for 1–2 weeks produced no clinical effect until the same rats were given 1000 mg/kg on days 21 and 23 and, 2 days thereafter, they showed slight ataxia and stiff tails (Seawright et al., 1990). At all doses of BMAA, degenerative changes were observed in several populations of cells in the cerebellar cortex: GABA-ergic inhibitory stellate cells, basket cells, Purkinje cells, and Golgi cells, but not in glutamatergic excitatory granule cells or the cerebellar roof nuclei. No other changes were found in the nervous system. Seawright et al. (1990) deemed that BMAA causes posture

and movement related changes in rats as a result of excitotoxic actions selective to cerebellar neurons. Macaques (M. fascicularis) fed BMAA at 100– 315 mg/kg daily for up to 12 weeks showed neurological impairments consistent with a disorder involving upper and lower motor neurons and the extrapyramidal system (Spencer et al., 1987). At doses 200 mg/kg, motor neuron dysfunction in the forelimbs was followed by muscle weaknesses and loss of muscle mass. After a month of BMAA administration, all animals displayed stooped posture, tremors, and weakness in extremities. Longterm treatment resulted in periods of immobility with a blank stare and crouched posture. The overt clinical signs did not progress after exposure ended (Spencer et al., 1991). Interestingly, two of the animals responded to oral antiParkinsonian drugs within 30 min. Morphologically, there was disruption in the motor cortex as well as displacement of Nissl bodies, chromatolysis of giant Betz cells, and similar changes in the large anterior horn cells of the spinal cord. At necropsy, vervet monkeys (Chlorocebus sabaeus) exposed to BMAA for 140 days at 210 mg/kg/day had more neurofibrillary tangles than those given 21 mg/kg/day or controls (Cox et al., 2016). b-Amyloid deposits were found in some animals in each of the treated groups.

14.5. Mechanism of Action BMAA has been reported to act as a glutamate agonist causing excitotoxicity in neurons, such that neurons containing glutamate receptors are a main target of BMAA. Allen et al. (1993) reported that BMAA causes depolarization of postsynaptic neurons, thereby relieving magnesium blockade of calcium channels, resulting in an influx of calcium. This, in turn, was associated with postsynaptic swelling and neuronal degeneration. Such a mechanism is similar to that of domoic acid, an excitatory neurotoxin produced by marine diatoms (see previous section on domoic acid) (Brownson et al., 2002). In addition to its excitotoxic potential, BMAA has been implicated in protein misfolding and aggregation, inhibition of specific enzymes, and neuroinflammation, all hallmark features of neurodegenerative diseases. An earlier proposed

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mechanism for BMAA bioaccumulation, misincorporation into proteins in the place of the canonical amino acid L-serine (Dunlop et al., 2013) has been disputed. More recently, van Onselen and Downing (2018) showed that BMAA binds strongly to proteins and cannot be removed by protein precipitation or denaturation, can inhibit activity of enzymes with functional hydroxyls in their active sites, and interferes with protein folding in the absence of de novo protein synthesis.

14.6. Human Exposure and Disease Although many reports have implicated BMAA in human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis–parkinsonismdementia complex (ALS/PDC) (Spencer 1987; Cox et al., 2005, 2018), whether human neurotoxicity results from BMAA in contaminated food or water is still controversial (Meneely et al., 2016; Chernoff et al., 2017). ALS and Parkinson’s disease are commonly diagnosed throughout the world. However, in a few locations in the Western Pacific, the symptoms, signs, and lesions of these diseases coexist in the fatal disorder known as ALS/PDC, also termed ‘lytico-bodig’ disease locally. This otherwise rare disease has been documented in Auyu and Jaquai linguistic groups of Papua (formerly Irian Jaya) in Indonesia, in people of the Kii Peninsula in Japan, and among the Chamorro population of Guam (Spencer, 1987; Duncan et al., 1988; Brownson et al., 2002). In association with the Chamorro cases, it was reported that symbiotic filamentous cyanobacteria species in the genus Nostoc, associated with the roots of cycads, produce BMAA which accumulates in seeds of the plants. Much higher concentrations of BMAA were found in Nostoc growing on the roots of the cycads than in free-living Nostoc. However, no correlation was found between the concentrations of the free amino acid in cycad flour and ALS/PDC. The indigenous Chamorro people ate not only the cycad seeds, but also megabats known as flying foxes (Pteropus mariannus mariannus). Those bats consume the cycad seeds, and it was proposed that they could biomagnify BMAA and become a vector of the toxin, as happens with Daphnia and marine invertebrates

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and fish. Eating of bats was proposed to lead to human cases of BMAA toxicosis manifested as ALS/PDC (Cox et al., 2003; Murch et al., 2004). In a recent study, however, Foss et al. (2018) were unable to detect BMAA from flying fox skin and preserved fur specimens from Guam similar to those analyzed earlier by Banack and Cox (2003). Although many possible etiologic factors have been investigated, the cause of ALS/PDC still remains undetermined. Cox et al. (2003) examined the superior frontal gyrus from the brains of 6 deceased ALS/PDC patients from Guam, 2 Alzheimer’s patients from Canada, 2 asymptomatic Chamorros, and 13 individuals with no signs of neurodegeneration. They found BMAA in the brain tissues of the 6 Chamorro ALS/PDC patients and in the 2 patients from Canada, but none in the brain tissues of the 13 individuals without neurodegenerative disease. However, when Montine et al. (2005) sampled brains from a similar group of individuals, they did not find free BMAA in any of the specimens. Rauk (2018) used quantum mechanical calculations and molecular dynamics simulations to examine whether BMAA would form covalent bonds with b-amyloid peptide and concluded that the toxin would not change the conformation of the b-amyloid peptide and that it was not involved in Alzheimer’s disease. Despite the discovery of BMAA in 1967 followed by several hundred publications, there remain important disagreements about its occurrence and roles in neurodegenerative diseases (Nunn, 2017). To our knowledge, no research has conclusively shown that BMAA from drinking water has impacted human health. Moreover, scientists, including those from US EPA, agree that there is insufficient evidence to link BMAA with neurological diseases (Chernoff et al., 2017).

14.7. Analytical Methods for Detection and Quantification A review of analytical detection methods for BMAA shows major discrepancies between two common methods for BMAA detection, reverse phase liquid chromatography and hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC MS/MS) (Bishop and

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Murch, 2020). Spencer et al. (2018) urged “cautious interpretation of previous BMAA analyses of bacterial, plant, animal and human material performed by HPLC-fluorescence detection of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC)-derivatized samples.”

14.8. Conclusion Additional research is needed on this topic to address concerns that multiple cyanobacteria may produce BMAA; the toxin may biomagnify through trophic levels; it was reported in some studies in the brain of humans with neurodegenerative diseases; and brain lesions occurred in vervet monkeys given a diet containing BMAA. Studies that estimate BMAA-related risks from environmental sources, including cyanobacterial synthesis, transfers through food webs, and uptake by different species of animals are warranted. Greater insights on neurotoxic mechanism(s) of action of BMAA are also needed to make definitive predictions regarding its role, if any, in common neurological diseases.

15. EMERGING PHYCOTOXINS 15.1. Vacuolar Myelinopathy and Aetokthonotoxin Avian vacuolar myelinopathy (AVM) has killed large numbers of coots (Fulica americana) and bald eagles (Haliaeetus leucocephalus). The syndrome was first recognized when 55 bald eagles died in an area of Arkansas in the winters of 1994–95 and 1996–97 (Thomas et al., 1998). Eagles were notably ataxic and were seen crashing into trees and rocky bluffs. Since that outbreak, AVM in coots and bald eagles has occurred repeatedly at many manmade lakes and reservoirs of the southeastern and southcentral United States. Coots spend their summers in Canada and northern states of the United States and are present in the southern states during colder times of the year. Bald eagle deaths at J. Strom Thurmond Reservoir on the border of South Carolina and Georgia have totaled more than 100, such that local news reports have called the area an ecological trap for the species. In addition to large numbers of coots and bald eagles, mallards (Anas

platyrhynchos), Canada geese (Branta canadensis), ring-necked ducks (Aythya collaris), lesser scaup (Aythya affinis), buffleheads (Bucephala albeola), and other waterfowl, as well as great horned owls (Bubo virginianus) and killdeer (Charadrius vociferus), have been found to have AVM in the wild. Lesions in the AVM-affected birds included marked, widespread, spongy degeneration of myelinated tracts most prominently in the optic tectum, but also in the cerebellum, medulla, and spinal cord, especially near the gray matter (Figures 5.39 and 5.40). Occasional swollen axons were also present. However, no vacuoles have been found in the retina or peripheral or autonomic nerves. A series of feeding trials definitively linked AVM to: (i) direct ingestion by waterfowl of substantial amounts of an exotic invasive submerged aquatic macrophyte, Hydrilla verticillata that was contaminated by the epiphytic cyanobacteria, Aetokthonos hydrillicola, and (ii) relay poisoning when birds that were clinically affected or had died with AVM were the food source for raptors (Wilde et al., 2014; Haram et al., 2020). Waterfowl may display neurologic abnormalities within 5 days of initial exposure. Chickens, mallards, and red-tailed hawks have been found to be susceptible to AVM in feeding trials, and chickens are relied upon as an experimental model species (Lewis-Weis et al., 2004).

FIGURE 5.39 Coronal section of the midbrain and vermis of the cerebellum from a bald eagle with avian vacuolar myelinopathy. Nearly all white matter tracts are lightly stained due to intramyelinic edema (arrows). Courtesy of Kevin Keel.

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FIGURE 5.40 Optic tectum from a bald eagle with avian vacuolar myelinopathy. The stratum opticum (arrows) is greatly rarefied due to intramyelinic edema. Vacuolation occurs in white matter of various locations in the brain, but it is most severe in the optic tectum. Courtesy of Kevin Keel.

Feeding the digestive tract and contents from affected coots to susceptible species has repeatedly resulted in neurologic signs and lesions of AVM. However, whether tissue residues of the toxin(s) associated with AVM play a significant role in relay poisonings remains unclear. H. verticillata that does not harbor A. hydrillicola has not been associated with AVM either in the wild or in various feeding trials, and it has often been used for control diets. Bioassays with water fleas (Ceriodaphnia), nematodes (Caenorhabditis elegans), larval zebrafish (Danio rerio), and chickens, in concert with toxin purification, mass spectrometry, and infrared and nuclear magnetic resonance spectroscopy, recently culminated in elucidation of the structure of aetokthonotoxin (AETX) as the cause of AVM (Breinlinger et al., 2021). AETX is an unusual pentabrominated biindole alkaloid (Figure 5.41). The authors also reported that a geologic or anthropogenic source of bromine is required for significant biosynthesis of AETX by A. hydrillicolla. The potential value of preventing anthropogenic inputs of bromine from coal-fired power plants, bromine used in water disinfection, and the aquatic herbicide diquat dibromide to reduce production of AETX warrants further investigation. Coots seem to be highly predisposed to AVM, probably due to their seasonal migration patterns and tendency to consume large amounts of H. verticillata, including when it is highly contaminated (Haram et al., 2020). Affected coots are often found dead, and scavenged coot carcasses

N Br

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FIGURE 5.41 Structure of aetokthonotoxin from Aetokthonos hydrillicola, an epiphytic cyanobacteria on the exotic invasive macrophyte Hydrilla verticillata. The toxin is a pentabrominated diindole alkaloid. Courtesy of Timo Niedermeyer. Figure reproduced from Breinlinger S, Phillips TJ, Haram BN, Mares J, Martı´nez Yerena JA, Hrouzek P, Sobotka R, Henderson WM, Schmieder P, Williams SM, Lauderdale JD, Wilde HD, Gerrin W, Kust A, Washington JW, Wagner C, Geier B, Liebeke M, Enke H, Niedermeyer THJ, Wilde SB: Hunting the eagle killer: a cyanobacterial neurotoxin causes vacuolar myelinopathy, Science 371:1335, 2021. https://doi.org/10.1126/science. aax9050, eaax9050, Fig. 3A.

as well as feather piles have been associated with outbreaks of AVM. Clinical signs in coots frequently include listlessness, ataxia, incoordination, inability to fly, swim, or walk, reduced withdrawal reflexes, proprioceptive deficits, beak or tongue weakness, head tremors, absent pupillary light reflexes, and less often anisocoria, apparent blindness, nystagmus, and strabismus (Larsen et al., 2002; Haram et al., 2020).

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There are no specific gross lesions of AVM in birds, but microscopic examinations of the brain reveal severe vacuolation of the white matter, especially in the optic lobes (Larsen et al., 2002; Lewis-Weis et al., 2004). AVM lesions have been documented in normal appearing coots during outbreaks of AVM. Vacuole formation has been associated with intramyelinic edema and splitting of myelin at the intraperiod line. In experimental chickens, lesions were consistently present in the optic tectum and brain stem, and they were often noted in the cerebrum and cerebellum. In surviving birds, AVM lesions may persist after resolution of clinical abnormalities. With prolonged supportive care, some coots have been able to recover from signs of AVM. Other species that have been documented to have fed on contaminated H. verticillata harboring A. hydrillicola in various studies include exotic herbivorous aquatic snails (Pomacea maculata), grass carp (Ctenopharydon idella), tadpoles of frogs of the genus Lithobates (formerly Rana); e.g., bullfrog (L. catesbeiana), greenfrog (L. clamitans), and southern leopard frog (L. sphenocephela), and painted turtles (Chrysemys picta). The roles, if any, of such species as potential victims or vectors of toxins that cause VM to predators and scavengers are only partially understood. The finding of AVM in chickens fed P. maculata that had been given a diet of the contaminated Hydrilla suggests that additional studies of potential relay toxicoses are warranted (Dodd et al., 2016). The potential risk of AVM in the endangered Florida snail kite (Rostrhamus sociabilis), which feeds on P. maculata in the wild, is of particular concern. Species in addition to birds have been found to be susceptible to AETX and, because the lesions are similar across species, the encompassing term, vacuolar myelinopathy (VM) is now recommended (Breinlinger et al., 2021). Although triploid (sterile) grass carp can help control contaminated Hydrilla, and a feeding trial revealed no clinical abnormalities in the tested fish, they had developed vacuoles in the central nervous system, including the optic tectum. Nevertheless, chickens that were fed the grass carp did not develop signs or lesions of AVM (Haynie et al., 2013). Additional studies may be warranted to ensure that grass carp used for

control of contaminated Hydrilla are not a significant source of toxin for predatory or scavenging species. When fed contaminated Hydrilla after it was collected in October–November, tadpoles of green treefrogs (Hyla cinerea) were spared, but tadpoles of L. catesbeiana, L. clamitans, and L. spenocephala were clearly susceptible to poisoning (Maerz et al., 2019). Incoordination in the susceptible species of tadpoles was followed by high mortality and severe vacuolation in the central nervous system. The late season association with toxic impacts in the larval amphibians was consistent with AVM outbreaks in waterfowl. Amphibian life cycles are highly variable, and aetokthonotoxin poisoning seems likely to be a more important concern for species that give rise to grazing tadpoles in contaminated sites during the fall and winter seasons. In the southeastern United States, painted turtles and other turtle species may rely on a diet largely consisting of Hydrilla. After approximately 3 months of feeding upon Hydrilla contaminated with A. hydrillicola, painted turtles developed mild gait asymmetry to severe limb dragging and loss of the ability to right themselves (Mercurio et al., 2014). Lesions in the turtles included severe, diffuse vacuolation of white matter of the cerebrum, cerebellum, brain stem, and spinal cord. VM presents a precautionary tale of the need to be far more purposeful in preventing introductions of exotic species. In this case, an invasive exotic aquatic plant supported epiphytic toxigenic cyanobacteria that caused direct and relay poisonings of diverse indigenous wildlife, including the national bird of the United States.

15.2. Palytoxins Palytoxins (PLTXs) are complex polyketides that were first isolated from a species of coral also, referred to as an anemone, (Palythoa toxica) in Hawaii. Corals of that genus, dinoflagellates of genus Ostreopsis, and tropical and subtropical marine cyanobacteria of the genus Trichodesmium are reported to be important producers of PLTXs and related compounds. The PLTXs include some of the most potent toxins in existence (Tubaro et al., 2012; Kerbrat et al., 2011) (Figure 5.42). PLTXs include the prototype PLTX as well as 42-hydroxy-PLTX, and other

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FIGURE 5.42 Structure of palytoxin that was isolated from the soft coral Palythoa toxica but had been produced by the marine dinoflagellate Ostreopsis siamensis. Author, Charlesy. This image of a simple structural formula is ineligible for copyright and therefore is in the public domain. It consists entirely of information that is common property and contains no original authorship, Own work, https://commons.wikimedia.org/wiki/File:Palytoxin.svg, downloaded March 22, 2021.

analogues that are termed ovatoxins, ostreocins, ostreotoxins, and mascarenotoxins (Kerbrat et al., 2011; Patocka et al., 2018). A number of studies have examined biochemical mechanisms of PLTXs. After intravenous administration, PLTX disrupts Na/K-ATPase, so that these essential ion pumps instead behave as monovalent ion channels, resulting in increased intracellular sodium and decreased intracellular potassium, with depolarizations that cause contractions of smooth, skeletal, and cardiac muscles, as well as increased glandular secretions (Munday, 2011; Seymour et al., 2015). Studies in vitro have also revealed secondary activation of voltage-dependent calcium channels resulting in increases in intracellular calcium (Patocka et al., 2018). Actin microfilaments become disrupted and apoptosis ensues (Louzao et al., 2011). PLTXs are also severely irritating to eyes and skin. Although PLTX was not mutagenic in a series of assays, it is a tumor promoter, as demonstrated in two laboratory animal studies that employed polyaromatic hydrocarbons as mutagenic initiators (Munday, 2011). However, the concentrations of PLTX that elicited tumor promotion were quite high relative to those that humans are likely to encounter.

PLTX is very toxic when administered intratracheally or sublingually (Munday, 2011). The onset of toxic effects after such dosing may be delayed by several days, but effects can be severe. Pulmonary alveolar hemorrhage and edema may be prominent. It was suggested that sublingual administration of PLTX in laboratory animals might result in inhalation of the toxin. Mice given a lethal intraperitoneal dose of PLTX rapidly became ataxic and had piloerection. After 30–60 min, they extended their hindlimbs, their respiratory rate slowed, and they appeared to be paralyzed, although they had gasping respiration just before death. PLTX is more toxic when given intravenously than after oral administration. Rats, rabbits, dogs, and rhesus monkeys are highly susceptible to the toxin when they are dosed intravenously (Munday, 2011). Postmortem examinations of mice given a lethal intravenous dose of PLTX revealed extreme dilation of the right ventricle. Anesthetized dogs and cats given a lethal iv dose of PLTX became bradycardic and had ectopic beats, ventricular arrhythmias, cessation of coronary blood flow, and cardiac arrest. Deaths from high iv doses of PLTX result from cardiac effects, whereas deaths that occur after

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a period of hours have been linked to cessation of respiration with cyanosis. Corticosteroids and intracardiac vasodilators were protective against intravenously administered PLTX (Munday, 2011). PLTXs have been associated with poisonings of humans after oral, inhalation, and cutaneous exposures (Deeds and Schwartz, 2010; Verma et al., 2019). Lethal or life-threatening PLTX toxicoses in humans have occurred in Japan, the Philippines, Madagascar, Brazil, and the United States, and suspected human poisonings with PLTXs have occurred in coastal areas in many parts of the world (Patocka et al., 2015). Ingestions of PLTXs in crustaceans, such as crabs, sea urchins, and finfish, including parrotfish, have been important in human poisonings. Toxic effects believed to be attributable to PLTXs in such cases have included a bitter, metallic taste in the mouth, nausea, abdominal cramps, vomiting, diarrhea, paresthesias, muscle cramps, myalgia, rhabdomyolysis, hemolysis, renal failure, cyanosis, angina-like chest pain, electrocardiographic abnormalities with an elevated T wave, bradycardia, and heart injury and failure leading to death (Tubaro et al., 2012; Seymour et al., 2015; Patocka et al., 2015, 2018). Therapy after oral exposures has included gastric lavage, forced diuresis, fluid administration, and artificial respiration, but sometimes patients have died despite such intervention (Tubaro et al., 2012). Other aspects of treatment for orally induced PLTX toxicosis were described by Patocka et al. (2015). Further research on potential therapies for PLTX poisoning is needed. Exposures of humans to PLTXs from aerosols of seawater that had blooms of Ostreopsis, and handling or pouring hot water onto zoanthid corals while tending saltwater reef aquaria, have been associated with respiratory distress, rhinorrhea, cough, fever, ocular irritation, and dermatitis (Deeds and Schwartz, 2010; Deeds et al., 2011; Hall et al., 2015; Pelin et al., 2016). Cleaning PLTX-containing aquaria has also been linked to tachypnea, wheezing, hemoptysis, radiographically evident pulmonary opacities, hypoxemia, tachycardia, electrocardiographic abnormalities, leukocytosis, myalgia, weakness, muscle spasms, nausea, paresthesias, ataxia, and tremors. Individuals with respiratory toxicity

have sometimes been treated as inpatients and have responded to supplemental oxygen and inhaled corticosteroids. A mouse bioassay has been employed to reveal the potential presence of PLTXs in seafood, but other methods are used for this purpose as well, including cell-based assays (European Food Safety Authority, 2009). In either case, positive results require analytical confirmation, and available methods include HPLC with fluorescence detection and LC-MS/ MS. Analyses for PLTXs require optimization and validation as well as certified reference materials. PLTXs may coexist with CTXs, STXs, and tetrodotoxin, which can complicate both diagnostic assessments and targeted therapeutic interventions (Deeds and Schwartz, 2010). PLTX concentrations in seafood are a concern. The European Food Safety Authority (2009) recommended that the sum of PLTX plus ostreocinD should not exceed 30 mg/kg of shellfish meat. This concentration was based on an oral acute reference dose of 0.2 mg of PLTXs/kg body weight, and a 60 kg adult who would ingest a 400g portion. Despite that recommendation and ongoing concerns regarding these and other PLTXs, to our knowledge, there are no current regulations on the concentrations of these toxins allowed in seafoods. Octopuses, sea stars, and sea urchins can be lethally poisoned by these compounds. Since these species as well as other echinoderms, anemones, polychaetes, mollusks, crustaceans, and finfish can bioaccumulate PLTXs, potential poisonings of aquatic species that rely on them as prey should be explored as a priority for future research (Munday, 2011; Patocka et al., 2015; Cen et al., 2019).

15.3. Yessotoxins Yessotoxins (YTXs) are sulfated polyether toxins with around 100 analogs, most of which are yet to be studied. They are produced by species of dinoflagellates of the genera Gonyaulax, Protoceratium, and Lingulodinium (Paz et al., 2008; Seymour et al., 2015). YTXs accumulate in bivalves, especially scallops and mussels, and contaminated shellfish have been found along the coasts of Japan, New Zealand, Chile, Spain, Italy, Ireland, Norway, Russia, and the United States (California) (Howard et al., 2008). They

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are cytotoxic and cardiotoxic (Howard et al., 2008). In mice, YTXs were much more toxic intraperitoneally than orally (Tubaro et al., 2012). Diarrhea is unlikely to result from YTX ingestion (Paz et al., 2008). A recent report indicated that YTXs were implicated in degeneration and necrosis of gills and the deaths of millions of cultured abalone near the coast of South Africa (Pitcher et al., 2019). Although we and other authors, e.g., Tubaro et al. (2012), are not aware of human YTX poisonings, these toxins and their producers are widely distributed around the world, and they may warrant ongoing studies in regard to potential effects on mollusks and on species that ingest them, including humans. YTXs are detected by ELISA and LC-MS/MS. Shellfish are routinely monitored for YTXs in New Zealand, Europe, and Japan. The European Commission has set a regulatory limit for YTXs of 1 mg/kg of shellfish meat for human consumption (Howard et al., 2008).

16. CONCLUSIONS AND FUTURE NEEDS HABs are ancient phenomena, but they are increasing in many places today due to nutrient pollution, degraded plant communities, previous and ongoing water impoundments, wasteful abstractions, and climate change. In addition to phycotoxin problems, many HABs result in hypoxic or anoxic waters that cause sublethal effects and death losses in fish and aquatic invertebrate populations. Due to overfishing and dead zones, the historic supply of naturally produced aquatic animals that have served as vital sources of high-quality nutrients for millennia is smaller now, and too often it is fouled by, not only pesticides, industrial chemicals, petroleum, and mercury, but also phycotoxins. As shown in this chapter, phycotoxins have prominent, often life-threatening effects on the nervous system and liver, but they also impact other key components of the digestive system, kidneys, heart, lungs, skin, and immune functions. Moreover, many of them are tumorpromoting cocarcinogens, and some are genotoxic. The future research agenda should be broadened to better understand the range of

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effects of phycotoxicoses on more cell types and all of the body systems. While great strides have been made in development of methods of phycotoxin analyses of specimens from animals in focused research studies, a focus on practical methods for comprehensive, fast, and affordable identifications of phycotoxin producers and quantifications of phycotoxins for wide application in diagnostic and medical laboratories is overdue (Loftin et al., 2016; Mishra et al., 2020). The current situation presents a timely opportunity for a new globally integrated system of research linked to comprehensive diagnostics, surveillance, and monitoring, based on: (I) gathering and interpreting satellite and aerial remote sensing data for characteristic pigments in waters; (II) collecting targeted water samples and using them to (A) identify and count potential phycotoxins producers, (B) determine genomic and epigenetic indicators of the capacity for phycotoxin production, and (C) quantify diverse phycotoxins; and (III) integrating the above data with antemortem and postmortem findings in animals and humansdat the individual and population levels. More studies of animals and people should compare exposure histories with clinical symptoms and/or signs, clinical pathology changes in blood, serum and urine, and gross, microscopic, and ultrastructural lesions. They should also deduce how residues of phycotoxins and their metabolites in digestive tract contents, blood, tissues, urine, and feces relate to the likelihood, character, severity, and time course of poisoning, as well as the probability of partial to full recovery. An additional need is for new studies of therapies to limit phycotoxin absorption, to promote detoxification, and to protect essential organ/ system functions. Optimally, there should be a repository of clinical information to form a foundation for effective case management provided to emergency rooms and poison control centers around the world. Managers at the local, state, and national levels have begun to address some of the problems caused by HABs (e.g., Lammers and Bledsoe,

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2017), but in many places, the funding and the scale of efforts are not nearly up to the challenges at hand. Current and future researchers should more effectively engage with governments, businesses, agricultural producers, and citizens to identify, implement, and refine incentives and disincentives that will rapidly reduce carbon dioxide, methane, and nutrient pollution, rehabilitate hydrologically impaired water bodies, and protect ponds, lakes, streams, reservoirs, estuaries, and oceans from the impacts of dead zones and phycotoxin poisonings. It is strongly emphasized that prevention of future HABs depends upon appropriately scaled multisectorial efforts that effectively and consistently reduce nutrient inputs and rehabilitate watersheds and riparian zones of all water suppliesdfresh, brackish, and marine.

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WHO (World Health Organization): Aquatic (marine and freshwater) biotoxins, Environ Health Criteria 37:1–68, 1984. World Health Organization Geneva, Switzerland, https:// apps.who.int/iris/bitstream/handle/10665/37292/92415 40974-eng.pdf. WHO (World Health Organization): Cyanobacterial toxins: anatoxin-a and analogues, Background document for development of WHO Guidelines for drinking-water quality and Guidelines for safe recreational water environments, Geneva, Switzerland, 2020a, WHO/HEP/ECH/WSH/2020.1. https://apps.who.int/iris/bitstream/handle/10665/33806 0/WHO-HEP-ECH-WSH-2020.1-eng.pdf?sequence=1&isA llowed=y. WHO (World Health Organization): Cyanobacterial toxins: cylindrospermopsins, Background document for development of WHO Guidelines for drinking-water and Guidelines for safe recreational water environments, Geneva, Switzerland, 2020b, WHO/HEP/ECH/WSH/2020.4. https://apps.who.int/ iris/bitstream/handle/10665/338063/WHO-HEP-ECH-W SH-2020.4-eng.pdf?sequence=1&isAllowed=y. WHO (World Health Organization): Cyanobacterial toxins: microcystins, Background document for development of WHO Guidelines for drinking-water and Guidelines for safe recreational water environments, Geneva, Switzerland, 2020c, WHO/HEP/ECH/WSH/2020.6. https://apps.who.int/ iris/bitstream/handle/10665/338066/WHO-HEP-ECH-W SH-2020.6-eng.pdf. WHO (World Health Organization): Cyanobacterial toxins: microcystin-LR in drinking-water. Background document for development of WHO guidelines for drinking-water quality, Geneva, Switzerland, 2003, WHO/SDE/WSH/03.04/57. https://cdn.who.int/media/docs/default-source/wash-d ocuments/wash-chemicals/2003-microcystin-backgrounddocument.pdf?sfvrsn=ced679e7_7. WHO (World Health Organization): Cyanobacterial toxins: saxitoxins, Background document for development of WHO Guidelines for drinking-water quality and Guidelines for safe recreational water environments, Geneva, Switzerland, 2020d, WHO/HEP/ECH/WSH/2020.8. https://apps.who.int/ iris/bitstream/handle/10665/338069/WHO-HEP-ECH-W SH-2020.8-eng.pdf?sequence=1&isAllowed=y. Wickstrom ML, Khan SA, Haschek WM, et al.: Alterations in microtubules, intermediate filaments, and microfilaments induced by microcystin-LR in cultured cells, Toxicol Pathol 23:326–337, 1995. Wiese M, D’Agostino PM, Mihali TK, Moffitt MC, Neilan BA: Neurotoxic alkaloids: saxitoxin and its analogs, Mar Drugs 8:2185–2211, 2010. https://doi.org/10.3390/md8072185. Wilde SB, Johansen JR, Wilde HD, Jiang P, Bartelme BA, Haynie RS: Aetokthonos hydrillicola gen, et sp. nov.: Epiphytic cyanobacteria on invasive aquatic plants implicated in Avian Vacuolar Myelinopathy, Phytotaxa 181:243–260, 2014. Williams CD, Burns J, Chapman A, Pawlowicz M, Carmichael W: Assessment of cyanotoxins in Florida’s surface waters and associated drinking water resources. Final Report. [Report]. St. Johns River Water Management District

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C H A P T E R

6 Mycotoxins Genevieve S. Bondy1,a, Kenneth A. Voss2,a, Wanda M. Haschek3 1

Formerly Food Directorate, Health Canada, Ottawa, ON, Canada, 2Formerly United States Department of Agriculture, Agricultural Research Service, Athens, GA, United States, 3University of Illinois at Urbana-Champaign, Urbana, IL, United States

O U T L I N E 1. Introduction

394

2. Aflatoxins 2.1. Source/Occurrence 2.2. Toxicology 2.3. Manifestations of Toxicity in Animals 2.4. Human Risk and Disease 2.5. Diagnosis, Treatment, and Control

401 401 401 406 409 411

3. Ochratoxins 3.1. Source/Occurrence 3.2. Toxicology 3.3. Manifestations of Toxicity in Animals 3.4. Human Risk and Disease 3.5. Diagnosis, Treatment, and Prevention

411 411 412 414 416 417

4. Patulin 4.1. Source/Occurrence 4.2. Toxicology 4.3. Manifestations of Toxicity in Animals 4.4. Human Exposure and Disease 4.5. Diagnosis, Treatment, and Control

417 417 417 418 419 419

5. Trichothecene Mycotoxins 5.1. Sources/Occurrence 5.2. Toxicology 5.3. Manifestations of Toxicity in Animals 5.4. Human Risk and Disease 5.5. Diagnosis, Treatment, and Prevention

419 419 420 425 430 433

6. Zearalenone 6.1. Source/Occurrence 6.2. Toxicology 6.3. Manifestations of Toxicity in Animals

433 433 434 436

6.4. Human Risk and Disease 6.5. Diagnosis, Treatment, and Prevention

439 440

7. Fumonisins 7.1. Source/Occurrence/Exposure 7.2. Toxicology and Mode of Action (MOA) 7.3. Manifestations of Toxicity in Animals 7.4. Human Risk and Disease 7.5. Diagnosis, Treatment, and Prevention 7.6. Regulations and Guidances

440 440 441 446 452 454 454

8. Ergot Alkaloids 8.1. Introduction 8.2. Source/Occurrence 8.3. Toxicology 8.4. Manifestations of Toxicity in Animals 8.5. Human Risk and Disease 8.6. Pharmaceutical Use 8.7. Diagnosis, Treatment and Prevention

455 455 455 457 460 463 464 465

9. Emerging Mycotoxins 9.1. Introduction 9.2. Alternaria Toxins 9.3. Aspergillus and Penicillium Toxins 9.4. Tremorgenic Mycotoxins 9.5. Fusarium Toxins 9.6. Diagnosis, Treatment, and Control

465 465 466 468 471 472 476

10. Summary/Conclusion

477

Acknowledgments

477

References

477

a

Retired.

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00006-X

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Copyright Ó 2023 Elsevier Inc. All rights reserved.

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1. INTRODUCTION Numerous species of fungi colonize food crops such as rice, corn (maize), wheat, barley, oats, peanuts, cottonseed, and soybeans, all of which are the basic ingredients of many human and animal foods, including livestock, companion, and laboratory animal diets. Under certain conditions, fungi produce mycotoxins that can cause adverse effects in other living organisms following exposure. Exposure is generally through the diet although inhalation or skin contact may be a route of exposure in occupational (e.g., during harvesting) or indoor (e.g., moldy homes) settings. Mycotoxins can also be produced by endophytic fungi, which are endosymbionts that live within a plant for at least part of its life without causing apparent disease. Toxins produced by these fungi have been shown to be responsible for toxicity that was previously attributed to the plant itself. For example, swainsonine found in both Astragalus and Oxytropis spp. is produced by the plant-associated endophyte Undifilum oxytropis (Cook et al., 2014; Wu et al., 2016a) (see Poisonous Plants, Vol 3, Chap 7). Mycotoxins are designated secondary fungal metabolites because they are not considered essential to the normal growth and reproduction of the fungus. Mycotoxins do, however, cause biochemical, physiologic, and/or pathologic changes in many species, including humans, animals, plants, and other microbes. These effects can range from poor growth/production parameters to death. The word mycotoxin is derived from “myco,” meaning mold, and “toxin,” a poison produced by a living organism. A great number of fungal metabolites have been designated as mycotoxins; however, only a small number are known to have significant animal/human health and economic significance. Mycotoxicosis is the term used to denote a syndrome resulting from poisoning of a biological system by a mycotoxin (IARC, 2012). Many factors can affect both the growth of fungi and the production of mycotoxins, including temperature and humidity, substrate moisture and nutrient content, level of fungal

inoculation, and microbial interactions. Temperature and moisture variations affect the growth rate of fungi and the types and amounts of toxins produced. Individual fungi often produce several different mycotoxins so that combinations of mycotoxins are frequently present, with the possibility of interactive effects. These interactions have been reviewed elsewhere and will not be discussed further in this chapter (Alassane-Kpembi et al., 2017). Fungi are aerobic organisms, but significant differences in oxygen requirements exist among different species. Fungal interactions with plants are complex: fungi can be saprophytes (fungi that obtain nutrients from dead organic matter) or live as endophytes in the host plant. In either case, environmental stress, insect damage, and plant disease predispose to colonization, growth, and toxin production. In the field, fungi invade both developing and mature seed grains on the plant, and the optimal moisture content for growth is 22%–25%. Storage fungi invade grain after harvest while it is in storage; the optimal moisture for growth is 13%–18%. Advanced decay fungi (saprophytes) typically require moisture of 22%– 25% but rarely develop and grow on seed grain in the field. Generally, fungi grow readily between 20 and 30
C, but optimal temperature ranges can be from below 0
C to above 60
C (Agriopoulou et al., 2020; Bryden, 2012; CAST, 2003). Mycotoxicoses occur worldwide and have been recognized for centuriesdfor example, St Anthony’s fire due to ergot in the Middle Ages (Pitt and Miller, 2017). Many factors contribute to the occurrence of mycotoxicoses in humans, livestock, companion animals, and wildlife. For example, modern harvesting methods in which corn is handled at higher moisture concentrations, combined with damage caused by harvesting machinery, increase the number of kernels in which fungi can initiate postharvest growth. Also, feeding ground diets prevents food-producing animals from sorting out and avoiding damaged kernels. A partial listing of mycotoxins is provided in Table 6.1. Our knowledge of many mycotoxins is extremely limited. In many instances, few substantive surveys of the frequencies of occurrence have been done. In other instances, the

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1. INTRODUCTION

TABLE 6.1

TABLE 6.1

A Partial Listing of Mycotoxins

A Partial Listing of Mycotoxinsdcont’d

Alternariol

Rubratoxins A and B

Alternariol monomethyl ether

Sporodesmin

Atranones AeG

Sterigmatin

Aflatoxins B1, B2, G1, G2, M1, M2, Q1, and aflatoxicol

Sterigmatocystins

Aspertoxins

Versicolorins

Autocystins

Tremorgens

Averufarin

Aflatrem

Beauvericin

Penitrem A

Bipolarin

Roquefortine

Butenolide

Paspaline

Citrinin

Paspalanine

Citreoviridin

Paspalitrems A and B

Culmorin

Verruculogen

Curvularin

Fumitremorgen

Cyclopiazonic acid

Fumigaclavine Trichothecenes

Cyclosporine Diplonine

T-2 and HT-2 toxins

Enniatins

Diacetoxyscirpenol (DAS)

Ergot alkaloids

Deoxynivalenol (DON)

Ergotamine

Verrucarins

Ergonovine

Roridins

Ergovaline

Satratoxins

Fumonisins B1-4 and homologs

Slaframine

Fusaproliferin

Swainsonine

Fusaric acid

Tenuazonic acid

Gliotoxin

Zearalenone and zearalenol

Luteoskyrin

Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table 1, p. 646, with permission.

Moniliformin Mycophenolic acid Ochratoxin series, especially ochratoxin A Patulin Penicillic acid Phomopsins Pyrrocidines (Continued)

compounds have been investigated in some detail, but surveys do not indicate sufficiently frequent occurrence in the United States or other countries for these toxins to be considered of major concern. For example, T-2 toxin and diacetoxyscirpenol are infrequently encountered in North America, where T-2 toxin causes only occasional outbreaks of toxicosis in the midwestern United States and western Canada. In

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contrast, T-2 toxin produced by Fusarium sporotrichioides and Fusarium poae on overwintered grain was likely responsible for severe outbreaks of mycotoxicosis in humans and animals in the Soviet Union in the 1940s (Pitt and Miller, 2017). Similarly, the nephrotoxic mycotoxin ochratoxin A causes only occasional problems in poultry and swine in North America, while it has been responsible for widespread outbreaks of toxicosis in swine in Denmark (Szczech et al., 1973). Four groups of mycotoxins account for most confirmed diagnoses of mycotoxicoses in the midwestern United States. These are fumonisins, deoxynivalenol (DON), zearalenone, and aflatoxins. Other common mycotoxins occurring worldwide are ochratoxin A and trichothecenes other than DON, such as T-2 toxin. Another group of mycotoxins, the ergot alkaloids, causes significant losses in livestock production in the United States, as well as Australia, Argentina, and New Zealand (Alshannaq and Yu, 2017; CAST, 2003). The primary fungal species producing these toxins are listed in Table 6.2. New mycotoxins continue to be identified. This includes reclassification of previously identified toxins as mycotoxins, e.g., swainsonine, the cause of locoism, and discovery of new entities such as diplonine, a neurotoxin, produced by the ear-rot fungus Stenocarpella (previously Diplodia) maydis, and responsible for diplodiosis. The term “emerging mycotoxins” has been used to describe both newly discovered mycotoxins, and mycotoxins that are relatively well known but for which there are gaps in knowledge of

their occurrence and toxicology. These include secondary metabolites that can co-occur with major mycotoxins, such as sterigmatocystin with aflatoxins, or citrinin with ochratoxins. The term also encompasses undercharacterized toxins produced by specific fungal species, such as those produced by molds in the genus Alternaria. Addressing research gaps for these secondary classes of toxins is an ongoing endeavor on many fronts (Cook et al., 2014; Gruber-Dorninger et al., 2016; Snyman et al., 2011). Interpretation of the significance of mycotoxin residues in animal diets is straightforward when extremely high concentrations are present. However, when low concentrations are present in foods, the interpretation of the toxicologic significance of mycotoxin residues can be more difficult. This is because there are differences between mycotoxicoses induced in the laboratory and field cases of mycotoxin poisoning that preclude direct extrapolation of the experimental mycotoxicoses to the situation in the field. In the field, the identified mycotoxin may be consumed along with other related or unrelated fungal metabolites, the latter including as yet unidentified mycotoxins. Fungal damaged, stressed grains may be of lower nutrient value and altered palatability. The toxin(s) is (are) unevenly distributed in the diet and the concentration(s) is (are) generally so variable that multiple samples are required to estimate the concentration(s) present. If sufficient moisture and appropriate temperatures occur during transit, additional fungal growth and toxin production may occur before analysis,

TABLE 6.2 The Major Mycotoxins and Fungi Producing Them Major mycotoxins

Major fungal species

Aflatoxins

Aspergillus parasiticus, Aspergillus flavus

T-2 toxin

Fusarium sporotrichioides

Deoxynivalenol (DON), zearalenone

Fusarium graminearum, Fusarium culmorum

Fumonisins

Fusarium verticillioides, Fusarium proliferatum, Aspergillus niger

Ochratoxin A

Penicillium verrucosum, Aspergillus ochracoeus

Ergot alkaloids

Claviceps spp., Neotyphodium coenophialum

Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table 2, p. 647, with permission.

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1. INTRODUCTION

potentially confounding analyses. The toxin may be bound to, or otherwise associated with, grain or food matrix components in a manner precluding detection or accurate quantification (referred to as hidden or “masked” mycotoxins). Some masked mycotoxins, such as deoxynivalenol-3-D-glucopyranoside, are produced by affected plants, which have the capability to metabolize or transform mycotoxins as part of their defense against pathogens. Finally, stressors, such as infectious agents, reproduction, lactation, crowding, and temperature variation, overlap and interact with effects of mycotoxins (Berthiller et al. 2013; Bryden, 2012; CAST, 2003). During experimental mycotoxin administration a single purified toxin is usually administered to experimental animals in a diet which is generally balanced and contains undamaged grains, and in a controlled, high-quality environment. The toxin is evenly mixed in the diet at a known concentration, and the diet sample is presented to the laboratory without additional fungal growth or toxin production occurring. However, it is the authors’ experience that interpretation and comparison of experimental work presented in the mycotoxin literature can be difficult because of the nature of the toxin administered (e.g., purified toxin vs. crude extract vs. contaminated feed), the vehicle used, the route and regimen of administration, the numbers and types of animal (species, sex, age, disease status) used, the endpoints examined, the existence of data gaps, and the possibility of multiple plausible interpretations of available data. Although purified toxin is the preferred form of toxin for most experimental studies, limitations on the amount of toxin available (especially when working with larger species) often require the use of naturally contaminated feed, culture material (grain that has been inoculated with a known toxin-producing fungal strain), or semipurified toxin. Culture materials and naturally contaminated feed may contain other known or unknown toxins, and as previously mentioned, determination of the concentration of the toxin of interest may be confounded by binding of the toxin to feed components. Culture material may also have low palatability, which can be due to the taste and smell of the fungus itself, or to changes in the qualities of the feed due to fungal growth, or both. This can reduce food

397

intake, especially when it comprises a significant portion of the diet. When food intake is reduced, due either to unpalatability or to toxic effects in the exposed animal, nutritional deficiencies may result (see Nutritional Toxicologic Pathology, Vol 3, Chap 3). Mycotoxicoses may manifest as acute/ subacute, subchronic, or chronic disease. In addition, there may be effects such as growth suppression, decreased weight gain, increased susceptibility to nutritional disorders, or increased susceptibility to infection due to immunosuppression, that are not as easily identified as due to mycotoxicosis. Mycotoxins may be carcinogenic, mutagenic, or teratogenic. As can be seen in Table 6.3, mycotoxins can affect virtually all organ systems and all species; however, each mycotoxin group has limited toxicological targets with distinctive patterns though these are usually not pathognomonic (Bennett and Klich, 2003; CAST, 2003). Conversely, there is no one syndrome consistent with exposure to mycotoxins as an overall group. The manifestation of toxicity depends on the specific mycotoxin, including exposure dose and time; the species exposed, including age and physiological status; and other factors such as exposure to multiple mycotoxins (not covered in this chapter) and the presence of masked mycotoxins that are not detectable by standard analysis but that can contribute to the exposure. The ability of intestinal and ruminal microflora to metabolize mycotoxins has been recognized for decades (Kiessling et al., 1984; Swanson et al., 1988), but the complexity of mycotoxin–gut microbiome interactions is just beginning to be recognized. After ingestion, mycotoxin biotransformation by microbes in the intestinal tract and/or rumen may lead to detoxification and reduced bioavailability. Gut microflora may contribute to the hydrolysis of masked mycotoxins back to their toxic parents, thereby increasing mycotoxin bioavailability. The extent to which this occurs for different mycotoxins and for different species is largely unknown. Mycotoxins may also impact gut health by exerting antimicrobial effects on the gut microbiome (Guerre, 2020; Liew and Mohd-Redzwan, 2018). For additional information on the gut microbiome (see Digestive Tract, Vol 4, Chap 1).

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TABLE 6.3 Mycotoxins Classified as to Target Organ Toxicity Specific agent(s)

Species affected

Time of onset

Usual duration of toxicosis (if survive)

A. NERVOUS SYSTEM

1. Mycotoxins associated with central nervous system (CNS) stimulation or seizures Tremorgenic mycotoxins (e.g., penitrem, roquefortine, lolitrem B, paspalitrems)

All domestic species, especially dogs, cattle, sheep, horses

Minutes to days

One day to weeks; often lethal in dogs

2. Mycotoxins with mixed effects on the CNS Ergot alkaloids

Humans

Minutes to days

Days; sometimes lethal

Fumonisins

Horses

Days to months

Permanent damage likely in survivors; often lethal

Diplonine

Cattle, sheep

2e5 days

Few days, reversible

Cattle, horses, sheep

Hours

Up to 3 days; rarely lethal

3. Muscarinic agonists Slaframine

4. Mycotoxins that cause paralysis (may eventually include respiratory paralysis) Lolitrem B

Sheep, cattle, sometimes horses

Chronic

Chronic; rarely lethal

Citreoviridin

Cattle

Importance in the field is not well established

Patulin

Cattle

Toxicosis is very rare

B. HEART

Citreoviridin (rare) Moniliformin

Poultry

Fumonisin

Pigs, horses

Days

Fusaric acid

Rabbits, rats, cats, dogs, humans (E)

Hypotensive effect

Ochratoxins

Swine, poultry

Days to weeks

Days to permanent damage; often lethal in poultry

Citrinin (may potentiate effect of ochratoxins)

Swine

Fumonisins

Rabbits, lambs, calves, rats, and mice (E)

Days to weeks

Days to weeks to permanent damage in rodents

Cyclosporine

Humans (immunosuppressive use) Days to chronic

Weeks to permanent damage, potentially lethal

Acute form lethal

C. KIDNEY

D. LIVER

Aflatoxins

Most species, humans; trout, ducklings, and young poultry are highly susceptible

(Continued)

399

1. INTRODUCTION

TABLE 6.3

Mycotoxins Classified as to Target Organ Toxicitydcont’d Usual duration of toxicosis (if survive)

Specific agent(s)

Species affected

Time of onset

Sterigmatocystin

Most species

Weeks to chronic

Weeks to months; toxicoses very rare

Rubratoxins A and B

Chickens; possibly cattle and swine

Days to chronic

Unknown; toxicoses rare

Sporodesmin

Cattle, especially sheep

Chronic

Chronic; primarily Australia, New Zealand

Penicillic acid

Swine

Chronic

Chronic; toxicoses very rare

Fumonisins

All species (evidence not available for humans)

Days

Weeks; may be lethal in horses

3-Substituted furans, e.g., ipomeanol

Ruminants

Hours to days

Days; often lethal

Fumonisins

Swine

Days

Usually lethal

Stachybotrys toxins, e.g., satratoxins

Humans, horses, cattle, pigs, sheep, poultry (when inhaled)

Days to weeks

Days to weeks, permanent reproductive damage rare

Acute (Clavicep s sp.)/chronic (fescue)

Chronic

Cyclopiazonic acid (rare) E. RESPIRATORY SYSTEM

F. IMMUNE SYSTEM

Aflatoxins

All species

Trichothecenes

All species

Fumonisins

Pigs, rodents (E)

Ochratoxin A

Pigs (E)

G. CARCINOGENIC MYCOTOXINS

Aflatoxins

Most species, trout, humans (liver)

Ochratoxin A

Mice, rats (kidney and urinary tract) (E)

Fumonisins

Rat (kidney and liver), mouse (liver) (E)

Sterigmatocystin

Rat (liver) (E)

H. ENDOCRINE, REPRODUCTIVE, AND MAMMARY GLAND

1. Mycotoxins that are estrogenic Zearalenone, zearalenol

Swine, cattle, sheep

Ochratoxin A

Rodents (E)

2. Other mycotoxins that affect reproduction Ergot alkaloids

Cattle, horses

(Continued)

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TABLE 6.3 Mycotoxins Classified as to Target Organ Toxicitydcont’d Specific agent(s)

Species affected

Time of onset

Usual duration of toxicosis (if survive)

3. Mycotoxins that affect the mammary gland or lactation Ergot alkaloids

Swine, cattle, horses

Days to months

Days to months; rarely lethal

Zearalenone

Swine, cattle

Days to weeks

Days to weeks (not lethal)

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Deoxynivalenol (DON)

Swine, cattle, dogs, and poultry

T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), and other trichothecenes

Cattle, swine, small animals, poultry, humans

Patulin

Rodents, poultry (E)

J. SKIN

T-2 toxin (dermal exposure)

All species

K. PERIPHERAL VASCULATURE (MAY CAUSE SLOUGHING)

Ergot alkaloid (gangrenous ergotism)

Cattle, sheep

Days to weeks

Weeks; potentially lethal

Ergot alkaloids in tall fescue (fescue foot)

Cattle

Weeks to months

Weeks to months

E, experimental. Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table 3, pp. 648–650, with permission.

Mycotoxins continue to attract worldwide attention because of their real and perceived impact on human health, the economic losses accruing from condemned foods and decreased animal productivity, the costs of quality control and monitoring, and the serious impact of mycotoxin contamination on internationally traded commodities. Although preharvest control of mycotoxin production is difficult, much effort has been expended to develop fungal-resistant crop strains by both breeding and direct genetic modification. Biocontrol technology, in which a nontoxigenic organism competes with a toxigenic fungus for a specific ecological niche (competitive exclusion technology) or otherwise inhibits fungal growth, is being developed. Postharvest control of mycotoxin production is aimed primarily at effective drying and storage regimens. Approaches utilized to limit exposure,

bioavailability, and toxicity of mycotoxins in foodstuffs for animals include the identification and segregation of contaminated material, use of chemical sorbents as sequestering agents, and chemical destruction (detoxification). The addition of various mycotoxin adsorbents to feed is a current area of interest, as are strategies for the removal of mycotoxins from food materials (Agriopoulou et al., 2020; CAST, 2003). Despite the best efforts of the agricultural community, mycotoxins continue to be present in a wide range of foods. In fact, mycotoxins may increase in the food supply due to ongoing climate changes that contribute to fungal colonization and toxin production on crops, feeds, and foods. Mycotoxins will continue to be a threat to human and animal health and food security worldwide (see Environmental Toxicologic Pathology and Human Health, Vol 3, Chap 1; Food and Toxicologic Pathology, Vol 3, Chap 2), and this chapter

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2. AFLATOXINS

addresses the most important mycotoxins that cause significant disease in animals and/or humans. Table 6.4 summarizes the exposure sources, species affected, manifestations, and mechanisms.

2. AFLATOXINS 2.1. Source/Occurrence Aflatoxins are a group of carcinogenic furanocoumarins mainly produced by Aspergillus flavus and Aspergillus parasiticus. These saprophytic fungi are distributed worldwide and are common in the southeastern United States, southern Asia, and Africa, where warm subtropical climates are conducive to fungal growth. These fungi reside in the soil, but under favorable conditions invade the host plants by various routes. Heat, drought, and insect damage to the plants are additional factors favoring infection and aflatoxin production in the field. Fungal growth and aflatoxin production may also begin or, if infection has been established in the feed, continue after harvest in grain that has not been properly dried or is otherwise improperly stored. Aflatoxins can be produced under conditions of 85% relative humidity (e.g., corn moisture content of 15%–28%) and temperatures over 25
C that persist for over 48 h. Under these or other improper conditions, aflatoxin concentrations can become extreme; therefore, proper grain drying and storage are of great importance to minimize hazards to animals and associated economic losses (Mannaa and Kim, 2017; Rushing and Selim, 2019; Smith et al., 2019). Corn, peanuts, cottonseed, and tree nuts are common aflatoxin sources; however, aflatoxin also may be found in other foodstuffs such as wheat, rice, copra, figs, some spices, eggs, and milk (Rushing and Selim, 2019). Meats and poultry are not significant sources of aflatoxin; however, aflatoxin M1 (a metabolite of aflatoxin B1) and other residues can be found in the muscle of exposed animals. The presence of aflatoxin M1 in milk is of concern as a potential exposure source to infants and children consuming milk, cheese, and other dairy products (Becker-Algeri et al., 2016). Aflatoxins were first identified as the causative agent of an acute and fatal disease in turkeys,

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called Turkey X disease. This outbreak occurred in England in 1960 and affected over 100,000 birds that were fed moldy groundnut (peanut) meal from Brazil. Since then, the aflatoxins, especially aflatoxin B1, have been the most thoroughly studied mycotoxins, both in the laboratory and epidemiologically in humans (Pitt and Miller, 2017). Although classically regarded as hepatotoxins and hepatocarcinogens, aflatoxins also exert adverse effects on other tissues, most notably the kidneys and the hematopoietic and immune systems. Exposure has been associated with impaired growth and malnutrition in neonates and children (IARC, 2015).

2.2. Toxicology The European Food Safety Agency (EFSA; https://www.efsa.europa.eu/en) has extensively reviewed the toxicology of aflatoxins and their recent document should be accessed for additional information (EFSA et al., 2020a). Toxin At least 13 aflatoxins have been identified. Aflatoxins B1, B2, G1, and G2 are the most common, with aflatoxin B1 being the most studied because of its importance as a potent toxin, mutagen, and carcinogen (Figure 6.1). The designations B and G refer to bluish (B) or greenish (G) fluorescence that is emitted under ultraviolet light in peanuts, corn, and other commodities. Aflatoxins G1 and G2 are produced by Aspergillus parasiticus. Aflatoxins B2, G1, and G2 are less potent than aflatoxin B1, and the metabolites M1 and M2 are considerably less potent than their precursors (Kensler et al., 2011; Rushing and Selim, 2019). Species Susceptibility Aflatoxin B1 is toxic to some degree in all domestic and experimental animals, although significant differences in sensitivity occur. Among agriculturally important mammals, pigs are generally more sensitive than cattle, which in turn are more sensitive than sheep. Ducklings and turkey poults are more sensitive than quail or chicks. Young animals are, as a rule, more susceptible than adults. Adult mice are less sensitive than neonates as well as adults of other laboratory species including

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TABLE 6.4

Major Mycotoxins, Sources, Species, Manifestations, and Mechanisms Primary pathology or manifestation

Main fungal producer(s)

Exposure source (ingestion)

Species affected

Acute

Chronic

Mechanism(s)

Aflatoxins, especially B1

Aspergillus spp., e.g., Aspergillus flavus, Aspergillus paraciticus

Corn, peanuts, cottonseed, tree nuts, contaminated pet food

Humans, cattle, pigs, dogs, horses, birds, fish, all susceptible

Liver: Zonal necrosis, lipidosis, biliary hyperplasia

Liver: Megalo cytosis, biliary fibrosis, cirrhosis, cancer

Electrophile, cytochrome P450 bioactivation, DNA and macromolecule adducts

Fumonisins, especially B1

Fusarium Corn verticillioides, Fusarium proliferatum

Horses, pigs, all laboratory animals (mammals) susceptible

Liver: Apoptosis, necrosis

Mycotoxin

Pigs

Ochratoxins, especially A

Penicillium ochraceus Grain, grapes, and Aspergillus coffee, pork verrucosum products

Experimental: rat, rabbit, lamb, calf

Kidney: Tubular Kidney: Cancer (rats) epithelial apoptosis

All species susceptible, pigs most commonly exposed

Kidney: Tubular degeneration/ necrosis, proximal tubules

Kidney: interstitial fibrosis (pig, ?human); cancer (rodents, poultry)

Cytochrome P450 bioactivation, free radical production, induction of oxidative stress; inhibition/ disruption of mitosis; proapoptotic; induction of cell cycle arrest

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Equids: horses, donkeys

Liver: fibrosis (pigs, Disruption of horses); neoplasia sphingolipid (rodents) metabolism and function; induction of oxidative stress, Lung edema due to Liver: fibrosis activation of ER cardiotoxicity stress and MAPKs, Central nervous Liver: fibrosis modulation of system: Vasogenic autophagy, alteration edema, leukoof DNA methylation encephalomalacia (ELEM)

Deoxynivalenol

Fusarium graminearum, Fusarium culmorum

Grain, especially wheat and barley

Pig most sensitive, all susceptible

No specific pathology. High dose, emesis; lower dose, feed refusal

T-2 toxin

Fusarium sporotrichioides, Fusarium poae

Grain, especially barley and wheat

Pig and cat most sensitive, all susceptible

Infection due to Inhibit protein Systemic: immune suppression synthesis and Hematopoetic, mitochondrial lymphoid, gastrointestinald function, activate apoptosis, necrosis. MAPKs, alter Local: Skin, oral neurotransmitters mucosadirritation. ATA (human)

Macrocyclic trichothecenes

Stachybotrys, Myrothecium spp.

Straw, hay (ingestion, contact)

Human, horse, (stachybotryo toxicosis)

Systemic: Hematopoetic, lymphoid, gastrointestinald apoptosis, necrosis. Local: Skin, oral mucosadirritation

Stachybotrys chartarum

Water damaged buildings (inhalation)

Human; experimental: monkey, laboratory animal

Respiratory, central nervous system. “Sick building syndrome”

Fusarium graminearum

Corn, wheat

Human, pig most sensitive, all presumed susceptible

Reproductive and mammary gland/ breast: estrogenic effects

Hormonal and cytokine dependent dysregulation of appetence/satiety involved

Assume similar to T-2 toxin

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Zearalenone

Reduced feed intake and weight gain, immune dysregulation (all species likely); IgA glomerulonephritis (mouse, ? human)

Endocrine disruption: Acts on estrogen receptors a and b (Continued)

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TABLE 6.4

Major Mycotoxins, Sources, Species, Manifestations, and Mechanismsdcont’d Primary pathology or manifestation

Mycotoxin

Main fungal producer(s)

Exposure source (ingestion)

Ergot alkaloids

Claviceps spp.

Grain, especially rye All species susceptible

Fescue grass

Cattle and horses mainly

Acute

Chronic

Ergotism. Gangrene Similar to acute if lower exposure dose (vascular smooth muscle hyperplasia/ hypertrophy), hyperthermia, reproductive (abortion, agalactia), neurologic and enteric syndromes

Mechanism(s) Endocrine disruption due to neurohormonal effects: Agonist/ antagonist action on adrenergic, dopaminergic and serotonergic receptors resulting in vasoconstriction and uterine smooth muscle contraction

Endocrine disruption Fescue toxicosis. and vasoconstriction Cattle: gangrene as above; D2 (fescue foot), hyperthermia with dopaminergic temperature stress. agonism results in Horses: reproductive decreased prolactin leading to agalactia (delayed parturition, agalactia). Vascular smooth muscle hyperplasia/ hypertrophy in bovine peripheral tissues and equine placenta

ATA, alimentary toxic aleukia. Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Table 39.4, pp. 1192–1194, with permission.

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Ergot alkaloids, Neotyphodium mainly ergopeptine coenophialum alkaloids

Species affected

2. AFLATOXINS

O

H

O

O

O H

O

O

CH3

Aflatoxin B1 FIGURE 6.1 Chemical structure of aflatoxin B1. Figure reproduced from Handbook of Toxicologic Pathology, third Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Figure 39.1, p. 1198, with permission.

rats, guinea pigs, and rabbits (Newberne and Butler, 1969; Rawal et al., 2010). Aflatoxin B1 induces hepatocellular carcinoma in rats (Newberne and Wogan, 1968) whereas adult mice are resistant. However, lung tumors have been induced in mice following aflatoxin B1 administration (Weider et al., 1968). Trout have been shown to be very sensitive to the hepatocarcinogenic effects of aflatoxin and have served as a model for large-scale carcinogenicity studies (see Animal Models in Toxicologic Research: Nonmammalian, Vol 1, Chap 22) (Williams, 2012). More recently, zebrafish have been used as a disease model for studying human hepatocellular carcinoma (Lu et al., 2015). Biodistribution, Metabolism, and Excretion Aflatoxins B1, G1, and others are procarcinogens subjected to both Phase I and Phase II metabolism in the liver and other tissues. Cytochromes P450 (CYP) including CYP1A2, CYP3A5, and CYP3A4 are responsible for their Phase I metabolism in mammals. Phase I metabolism leads to formation of less toxic molecules as well as more reactive, electrophilic species that readily react with cellular macromolecules. For example, CYP1A2-catalyzed conversion of aflatoxin B1 to aflatoxin M1 results in formation of a 10 times less potent carcinogen. Aflatoxins P1 and Q1 are further examples of less toxic oxidative metabolites (see Pharmacodynamics and Toxicodynamics, Vol 1, Chap 5) (Deng et al., 2018; Kensler et al., 2011; Rushing and Selim, 2019).

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Aflatoxin B1 is bioactivated by its conversion to exo-aflatoxin B1-8,9-epoxide which, like other bioactive epoxides, undergoes further reactions, including Phase II glutathione-S-transferase mediated conjugation to glutathione and subsequent urinary excretion as aflatoxin–mercapturic acid, conversion to dihydrodiols or dialdehydes, and covalent binding to macromolecules. Covalent binding of aflatoxin epoxide to DNA occurs, resulting in adduct formation. The N7 sites of guanine nucleosides are particularly susceptible. The aflatoxin–N7 guanine adduct, which is a depurination product formed during DNA repair, can be found in blood and urine and has proven to be an extremely useful biomarker of recent exposure in epidemiological and other investigations. Aflatoxin–albumin adducts found in serum and aflatoxin–mercapturic acids in urine are additional metabolic products that are also useful biomarkers of exposure (Turner et al., 2012). Differences among species and gender in susceptibility to aflatoxicosis are explained in part by differences in metabolism rates and the amounts and types of Phase I and Phase II metabolites formed. For example, the relative insensitivity of adult mice to hepatocarcinogenicity can be attributed to their significantly higher levels of glutathione-S-transferase activity compared to rats and other more sensitive species. However, cytosolic Phase II metabolism of aflatoxin is significantly less efficient in humans than in mice. The importance of Phase II metabolism for detoxification is illustrated further by the protective effects observed in animals treated with agents such as Oltipraz (a dithiolethione) that induce glutathione-S-transferase activity (Degen and Neumann, 1981; Eaton and Gallagher, 1994; Kensler et al., 2011). Mechanism of Action Aflatoxins are acutely hepatotoxic. The underlying mechanisms are nonspecific interactions between aflatoxins or their activated metabolites and various cell proteins, leading to disruption of basic metabolic processes and protein synthesis that, in turn, can cause cell death. Aflatoxins are mutagenic and genotoxic. The correlation between aflatoxin exposure and the appearance of N7 guanine–aflatoxin adducts in urine has been repeatedly demonstrated. After DNA adduction, mutations occur during DNA repair or replication and, if involving critical

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genes, they can significantly alter cell functions. One example with implications for human health is the high correlation between aflatoxin exposure and a characteristic point mutation found at the third base of codon 249 of the TP53 tumor suppressor gene. This mutation, a transversion of guanine to thymidine (AGG to AGT), is present at relatively high frequency in Chinese and African liver cancer patients. A characteristic mutational signature of aflatoxin-induced hepatocellular carcinoma that includes the TP53 mutation and other novel mutations has been identified in aflatoxin-induced hepatocellular carcinoma cases in a high-risk community in Qidong, China (Gouas et al., 2009; Woo et al., 2011; Zhang et al., 2017). In addition, epigenetic modifications including DNA methylation, histone modifications, and regulation of noncoding RNA may play an important role in aflatoxin B1–induced disease and carcinogenesis (Ferreira et al., 2018). There is a body of evidence that other factors, along with exposure to aflatoxins, contribute to the increased risk of development of hepatocellular carcinoma. In this regard, the role of hepatitis B as a comorbidity for aflatoxicosis has gained much attention, as infection with hepatitis B virus is common in areas where aflatoxin exposure is high, such as Africa and Southeast Asia. Some epidemiological studies using hepatitis B surface antigen as a biomarker for viral exposure indicate that carcinogenesis is likely related to an interaction between hepatitis B virus and aflatoxin. For example, results of a cohort study in China indicated that the relative risk of liver cancer was significantly greater among men who were positive for both urinary N7 guanine adducts and serum hepatitis B surface antigen than in men having only the viral surface antigen. Nutritional considerations and oxidative damage to DNA and other macromolecules caused by aflatoxininduced lipid peroxidation also may play an important mechanistic role (El-Serag, 2012; Liu et al., 2012; Williams et al., 2004).

2.3. Manifestations of Toxicity in Animals Overview The aflatoxin literature is extensive, more so than for any other mycotoxin. A comprehensive treatment of the subject is therefore beyond the scope of this chapter. The reader is referred to the extensive reviews of the toxicity and pathology of aflatoxins, especially the hepatic

histopathology, by Kensler and colleagues and by others (EFSA et al., 2020a; Kensler et al., 2011; Newberne and Butler, 1969). Acute aflatoxin poisoning occurs less commonly than chronic aflatoxicosis. However, acute toxicosis does occur in humans, especially in Africa, and periodically in dogs in the United States due to contaminated corn-based dog food. The principal target organ is the liver, with hepatocellular fatty change, degeneration, and necrosis. Loss of liver function results in icterus, decreased synthesis of serum proteins (hypoproteinemia), and coagulopathy, due to decreased synthesis of clotting factors. Coagulopathy can lead to extensive hemorrhaging and anemia. Clinical signs of chronic aflatoxicosis are nonspecific, but biochemical evidence of hepatocyte and biliary damage can be obtained from serum. Chronic intoxication is associated with decreased weight gain or weight loss, decreased food consumption and conversion, and decreased reproductive performance, including abortion. Laying hens exhibit reduced egg production, and milk production of cows declines. Affected animals have increased susceptibility to infection, presumably due to immunosuppression: aflatoxins have an adverse effect on cell-mediated immunity, principally by affecting the reticuloendothelial system, macrophages, and T cells (Kemboi et al., 2020; Rawal et al., 2010). Although affecting other tissues, aflatoxins usually are thought of in terms of hepatotoxicity and hepatocarcinogenicity (see Liver, Vol 4, Chap 2). There is a reasonable degree of similarity in the type of liver lesions seen in different species, both on the gross and microscopic levels and in accompanying clinical pathology changes. Some variability exists, for example, the distribution of hepatic necrosis tends to be centrilobular in some species (guinea pig, dog, pig, cattle) and more periportal in others (rat, poultry, cat). Qualitatively, the lesions caused by the various aflatoxins appear similar, although there is a difference in potency: aflatoxin B1 is more potent than aflatoxin G1 and far more so than aflatoxin M1 (Newberne and Butler, 1969). Grossly, the liver has been described as enlarged, swollen, or fatty; it tends to be pale with gray to yellow or orange discoloration (Figure 6.2). Congestion or petechial hemorrhages are sometimes evident. The texture is variable, and may be firm, fibrous, friable, or fatty, particularly in chickens and dogs. The gall bladder may be enlarged and turgid with

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FIGURE 6.2 Liver from a dog fed aflatoxin-contaminated pet food. The liver is swollen and yellow because of hepatocellular lipidosis. The gall bladder is distended. Photograph courtesy of Dr B. Summers, Cornell University. Figure reproduced from Fundamentals of Toxicologic Pathology, second Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), Figure 9.11A, p. 215, with permission.

mucosal hemorrhage. Splenic or renal enlargement, hydrothorax, hydropericardium, or ascites are also reported (Newman et al., 2007). Microscopic lesions caused by acute or subchronic exposure in most species include hepatocyte degeneration, necrosis, hepatocellular vacuolation (fatty change), cellular pleomorphism with variability in cell (anisocytosis) and nuclear (anisokaryosis) size, bile duct or oval cell proliferation (Figure 6.3), and nodular regeneration, which may progress to cirrhosis or cancer. Hepatic lesion distribution is not consistent from species to species, and may be primarily periportal (rats, ducklings), centrilobular (guinea pigs, swine), periportal and centrilobular (dogs), or midzonal (rabbits) (Newberne and Butler, 1969). The predominance of individual findings such as bile duct proliferation, distribution of necrotic hepatocytes, degree of lobular architectural disruption, and time course of lesion development also vary by species and mode of exposure. Laboratory Animals Rats are sensitive to the acute hepatotoxic effects of aflatoxin, but less so than guinea pigs and more so than mice. Lesions are progressive

FIGURE 6.3 Chronic aflatoxicosis; liver from a dog fed aflatoxin-contaminated pet food. H&E stain. (A) Hepatocytes are pale due to cytoplasmic vacuoles (lipid) except for a focal area of hepatocellular hyperplasia (lower right). Bile duct proliferation is noted in periportal areas (arrows). A centrilobular vein is present in the center of the field. (B) Higher magnification demonstrates bile duct hyperplasia and hepatic lipidosis. Multiple bile ductules (arrows) extend from the portal area. Adjacent hepatocytes contain variably sized cytoplasmic vacuoles (lipid). Figure reproduced from Handbook of Toxicologic Pathology, third Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Figure 39.3, p. 1199, with permission.

in rats given a single dose at the median lethal dose (LD50) concentration. Shortly after dosing, there is periportal degeneration and necrosis of hepatocytes with biliary and oval cell proliferation. Later, hepatocytes become more pleomorphic with nuclear hyperchromasia and differences in nuclear size and shape.

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Regeneration becomes more obvious until overt nodular regeneration is present. Fibrosis may not be extensive, and hepatocellular tumors, which morphologically resemble those routinely induced by other rodent hepatocarcinogens, develop in the absence of cirrhosis. Similar nonneoplastic lesions are found in mice, guinea pigs, and rabbits; however, hepatic necrosis in guinea pigs is typically centrilobular and in rabbits is centrilobular to midzonal (Kensler et al., 2011; Newberne and Butler, 1969). Poultry Poultry, particularly ducklings, are sensitive to aflatoxins with effects on the liver, leading to decreased growth, as well as decreased egg production and immune response (Yang et al., 2020). As in mammals, biliary hyperplasia is a predominant histologic feature. Hepatocellular degeneration and necrosis occur. Necrotic cells may be scattered throughout the hepatic lobules or, more commonly, are found in the periportal zone. In ducklings, periportal hepatocellular necrosis may be accompanied by the development of lesions described in the literature as “lakes of fat,” representing intracellular lipid accumulation. Biliary hyperplasia and nodular regeneration become pronounced, and fibrosis progresses to cirrhosis (Arafa et al., 1981; Newberne and Butler, 1969). Livestock Aflatoxicosis is a problem in livestock, most notably swine and cattle (Yang et al., 2020). Young pigs are especially sensitive to acute exposures. Gross lesions include hepatic enlargement, congestion, yellow discoloration, and friability; petechiae or more generalized hemorrhage; and edema and ecchymotic or petechial hemorrhages of the gall bladder. Microscopic findings depend upon the dose and duration of exposure but are typical of aflatoxicosis, with hepatocellular degeneration and necrosis in the centrilobular zone. Cattle are more resistant than pigs, but the typical lesions of aflatoxicosis, as described above, can be found following exposure. Fibrosis and bile duct proliferation may be extensive and found together with sinusoidal obstructive syndrome (previously termed veno-occlusive disease) of the central veins. Sheep are relatively resistant to aflatoxin (Newberne and Butler, 1969). Chronic low-level exposure can lead to decreased growth and adverse effects on reproduction and the immune system (Yang et al., 2020).

Other Species Other species, including nonhuman primates, have shown varying degrees of sensitivity to aflatoxin and develop lesions of the type described above, particularly hepatocellular degeneration and necrosis, and biliary proliferation that progress to nodular cirrhosis. Dogs are quite sensitive, presumably in part due to low hepatocellular glutathione levels as compared to other species. Outbreaks of toxicoses associated with commercial pet food made with corn contaminated by high levels of aflatoxins are reported periodically in the United States. As recently as 2020–2021 a recall was issued by a prominent pet food manufacturer for all pet foods containing corn. In this incident, more than 130 dogs died and more than 220 were sickened after eating the contaminated pet food (FDA, 2021). Acute exposure to aflatoxin results in jaundice and liver and gall bladder lesions. Grossly, the livers can be enlarged and yellow (see Figure 6.2). Microscopic findings are consistent with those found in other species and include centrilobular hepatocyte necrosis, lipid vacuolation, and biliary hyperplasia (see Figure 6.3). Clinical findings in cases of acute or subacute exposure include elevated liver enzymes, hyperbilirubinemia, hypoproteinemia, hypocholesterolemia, and reduced clotting activity (Dereszynski et al., 2008). Aflatoxin toxicity is not confined to mammalian and avian species, but extends to fish, with trout being particularly sensitive. The acute and subchronic effects in fish include hemorrhage and hepatocyte necrosis. Biliary proliferation, regenerative nodules, and hepatocellular carcinoma are common findings after prolonged exposure (Bailey et al., 1996). Liver Cancer in Laboratory Animals The lesions associated with aflatoxin hepatocarcinogenesis were thoroughly studied and reported in the late 1960s and early 1970s (Newberne, 1973; Newberne and Butler, 1969). These investigations not only brought attention to the carcinogenic potential of aflatoxins, but also established mycotoxins as important environmental toxins. The morphology of hepatic neoplasms induced by aflatoxins is like those induced by other well-characterized carcinogens. However, in contrast to tumors induced by some other compounds, aflatoxin-induced hepatomas and hepatocellular carcinomas can arise in livers that are not cirrhotic.

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The sequence of lesions that occur in rats fed aflatoxin B1 (1 ppm in the diet) was described in detail by Newberne and Wogan (1968). In the early stages, there is bile duct proliferation, and foci of altered hepatocytes consisting of intensely stained cells with small nuclei or, alternatively, larger cells with clear to lightly staining cytoplasm. With continued exposure, hyperplastic nodules, hepatomas, and overt carcinomas develop. The hyperplastic nodules contain well-demarcated collections of hepatocytes undergoing mitosis or fatty change that compresses the surrounding parenchyma. Carcinomas range in appearance from well differentiated, with cells arranged in trabeculae, to poorly differentiated types. In the latter, cells display varying degrees of anaplasia and may be arranged in sheets, cords, cysts, or ductlike structures. The tumors invade the adjacent parenchyma and vasculature, and frequently metastasize to the lung. Cholangiofibromatous lesions and cholangiocarcinomas are rare (Newberne and Wogan, 1968).

2.4. Human Risk and Disease The aflatoxin literature and dietary exposure to aflatoxins in Europe was extensively reviewed during a series of risk assessments performed for the European Union (EFSA et al., 2020a). Acute aflatoxicosis following the ingestion of highly contaminated food continues to be documented in various locations, particularly in Africa and southeastern Asia. Diagnosis in humans is difficult, as symptoms are not specific for aflatoxin. Detection of high concentrations of aflatoxin in food, and clinical signs, such as anorexia, diarrhea, malaise, or depression, can be indicative of acute aflatoxicosis. Death may occur. Hepatobiliary involvement is indicated by jaundice, ascites, or tenderness when pressure is applied to the upper abdomen. Histopathological findings consistent with aflatoxin-induced injury, such as fatty infiltration and centrilobular necrosis, have been reported following acute outbreaks of suspected human aflatoxicosis (Benkerroum, 2020; Gong et al., 2012; IARC, 2015). Severe outbreaks of acute aflatoxicosis have occurred from time to time. In India, over 100 fatalities occurred in 1974 when unseasonable rain and a scarcity of food resulted in

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consumption of corn that was heavily contaminated with aflatoxin. Kenya has had periodic outbreaks of aflatoxicosis since 1972, including the severe episode of January through July 2004 that involved 317 known cases and claimed the lives of 125 individuals. In one survey, aflatoxin levels of up to 58 ppm were found in corn samples from the outbreak area and concentrations exceeding 1 ppm were found in more than half of corn samples that were associated with illness. While cases were associated mostly with consumption of homegrown corn, aflatoxin contamination in corn sold at local markets was also extensive and contributed to exposure: more than 50% of the market samples exceeded the Kenyan regulatory standard of 20 ppb. Additional cases of acute aflatoxicosis in Kenya were identified in 2005 and in Tanzania in 2016 (Azziz-Baumgartner et al., 2005; IARC, 2015; Kamala et al., 2018; Williams et al., 2004). Aflatoxin B1 has been classified as a Group I human carcinogen by IARC, with hepatocellular carcinoma being the major concern. Liver cancer is an emerging global health issue with Guatemala experiencing the highest rates of this disease in the Western Hemisphere (Smith et al., 2017). While there is considerable uncertainty due to methodological limitations, it has been estimated that aflatoxin is involved in up to 155,000 cancer cases worldwide, corresponding to as much as 28% of all cases of liver cancer per year. The association between aflatoxin and liver cancer has been a focus of intense epidemiologic investigation in the developing world, particularly in China and various sub-Saharan African countries where high liver cancer incidence is found (Claeys et al., 2020). Significant exposure in these regions, as well as in Guatemala, has been unequivocally demonstrated using aflatoxin–lysine adducts in serum and aflatoxin B1–N7guanine adducts as biomarkers. Recent data from Qidong, China, show a causal link between aflatoxin exposure and liver cancer, with a decreasing liver cancer mortality rate associated with a decrease in aflatoxin–lysine adducts (Chen et al., 2021). However, nutritional and other conditions, such as viral hepatitis (HBV) or parasite infection, frequently exist in affected human populations, which confound the situation (Kensler et al., 2011; Liu and Wu, 2010).

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Of particular interest is exposure to hepatitis B virus, which is now generally accepted to be a potentially important cofactor, as well as hepatitis C virus. Epidemiological studies have yielded mixed results: some suggest that aflatoxin acts independently, as shown in Guatemala, some correlate liver cancer with hepatitis B or C virus exposure, and still others show an interaction between them. Studies in China and Thailand, in which biomarkers were used to assess exposures, indicate a strong likelihood that both aflatoxin B1 and hepatitis B virus are involved and that an interaction between the two contributes to risk. This is supported by the findings of an in-depth review and metaanalysis of multiple epidemiological studies on aflatoxin-related liver cancer risk: (1) aflatoxin is by itself a significant risk factor and (2) in regions where aflatoxin exposure is high and chronic hepatitis B virus is prevalent, the two factors work synergistically or “multiplicatively” to increase cancer risk. Other hepatotoxic mycotoxins, such as fumonisin, are often also found in countries such as Guatemala. The study in Thailand showed the strongest correlation with hepatocellular carcinomas occurring without concurrent cirrhosis (El-Serag, 2012; Liu et al., 2012). The recent assessment by EFSA et al. (2020a) of human risk due to aflatoxin exposure in food was based on a benchmark dose lower confidence limit (BMDL) for a benchmark response of 10% of 0.4 mg/kg bw/day for the incidence of hepatocellular carcinoma due to aflatoxin B1 exposure in male rats. Based on this BMDL10, the calculated margin of exposure (MOE) values for aflatoxin B1 across some dietary surveys and age groups were less than 10,000, which raised a human health concern. For aflatoxin M1, based on the BMDL10 for aflatoxin B1 and a potency factor of 0.1, calculated MOE values were less than 10,000 for some surveys, particularly for the younger age groups, which also raised a health concern (EFSA et al., 2020a). There is increasing evidence that other human health outcomes are associated with aflatoxin exposure, including growth impairment and immune disorders. Stunting of growth is associated with poor health in later life, including

decreased resistance to infection. Both crosssectional and longitudinal epidemiological studies in West Africa revealed significant correlations between growth impairment and serum aflatoxin–albumin adduct levels in young children. Transition to solid food was a critical contributor to exposure, as significantly greater adduct levels were found in fully weaned compared to partially weaned children. Prenatal exposure might also be critical and, in this regard, high levels of aflatoxin–albumin adduct in maternal blood during pregnancy have been associated with weight and height deficits in children during the 4 months following birth. While implicating aflatoxins, the issue is not resolved and any physiological mechanism by which aflatoxin, directly or indirectly (in conjunction with other risk factors), affects growth remains to be elucidated. Secondly, the aflatoxin findings might be serving as a surrogate marker for other conditions that impact growth, such as a vitamin or other nutrient deficiency, altered gut microflora composition, or parasite burdens (IARC, 2015; Khlangwiset et al., 2011; Turner et al., 2007; Watson et al., 2017; Wild and Montesano, 2009). The impact of aflatoxin exposure on the immune system is likewise not clear. Results of animal studies vary by species and experimental design but have nonetheless shown inhibition of cellular and humoral immune responses. Targets include T lymphocyte populations, monocytes, and macrophages; accordingly, cytokine and antibody production, as well as proliferation and phagocytic functions, were impaired in some experimental models. The number of studies evaluating aflatoxin and human immunity is limited, but the results do suggest that exposure is potentially detrimental. Among the reported effects associated with high levels of serum aflatoxin–albumin adducts in West African populations are reductions in the number of activated T (CD4þCD69þ) cells, including cytotoxic T cells staining positive for perforin or granzyme, decreased numbers of activated B (CD18þCD69þ) cells, and decreased salivary levels of secretory IgA in children. A preliminary positive correlation between serum aflatoxin–albumin adducts and high tissue burdens of HIV virus has been reported (IARC, 2015; Jiang et al., 2005; Turner et al., 2003).

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2.5. Diagnosis, Treatment, and Control When conditions are favorable for production of aflatoxin, grain elevators often use black light (ultraviolet) to screen commodities. While simple and quick, this test only indicates fungal growth and not toxin presence. All positive ultraviolet findings must be followed up with a specific test used to identify and quantify any aflatoxins present. USDA Grain Inspection, Packers, and Stockyards Administration (GIPSA)-approved test kits are available for both qualitative screening for aflatoxins in corn and quantitative determination of aflatoxin concentrations in corn and other commodities. Qualitative methods give a positive or negative result, with the “cut-off” in the United States being 20 ppb, the actionable limit concentration set by US Food and Drug Administration (FDA) for total aflatoxins in food and nuts for human consumption. Action levels for animal feeds vary from 20 (most species) to 300 ppb (beef cattle, nonbreeding) depending not only on the species but also on physiologic status. Aflatoxin M1, a hydroxylated metabolite of aflatoxin B1, is readily detected in the milk of exposed dairy cattle, and the action level is 0.5 ppb aflatoxin for the sale of milk (BeckerAlgeri et al., 2016; FDA, 2019). Diagnosis of aflatoxicosis in a clinical or field setting should not be based on lesions and clinical pathology findings alone. Confirmation of diagnosis requires direct evidence of aflatoxin exposure, such as the identification of aflatoxin–albumin adducts in serum, the presence of aflatoxin–N7 guanine adducts in the urine, identification of sufficient concentration of aflatoxin(s) in source materials, or, preferably, a combination of the above (Arce-Lopez et al., 2020; Wogan et al., 2012). Treatment of affected animals consists of changing the diet to an aflatoxin-free ration, increasing dietary protein, and supplementing the diet with Vitamin B12, Vitamin K, and selenium. Toxicity can be prevented to some extent by treatment of contaminated foodstuffs. NovaSil clay, a hydrated sodium calcium aluminosilicate (HSCAS), binds aflatoxin. When added to aflatoxin-contaminated feed, NovaSil and some other clays have been shown to decrease aflatoxin absorption and toxicity in swine, poultry, and humans. However, the efficacy of NovaSil clay in preventing residues of aflatoxin M1 in the milk of dairy cows is less than desirable. NovaSil7 has been approved by the FDA only as an anticaking agent at up to 2% of a ration.

Ammoniation of corn to reduce its aflatoxin concentration is a last resort since ammoniated corn turns brown. Ammoniation has been used extensively to detoxify aflatoxin in cottonseed intended for animal consumption. Ammoniation of corn for animal consumption has been approved by some states, but not by the FDA, and interstate shipment of treated corn is illegal. Both NovaSil7 and ammoniation can be used only for animal feed. However, while not approved for humans, NovoSil clay given in capsule form has been tested in a 3-month Phase IIa clinical intervention trial in Ghana. Treatment reduced the levels of aflatoxin– albumin adducts found in the serum compared to the group given placebo, suggesting that NovoSil clay can reduce the bioavailability of aflatoxin found in foods. Finally, the introduction of nonaflatoxigenic Aspergillus strains as competitive organisms in the field has been shown to reduce aflatoxin concentrations in cottonseed and peanuts (Phillips et al., 2019; Udomkun et al., 2017; Wu and Khlangwiset, 2010).

3. OCHRATOXINS 3.1. Source/Occurrence Ochratoxins A, B, and C are secondary metabolites of Aspergillus ochraceous, Aspergillus carbonarius, Aspergillus niger, Penicillium verrucosum, and related species. These ochratoxins as well as ochratoxin methyl esters, ethyl esters, and other analogs have been characterized. Ochratoxins A, B, and C contain a phenylalanine moiety attached to a dihydroisocoumarin group via an amide bond (Figure 6.4). Ochratoxin A is

FIGURE 6.4 Chemical structure of ochratoxin A and phenylalanine. Ochratoxins compete with phenylalanine for binding sites of enzymes. Figure reproduced from Handbook of Toxicologic Pathology, third Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Figure 39.4, p. 1203, with permission.

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most common and important from an animal and human health standpoint. It is nephrotoxic to multiple species, and is a potent renal carcinogen in rodents (Perrone and Gallo, 2017). Citrinin, another secondary metabolite, frequently co-occurs with the ochratoxins, and is also nephrotoxic, though at much higher concentrations. The focus of this section will be on ochratoxin, which is roughly 10 times more toxic and, thus, has a greater potential to cause illness in animals and humans. Humans and animals can be exposed via the diet, though both ochratoxin and citrinin are much lower in processed and baked foods than in raw products (Gupta et al., 2018b). The principal source of ochratoxins is cereal grain, including barley, rye, wheat, corn, sorghum, and oats, as well as coffee beans and grapes, including wine and raisins. They may also occur in other commodities, such as cottonseed, nuts, dried beans, or meats, especially kidney or pork products such as sausages, bacon, or ham. Geographically, ochratoxins are found in regions having temperate climates, with the northern European countries, the Balkans, and Canada being most affected. Optimal conditions for ochratoxin A production are a moisture content of 19%–22% and a temperature of 24
C (Duarte et al., 2011a; Kumar et al., 2020). Ochratoxin concentrations in grains are variable, but can periodically be high enough to cause outbreaks of porcine nephropathy or other animal diseases (Battacone et al., 2010). Ochratoxin A residues have been found globally in human serum, plasma, urine, and milk, indicating that exposure is not limited to the Balkans and northern Europe (Bui-Klimke and Wu, 2015; Duarte et al., 2011b).

3.2. Toxicology Species Susceptibility The toxicities of ochratoxin A and ochratoxin C are similar, although the former is much more commonly encountered. Ochratoxin B is a several times less potent toxin (Heussner and Bingle, 2015). Synergistic or additive effects have been described in animals coexposed to ochratoxin A and other mycotoxins such as aflatoxin, citrinin, and penicillic acid (Grenier and Oswald, 2011). Ochratoxins are potentially hazardous to livestock, and toxicity has been demonstrated in swine, horses, ducklings, chickens, turkeys, and dogs. Cattle are relatively resistant due to

metabolism of the mycotoxin by ruminal microflora (microbiome) (Duarte et al., 2011a). Young animals are in general more sensitive than adults. Older calves (46–69 d old) with functional rumens are less sensitive to orally administered ochratoxin A than 10- to 21-dayold preruminant calves (Sreemannarayana et al., 1988). The kidney is the major target organ in swine and other species, although the liver, immune system, and other organs also may be affected. Ochratoxin A is teratogenic in most species examined, and dose-dependent transfer across the placenta has been demonstrated in rodents. In contrast to rodents, ochratoxin A does not cross the placenta when given to sows at low levels and thus teratogenic effects do not occur (Duarte et al., 2011a). Biodistribution, Metabolism, and Excretion The pharmacokinetics of ochratoxin A in mammals vary depending on species and dose. In general, about 60% of an orally administered dose is absorbed from the gastrointestinal tract of rats and other monogastric animals. In rats, significant amounts bind to plasma albumin, with maximum serum concentrations (Cmax) occurring within 4 h of dosing (Tmax). Binding to serum albumin is also high in cattle, pigs, and humans. The oral plasma half-life (t1/2) varies from about 8 h in rabbits to about 120– 230 h in rats and to slightly over 500 h in monkeys. The t1/2 in pigs is about 90 h, and the maximum plasma concentration (Cpmax) reportedly ranges from 0.50 mg/mL or less in mice and monkeys to as high as 87 mg/mL in rats (Benford et al., 2001). Biliary excretion and enterohepatic circulation occur, and ochratoxins and their metabolites are excreted in urine and feces. The high level of binding to serum albumin and greater retention rates in pigs lead to the accumulation of residues in tissues, most notably the kidneys, where amounts of up to 100 ppb have been documented. Lower levels can accumulate in the tissues of chickens (Duarte et al., 2012). Although the kidney is the primary target, after repeated doses in rats, ochratoxin A is widely distributed to tissues and has also been detected in liver, lung, heart, and testes. Due to extensive binding to serum albumin, glomerular filtration of ochratoxin A is minimal. Toxicity occurs in the renal proximal and distal tubules, where it is reabsorbed. Differences in the relative sensitivity of individual species and sexes

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toward ochratoxin A may be due to variations in transport mechanisms and cellular uptake in renal cells. Ochratoxin A is a substrate for the family of organic anion transporter proteins (OATs) and organic anion transporting polypeptide (OATP) and can regulate their expression in rat kidney cortex or impair their activity. Kidney ochratoxin A uptake and handling by OATs is dependent on the ochratoxin A bound to plasma protein. These specific transporters may also be involved in the accumulation of ochratoxin A in organs other than kidney (Vettorazzi et al., 2014). Ochratoxin A is also excreted into mammalian milk (Ringot et al., 2006). Metabolism varies by species, sex, and strain of animal, occurs in the kidney, liver, and intestines, and is mediated by cytochrome P450s. Hepatic and renal metabolic pathways found in various species, including rat, mouse, rabbit, and monkey, convert ochratoxin A by hydrolysis to the less toxic metabolites, ochratoxin a, and ochratoxin B. Ochratoxin A is also metabolized to ochratoxin a, by carboxypeptidases found in the rumen and intestine; enteric bacteria are the likely source of the enzymes. Various hydroxylated metabolites, a metabolite with an open lactone ring, pentose or hexose mycotoxin conjugates, or other uncharacterized products are also formed (Wu et al., 2011). There is evidence of Phase II ochratoxin A conjugates in human urine samples (Mun˜oz et al., 2017). Conversion to reactive oxygen species (ROS) is also a possibility (see below). Mechanism of Action As a result of their structural similarity to phenylalanine (Figure 6.4), ochratoxins effectively compete with phenylalanine for the binding sites of enzymes that utilize the latter as a substrate. The biological effects of the inhibition of many potential metabolic targets, e.g., the inhibition of phenylalanine hydroxylase, are not known. Ochratoxins do, however, inhibit phenylalanine tRNA synthetase and, as a result, cellular protein synthesis is reduced. Ochratoxins also inhibit mitochondrial respiration, leading to depletion of cellular ATP, disrupted calcium homeostasis, lipid peroxidation, and oxidative damage of macromolecules. Phenylalanine and aspartame (which contains phenylalanine) are antagonistic to ochratoxins, presumably by competing for critical phenylalanine binding sites (Koszegi and Poor, 2016). The molecular mechanism(s) and mode of action underlying the toxicity and carcinogenicity of ochratoxin A, including the critical

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issue of whether ochratoxin A is genotoxic, are not yet clear. The supporting in vitro and in vivo studies have been extensively reviewed by EFSA et al. (2020b). Evidence supporting a genotoxic mode of action initially came from 32Ppostlabeling studies in which putative ochratoxin A– or ochratoxin A metabolite–DNA adducts comigrated chromatographically with a photochemically generated ochratoxin A–DNA reaction product, possibly a C8-deoxyguanosine–ochratoxin A compound (Kuiper-Goodman et al., 2010). It has been hypothesized that adducts form via cytochrome P450–mediated conversion of ochratoxin A to an electrophilic ochratoxin A quinone, by reductive dechlorination to an aryl radical, or by peroxidase- and glutathione-mediated conversion to a phenoxyl radical. In turn, these reactive metabolites might bind DNA. The detection of ochratoxin A-hydroquinone, which is formed by further reduction of ochratoxin A-quinone, in urine and kidney of rats as well as in human urine and blood from the Balkans has been reported. While this is consistent with the hypothesized mechanism, reactive metabolite formation appears to be a minor in vivo metabolic pathway (EFSA et al., 2020b; Wu et al., 2011). The issue of mutagenicity continues to be unresolved. Ochratoxin A does not elicit a mutagenic response in the Salmonella typhimurium T100-, T102-, and T102-related tester strains but is mutagenic in others, including T98, when the procedure is modified to include metabolic activation by mouse kidney microsomes. A comprehensive review of in vivo and in vitro genotoxicity studies concluded that there is insufficient evidence to support either direct DNA damage or indirect DNA damage by ochratoxin A (EFSA et al., 2020b). Results of genotoxicity tests such as sister chromatid exchange and unscheduled DNA synthesis have been inconsistent. Single-strand breaks, double-strand breaks, and chromosomal damage occur in cell lines and primary cell cultures exposed to ochratoxin A. Metabolic activation is not required to induce DNA breaks. Increased levels of single-strand DNA breaks have also been shown in vivo in rat and mouse kidneys, with mixed evidence of associated oxidative damage. Double-strand DNA breaks have been demonstrated in rat tubular epithelial cells. Ochratoxin A induces karyomegaly and aberrant mitoses at its primary site of carcinogenesis in the kidney outer stripe of the outer medulla, which links these lesions to tumors but has not been proven to be causal.

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Numerous nongenotoxic mechanisms of carcinogenicity have also been proposed. Studies using the comet and Fpg modified (with foramidopyrimidine-DNA-glycosylase, an enzyme that recognizes and repairs oxidative damage in DNA) comet assays suggest that ochratoxin A causes DNA damage indirectly via oxidative stress while other studies suggest that ochratoxin A interferes with Nrf2-dependent processes in kidney that have antioxidant activity. However, evidence of oxidative DNA damage or lipid peroxidation has not been found in other studies, including experiments in which ochratoxin A was given at nephrotoxic doses (up to 2 mg/kg body weight) to male F344 rats. Based on their review of ochratoxin A mechanism of action, EFSA theorized that ochratoxin A elicits cellular stress responses (including oxidative stress) that alter gene expression (in part mediated by epigenetic modifications), contributing to modulation of DNA repair enzymes, disruption of cell cycle, and an imbalance of apoptosis and cell proliferation. Impaired spindle function leads to aberrant alignment of chromosomes and the outcomes are increased ploidy, aneuploidy, and karyomegaly, leading to DNA replication stress and ultimately, DNA damage (EFSA et al., 2020b). As is the case for the roles of genotoxicity and adduction, these hypotheses of carcinogenicity continue to be the subject of debate.

3.3. Manifestations of Toxicity in Animals Laboratory animals, in particular rats, have been widely used to characterize the toxicity and mechanism of action of ochratoxin A. However, pigs, poultry, and dogs are more sensitive to acute ochratoxicosis. Kidneys are consistently the primary target organ in most species, but hepatotoxicity may also be observed after acute exposures. LD50s for oral ochratoxin A exposure range from 12.6 to 30.3 mg/kg bw in the rat, 8.1–9.1 mg/kg bw in the guinea pig, 16.5 mg/kg bw in Japanese quail, 5.9 mg/kg bw in turkeys, 3.4 mg/kg bw in White Leghorn chickens, b-zearalenol (EFSA, 2017b). Relative binding affinity of a-zearalenol to ERs is pig > rat > chicken, which correlates with species sensitivity (EFSA, 2017b). Zearalenone and its metabolites are considered mycoestrogens, a subset of naturally occurring estrogenic compounds or xenoestrogens, and are classified as endocrine-disrupting chemicals (EDCs) (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). The uterine

and mammary effects are induced by an interaction of zearalenone with estrogenic cytosolic receptors in these organs. Zearalenone also acts on the hypothalamic–hypophysial axis with release of prolactin and LH. Zearalenone affects a number of transcription factors. It can activate the pregnane X receptor (PXR), a human xenobiotic receptor member of ligand-activated nuclear transcription factors. PXR regulates the expression of genes involved in the metabolism of xenobiotics, such as the cytochrome P450 enzymes CYP3A4, and has a role in the transcriptional regulation of glutathione-Stransferases, sulfotransferases, and uridine diphosphate glucuronyltransferases (UGTs), as well as several transporters. In addition, zearalenone was shown to activate constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR) mRNA levels, as well as several CYP enzymes in human hepatocyte cultures. Genotoxicity data for zearalenone are largely negative. It does not cause gene mutations in bacterial test systems but is clastogenic and aneugenic in vitro and has been confirmed as an in vivo clastogen in mice. DNA adducts induced by high doses of zearalenone in rats and mice are thought to be secondary to oxidative damage

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since they were reduced by coadministration of the antioxidant a-tocopherol. Oxidative stress has been demonstrated in zearalenone-exposed mice and swine by increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD) and glutathione peroxidase (GSHPx). Zearalenone and its metabolites induced MDA formation and apoptotic cell death in several cell culture systems that could be inhibited by antioxidants. Oxidative damage may be responsible for nonestrogenic cytotoxicity induced by zearalenone at high doses. Species Susceptibility Zearalenone has little toxicity following administration of single oral or intraperitoneal doses in any species. Effects of oral administration for up to 90 days appear to be primarily dependent on the estrogenic activity of zearalenone and/or its metabolites. Pigs and dogs are sensitive to zearalenone, with LOEL/LOAELs of 17.6 and 25 mg/kg bw/day, respectively, and an NOEL of 10.4 mg/kg bw/day for pigs. In sheep, an LOAEL of 56 mg/kg bw/day and an NOAEL of 28 mg/kg bw/day have been derived based on depressed ovulation rates and lower lambing percentages. Cattle, horses, and poultry appear to be relatively resistant to the adverse effects of zearalenone (EFSA, 2017b). In mice and rats, NOAELs for nonneoplastic lesions are in the mg/kg bw/day range for zearalenone (Kuiper-Goodman et al., 1987). Biodistribution, Metabolism, and Excretion Species-specific differences in the disposition of zearalenone in farm animals and laboratory animals have been reviewed (Da¨nicke and Winkler, 2015; EFSA, 2011c, 2017b). Wide interspecies differences in zearalenone absorption, distribution, metabolism, and excretion (ADME) have been documented. The nature and the amount of the generated metabolites may affect the species sensitivity to the toxin. Zearalenone is readily and rapidly absorbed from the gastrointestinal tract, with about 85% absorbed in the pig after a single oral dose. Enzymatic reduction results in formation of a-zearalenol (more estrogenic than zearalenone) and b-zearalenol (less estrogenic) as well as smaller amounts of a- and b-zearalanol. Zearalenone is also monohydroxylated by CYP1A2 and, to a lesser extent, CYP3A4, with the major oxidative metabolites being catechols that can undergo

oxidation to quinones, which can then redox cycle and covalently modify biological macromolecules such as N-acetylcysteine. Zearalenone and its reduced metabolites undergo Phase II conjugation with glucuronic acid and sulfate in the intestine, liver, and other organs. There is significant species variation in the extent of zearalenone metabolism, which could explain differences in susceptibility. For example, greater amounts of a-zearalenol, the more estrogenic metabolite, are formed in man and pig compared to rodents. Ruminal metabolism by microbes has been documented in vitro with zearalenone reduced to a-zearalenol and to a lesser degree to b-zearalenol. Based on radiolabel studies in mice, zearalenone is distributed to estrogen target tissues such as uterus, ovarian follicles, and interstitial cells in the testis as well as adipose tissue. Placental transfer of zearalenone and a-zearalenol has been reported in rats and pigs. Zearalenone and zearalenol (as a combination of free and conjugated forms) are excreted relatively rapidly in feces, urine, and to a small extent in milk. Humans and rabbits excrete the zearalenone metabolites primarily in urine, and elimination from the blood is much slower than in rats, dogs, and monkeys; humans had a significantly higher peak fraction of the dose in plasma. Considerable enterohepatic recycling of glucuronidated metabolites occurs in swine and rodents, extending the half-life of plasma zearalenone and prolonging its estrogenic effects. Excretion in swine is primarily through the urine. Zearalenone residues do not persist in animal tissues, although a small amount (less than 1%) of the dose is excreted in milk. Based on monitoring of animals and animal-derived foods, zearalenone and related compounds in foodstuffs of animal origin probably do not pose a significant risk for the consumer (Da¨nicke and Winkler, 2015).

6.3. Manifestations of Toxicity in Animals The predominant toxicologic effect is related to the estrogenic activity of zearalenone and its metabolites resulting in adverse effects on the endocrine organs, male and female reproductive systems, and mammary tissue (see Endocrine System, Vol 4, Chap 7; Male Reproductive System, Vol 5, Chap 9; Female Reproductive System, Vol 5, Chap 10; Mammary Gland, Vol 5, Chap 8). Toxicity to the liver, hematopoietic system, and

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immune system (in vitro) has also been described in animals exposed to high doses. Anabolic activity has led to the use of a-zearalanol as a fattening agent in cattle and sheep in some countries. In domestic animals, zearalenone can interfere with the estrus cycle, ovulation, conception, and implantation; induce embryonic death; reduce fetal weight and litter size; and impair neonatal survival (see Embryo, Fetus and Placenta, Vol 5, Chap 11). Swollen vulvas and vaginal and rectal prolapses are common clinical signs of exposure, especially in the pig, which is the most sensitive species, with the female prepubertal pig the most sensitive (Figure 6.12). Many outbreaks of naturally occurring mycotoxicosis due to exposure to zearalenone in the diet have been reported in swine and less often in cattle and sheep. Naturally contaminated feed, often used to study zearalenone toxicity, is frequently also contaminated by trichothecenes. Although trichothecenes are not estrogenic, overall interpretation of study results must account for the presence of trichothecenes (EFSA, 2017b). Laboratory Animals Zearalenone has a low acute toxicity with oral LD50 values of greater than 2000 mg/kg bw in mice, rats, and guinea pigs. In female rodents, zearalenone has adverse effects on the reproductive

FIGURE 6.12 Vulval hypertrophy, edema and congestion, estrogenic effect, pig (right) as compared to unaffected (left). Zearalenone, a mycotoxin commonly found in corn, has estrogenic effects that result in vulval edema. Reproduced from Noah’s Arkive by courtesy of the Davis Thompson DVM Foundation. Image No. F33944, Marked vulvar edema and congestion, submitted by Sawang Kesdangsakonwut. Accessible at: https://davisthompsonfoundation.org/imagedetail?image¼F33944

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tract, fertility, and embryo survival, but teratogenic effects have not been observed. Effects are similar to those described below for swine but occur at much higher doses, in the range of 1–10 mg/ kg bw/day. In addition, there were changes in the weights of adrenal, thyroid, and pituitary glands, and in serum levels of progesterone and 17b-estradiol (Kuiper-Goodman et al., 1987). In males, adverse effects on testosterone synthesis, sexual behavior, accessory sex organ weights, testicular morphology, and spermatogenesis have been observed (see Male Reproductive System, Vol 5, Chap 9), but again, at much higher doses than in swine. In rats, germ cell apoptosis was identified at stages I–VI of spermatogenesis 12 h after dosing. This was proposed as the principal mechanism contributing to germ cell depletion and testicular atrophy following zearalenone exposure (EFSA, 2011c). Of particular interest are long-term studies on zearalenone, including subchronic and carcinogenesis bioassays using purified zearalenone conducted by the National Institute of Environmental Health Sciences (NIEHS) National Toxicology Program (NTP) using B6C3F1 mice and F344/N rats (NTP, 1992). The animals were fed zearalenone at concentrations of up to 3000 mg/kg diet (450 or 300 mg/kg bw per day in mice or rats, respectively, for 13 weeks) or up to 100 mg/kg of diet to mice and 50 mg/kg diet to rats for 103 weeks (carcinogenicity bioassay). In the 13-week study, several high-dose-treated female mice died. Most female mice had endometrial hyperplasia that was not dose related. Male mice had atrophy of the seminal vesicles and cytoplasmic vacuolization of the adrenal at 1000 mg/kg diet and squamous metaplasia of the prostate at the 3000 mg/kg diet. Doserelated osteopetrosis was seen at 100 mg/kg and myelofibrosis at 1000 mg/kg in both sexes. Similar changes were found in the seminal vesicles, uteri, and bones of rats. Fibromuscular hyperplasia of the prostate and atrophy of the testis were present at dietary levels of 1000 mg/ kg and above. In addition, in both sexes there was chromophobe hyperplasia of the pituitary at 1000 mg/kg and ductular hyperplasia of the mammary gland at the highest dose. In the mouse carcinogenicity bioassay, treatment-related nonneoplastic lesions were found only in females. These lesions were estrogen-related: uterine fibrosis, cystic ducts in

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the mammary gland, and myelofibrosis of the bone marrow. There was a dose-related increase in hepatocellular adenomas in females, and a dose-related increase in pituitary adenomas in both sexes. These tumors are believed to be due to the estrogenic effects of zearalenone. In rats, nonneoplastic treatment-related lesions included inflammation of the prostate, testicular atrophy, hepatocellular cytoplasmic vacuolation in males, and an increased incidence of chronic progressive nephropathy in both sexes. No treatmentrelated increase in neoplasms was found in these rats or in FDRL Wistar rats in a similar study. Because of the limited evidence of carcinogenicity, IARC allocated zearalenone to Group 3 (not classifiable as to its carcinogenicity to humans) based on inadequate evidence in humans and limited evidence in experimental animals. An NOEL of 0.1 mg/kg bw per day was derived in rodents based on the absence of increased uterine weight (IARC, 1993; NTP, 1992). When administered to dogs, zearalenone reduced the number of corpora lutea and caused uterine hyperplasia in females and arrested spermatogenesis in males. This is of interest since endometrial hyperplasia predisposes to pyometra, a life-threatening bacterial infection of the uterus that is not uncommon in the dog pet population. Experimentally, estrogen priming followed by progesterone stimulation can reproduce the cystic endometrial hyperplasia that precedes pyometra. An LOAEL of 25 mg zearalenone/kg bw/ day was estimated for dogs based on uterine and ovarian gross and microscopic changes, along with changes in the hematological and blood biochemical profiles (EFSA, 2017b). Swine Since swine are the most susceptible species to zearalenone toxicity, the adverse effects of zearalenone in mature and prepubertal male and female pigs have been extensively reviewed (EFSA, 2011c, 2017b; Liu and Applegate, 2020; Tiemann and Danicke, 2007; Zhang et al., 2018). The ovary, uterus, and vulva are important target organs in this species. Estrogenic effects may occur in swine fed diets containing >1 ppm zearalenone. In prepubertal gilts, the most sensitive physiological state, the effects include swelling and edema of the vulva (Figure 6.12), vaginal and rectal prolapse, uterine enlargement and edema, atrophy of the

ovaries, enlargement of the mammary glands, and a thin catarrhal exudate from the vulva. Experimental exposure of prepubertal gilts to estradiol, zearalenone, and F. graminearum–inoculated corn induces similar histological changes (see Female Reproductive System, Vol 5, Chap 10). Cervical changes consist of epithelial metaplasia, the normal double layer of columnar-type cells replaced by a stratified squamous cellular layer up to 15 cells thick and irregular in distribution (i.e., squamous metaplasia). Similar but more severe changes were seen in the vagina. Interstitial edema, together with cellular proliferation, account for the clinically observed tumefaction (swelling) of the vulva and enlargement of the uterine horns with hypertrophy of all uterine layers. This effect is the basis for the rat uterotrophic bioassay, which has proven to be a practical laboratory method for testing a variety of compounds for their potential estrogenic effects (Yamasaki et al., 2002). Ovarian changes in immature gilts include reduced proliferative activity of estrogen-sensitive cells, including ovarian granulosa cells and cells in the ovarian stromal connective tissues, which may contribute to ovarian follicular atresia (Gajecka et al., 2011). The mammary gland and nipples enlarge due to interstitial edema and ductal hyperplasia. Reproduction may be affected in sows, but higher levels of exposure are required. The changes induced by zearalenone depend on the time of dose administration in relation to the estrus cycle, as well as on the amount administered. Anestrus or nymphomania may be noted. In sows exhibiting nymphomania, ovaries are atrophic and lack corpora lutea and Graafian follicles, indicating follicular atresia. The effects of zearalenone on the uterus, cervix, vagina, and mammary gland in sows are similar to the effects in prepubertal gilts. Reduced litter size due to fetal resorption (mummification) and/or implantation failure occurs when exposure to dietary zearalenone occurs at 7–10 days postmating. Pigs may be weak or stillborn, and occasionally exhibit swollen vulvas at or shortly after birth (juvenile hyperestrogenism). Gilts may develop pseudopregnancy with multiple persistent corpora lutea in the ovary indicating a luteotrophic property of zearalenone. Uterine changes, characterized by both hyperplasia and hypertrophy, are indicative

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6. ZEARALENONE

of estrogenic effects from zearalenone as well as a progesterone effect due to the persistent corpora lutea (see Female Reproductive System, Vol 5, Chap 10). Squamous metaplasia of the vaginal lining as well as alveolar development and ductular squamous metaplasia are found in the mammary gland (see Mammary Gland, Vol 5, Chap 8). Field observations of zearalenone-induced abortions are thought to be largely erroneous since estrogens are luteotropic in swine. Instead, it is suspected that implantation failure followed by pseudopregnancy leads to a diagnosis of abortion. Zearalenone affects granulosa cell steroidogenesis, oocytes, and fertilized ova, based on in vitro studies. The NOAEL for zearalenone in pubertal female pigs (gilts) is 40 mg/kg bw/day. Prolongation of the luteal phase of the estrus cycle, accompanied by high serum progesterone levels, appears to be the most sensitive adverse effect, with a lowest-observed-effect level (LOEL) of 200 mg/kg bw/day. In prepubertal gilts, the LOEL for zearalenone varies with the criteria and study: 17.6 mg/kg bw/day based on vulval volume (swelling and lengthening) and >20 mg/kg bw/day based on histological evaluation of the uterus (cell proliferation and hyperemia). The overall NOEL for zearalenone is 10 mg/kg bw/day. In boars, adverse effects on testosterone concentrations, sexual behavior, testis and secondary sex organ weights, testicular morphology, and spermatogenesis have been observed following exposure to zearalenone or its metabolites (see Male Reproductive System, Vol 5, Chap 9). Adverse effects were observed at 20 mg zearalenone/kg or more but not at 1 mg/kg in feed (for 2 months). In prepubertal boars, zearalenone may reduce libido and plasma testosterone concentrations. In castrated or prepubertal males, there is also enlargement of mammary glands and swelling of the prepuce. Testicular atrophy has also been reported. Cattle and Sheep Cattle and sheep are much less sensitive than pigs to the estrogenic effects of zearalenone. Zeranol, in the form of an implant, is widely used in the United States as a growth promoter in beef cattle and is also used in sheep. In cattle, zearalenone toxicity may be associated with precocious udder development in heifers and reduced fertility in

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breeding animals; however, most animals exposed to zearalenone for brief periods of time will recover normal reproductive function. Severe toxicosis may be characterized by ovarian fibrosis and changes in the fallopian tube and uterus which could conceivably have more prolonged effects on reproduction. Vulval swelling, and decreased feed intake and milk production also have been described. Ewe infertility with a decrease in ovulation rate and lambing percentage has been reported (EFSA, 2011c, 2017b).

6.4. Human Risk and Disease EFSA established a TDI for zearalenone and its metabolites of 0.25 mg/kg bw in 2011. This TDI is above median chronic dietary exposures, for which the upper limits range from 16 to 182 ng/kg bw/day in European populations. The main source of exposure is corn (Zea mays) and corn products, although other grains and possibly eggs, which accumulate a zearalenone metabolite in the yolk, also contribute. Meat, including that from cattle implanted with a-zearalanol, and milk are not considered significant sources of exposure. In Europe, the highest concentrations of zearalenone were found in wheat bran and corn and products thereof. Significant amounts were also found in corngerm and wheat-germ oils. Zearalenone, like most mycotoxins, is heat stable. However, cooking under alkaline conditions and extrusion cooking significantly decreases the concentration (EFSA, 2011c). Because of the ability to interact with estrogen receptors, dietary zearalenone and metabolites can increase human exposures to estrogenic compounds (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). High concentrations of zearalenone identified in foods in cases of estrogen-related adverse effects in humans, such as precocious puberty and breast cancer, led to speculation regarding possible cause and effect. Zearalenone was suggested as a cause of premature thelarche (onset of secondary breast development) and precocious puberty in children in regions of Italy, Hungary, and Puerto Rico, partly based on detection of these compounds in blood and/or food samples. However, insufficient evidence is available to confirm this assertion (EFSA, 2011c). A study in healthy girls aged 9–10 years from New Jersey found that girls with detectable urinary zearalenone levels tended to be shorter

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and less likely to have reached the onset of breast development. The authors postulated an antiestrogenic effect of zearalenone or its metabolites. Urinary zearalenone levels were associated with beef and popcorn intake (Bandera et al., 2011). In China, zearalenone was extracted from buckwheat contaminated with Fusarium spp. that was linked to “endemic breast enlargement.” Because zearalenone is thought to be associated with breast enlargement, it is found in breast-enhancing dietary supplements. The suggested zearalenone link to human breast cancer is based on in vivo studies showing mammary gland enlargement and ductal hyperplasia, and on in vitro studies in which zearalenone stimulated the proliferation of human breast cancer MCF-7 cells containing human ER through regulation of the cell cycle and prevention of apoptosis. However, zearalenone decreased the incidence and multiplicity of 7,12–dimethylbenz(a)anthracene (DMBA)induced mammary tumors when given to prepubertal rats, possibly due to increased differentiation of the mammary epithelial tree (Hilakivi-Clarke et al., 1999; Pazaiti et al., 2011).

6.5. Diagnosis, Treatment, and Prevention In domestic animals, clinical signs together with the identification of zearalenone in the diet form a strong basis for diagnosis. The diagnosis usually is confirmed when normal reproductive function returns after withdrawal of the contaminated feed, and removal of contaminated feed is generally curative. Zearalenone in foodstuffs can be determined by HPLC coupled to fluorescence detection, triple quadrupole mass spectrometers, thin layer chromatography (TLC), or gas chromatography with mass spectrometry (GC-MS); ELISA-based tests can also be used and are readily available from commercial suppliers. Quantification can be achieved with matrix calibration or by using stable isotope–labeled standards. Cleaning and selection steps applied to grains after harvesting lead to a decrease in concentration of zearalenone in grains for food consumption (Alshannaq and Yu, 2017). No US FDA action, advisory, or guidance levels have been established for zearalenone in feed. The EU has guidance values in feedstuff with a moisture content of 12% of 0.1 mg/kg or ppm for piglets and gilts, 0.25 mg/kg for sows

and fattening pigs, and 0.5 mg/kg for calves, dairy cattle, sheep, and goats (EC, 2016). For human consumption, the EU has a maximum limit of 0.35 mg/kg in unprocessed corn and 0.4 mg/kg in refined corn oil (EC, 2006). There are no binders recommended as efficacious for zearalenone absorption by the US FDA. In addition to concern over the presence of zearalenone in human and livestock diets, diets prepared for laboratory animals and pets also need consideration. The potential of diets containing zearalenone to confound research on endocrine-disrupting chemicals (EDCs) or evaluation of estrogenic effects is especially problematic. In addition, with the expanded use of pigs and minipigs in research and for pharmaceutical safety evaluation (see Animal Models in Toxicologic Research: Pig, Vol 1, Chap 20), care must be taken to avoid the potential confounding effect of zearalenone in the diet of the most sensitive species.

7. FUMONISINS Fumonisin chemistry, occurrence, toxicology, and mode of action have been the focus of several authoritative reviews that have informed this section (EFSA 2018a,b; Smith, 2018; Voss et al., 2007; Voss and Riley, 2013).

7.1. Source/Occurrence/Exposure Fumonisins are mycotoxins produced by Fusarium verticillioides (formerly Fusarium moniliforme Sheldon), Fusarium proliferatum, and a few other Fusarium species. Production by other species, such as Aspergillus niger, has also been reported, Both the fungi and fumonisins are found worldwide in corn (Zea mays) as well as other cereals. They have, on occasion, been found in other crops, including cowpeas and asparagus, as well as spices, herbs, black tea, and corn-based beer. Fumonisins from contaminated corn may be concentrated in dried distillers’ grains with solubles (DDGS), a coproduct of ethanol production and used as a high protein animal feed. Fumonisins associated with corn and cornbased feeds or foods are an animal, and potentially human, health threat. More than 30 homologs of fumonisins have been identified, and the number continues to grow. Fumonisins B1, B2, and B3 are most

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common, with fumonisin B1 found at the highest concentration in food and feed. Fumonisin concentrations vary geographically, often differing between nearby locations. Factors favoring fungal growth and toxin production include heat stress, insect damage, high humidity, and a delay in harvest, as well as improper (wet) storage. Insects that damage corn, such as the European corn borer (Ostrinia nubilalis), provide a means for the fungus to invade the plant. Resistance to the corn borer, as found in some strains of genetically modified corn (GMO), transgenic corn expressing Bacillus thuringiensis kurstaki (Btk) toxin, decreases fungal invasion. Accordingly, fumonisin concentrations are lower in GMO corn than in unmodified cultivars that are grown under conditions favoring high amounts of insect damage. While fumonisins occur worldwide, especially high concentrations have been found in China and southern Africa. Fumonisin concentrations in “home grown” corn from Linxiang, China, and areas of the Transkei, southern Africa exceeding 100 ppm have been occasionally reported. Much lower concentrations of fumonisins, less than 1 to a few ppm, are generally found in corn from the USA and South America, while corn from Canada is virtually free of fumonisins. Concentrations in excess of 10 ppm are sometimes found and concentrations exceeding 100 ppm occur given appropriate climatic conditions, such as the 1989 drought in the midwestern USA. Because fumonisin concentrations periodically can be high, there is a continuing possibility of episodic outbreaks of fumonisin-related diseases in susceptible animals such as horses and pigs. Fumonisin concentrations in screenings and cracked, broken, or otherwise damaged kernels are generally higher than in whole grains. Normal-appearing corn may contain fumonisins since the fungus is endophytic.

7.2. Toxicology and Mode of Action (MOA) Toxins The chemical structure of the fumonisins was first reported in 1988 (Gelderblom et al., 1988) Fumonisins have a long chain carbon “backbone” with two tricarballylic acid moieties attached (Figure 6.13). Different classes of

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FIGURE 6.13 Chemical structure of fumonisin B1 and the sphingoid bases, sphingosine and sphinganine. Because of the structural similarity of the fumonisins to the sphingoid bases, they bind to and inhibit ceramide synthase. Figure reproduced from Voss et al. (2007) Fumonisins: toxicokinetics, mechanism of action and toxicity, Anim. Feed Sci. Technol. 137, Figure 1, p. 303, with permission.

fumonisins have been identified including A-, B-, C- and P-series. In the B-series, fumonisin B2, B3, and B4 lack one or more of the hydroxyl groups at the C5 or C10 positions. The primary amino function at C2 is important. The structural similarity of the fumonisin backbone and primary amino function to the sphingoid bases, sphinganine (Sa) and sphingosine (So), is critical to their ability to disrupt sphingolipid metabolism (Merrill et al., 2001), as discussed below. Modified forms of fumonisins have been identified and may arise via biotransformation in fungi, plants, and animals, occurring as phase I and II metabolites, or during food or feed processing. These include hydrolyzed fumonisins B1–4, partially hydrolyzed fumonisin B1–2, N-(carboxymethyl)-fumonisin B1–3, N-(1-deoxy-

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D-fructos-1-yl)-fumonisin B1, O-fatty acyl fumonisin B1, N-fatty acyl fumonisin B1 and N-palmitoyl-HFB1; overall, these are considered toxicologically similar but less potent than fumonisin B1 (EFSA, 2018a). Assays for fumonisins may not identify these modified forms. In some cases, for example N-(deoxy)-D-fructos-1yl) fumonisin B1, some of the conjugated form can be cleaved in the gut to liberate the parent fumonisin with the primary amino function. In addition, matrix-bound fumonisins resulting from covalent and noncovalent binding can result in poor recovery rates from food or feed so that chemical analysis might in some cases underestimate potential toxicity. Although fumonisins are heat stable, thermal processing of corn often results in a reduction of fumonisin content. Hydrolyzed fumonisins, which lack the tricarballylic acid functions, are produced by base hydrolysis and therefore may be found in alkaline cooked (nixtamalized) foods such as masa and tortillas. In its purified form, hydrolyzed fumonisin B1 elicited significantly reduced to negligible biological activity in various rodent studies. Extrusion, as well as nixtamalization, of fumonisin contaminated cornbased foods resulted in significantly reduced toxicity in rodent bioassays (Voss et al., 2017). Partially hydrolyzed forms can co-occur with the parent compounds and also have been identified in the feces, suggesting formation by bacteria in the intestinal tract.

TABLE 6.7

Species Susceptibility While causality between fumonisins and human disease is unproven, fumonisins elicit adverse effects in a broad range of species, including horses, swine, sheep, cattle, fish, poultry, nonhuman primates, mink, rabbits, and rodents (EFSA, 2018a,c; Smith, 2018; Voss et al., 2007). Hepatotoxicity is elicited in all species and nephrotoxicity in many of the species evaluated to date such as rodents, rabbits, and calves. Naturally occurring disease occurs in horses and pigs, with horses being considered the more susceptible species. Species-specific syndromes occur and are termed equine leukoencephalomalacia (ELEM) and porcine pulmonary edema (PPE), respectively, based on the primary clinical presentation (Table 6.7). Cardiovascular toxicity is proposed to be responsible for both these syndromes, which are usually lethal (Haschek et al., 2001; Smith et al., 2002). Importantly, and in contrast to other Fusarium verticillioidesproduced mycotoxins, the toxicity and lesions associated with corn molded with Fusarium verticillioides or Fusarium proliferatum have been experimentally reproduced with purified fumonisin B1 in horses and pigs (Foreman et al., 2004; Haschek et al., 2001; Kellerman et al., 1990). Likewise, the hepatic and renal effects of contaminated corn and culture materials were reproduced in rodents by fumonisin B1 and, albeit less potently, fumonisin B2 (Bondy et al., 2000).

Fumonisin-Induced Species-specific Target Organ Toxicity Species

Organ

Horse

Pig

Cattle (calves)

Sheep (lambs)

Rabbits

Rats

Mice

Nonhuman primates

Liver

þþ

þ

þ

þ

þ

þþ

þþþ

þ

Kidney

þ/

þ/

þþþ

þþþ

þþþ

þþþ

þ

þ

Brain

þþþ















Cardiovascular system

þþ

þþ











þþ

Lung



þþþ













þ/, reported in some studies. Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table 4, p. 675, with permission.

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Among farm animals, horses and donkeys are considered most susceptible to naturally occurring disease although experimentally pigs may develop mild lesions at lower doses (Table 6.8). The level of exposure required to induce ELEM, a disease unique to equidae, has not been established, but is certainly quite low, perhaps less than 5 ppm fumonisins in feed. Limited experimental work with purified fumonisin B1 is available in horses, but only following intravascular administration (Foreman et al., 2004). Swine are also susceptible, although much more highly contaminated feed is generally required to induce PPE in swine than to cause ELEM in horses. Cattle and poultry are more resistant. Studies with fumonisin B1 in poultry demonstrate hepatic toxicity, decreased feed intake and adverse effects on the immune system. Biochemical evidence of mild hepatic injury has been reported in cattle, with nephrotoxicity in calves. The relative resistance of bovine adults may be due to degradation of fumonisins by ruminal microflora. Limited information is available in fish from feeding studies. Among laboratory species, mice and rats differ in target organ sensitivity with mice more sensitive to hepatotoxicity and rats to nephrotoxicity. Male Sprague–Dawley (SD) and F344 rats are extremely sensitive to the nephrotoxic effects of fumonisins. Diets containing 9 ppm fumonisin B1 elicited renal apoptosis in male SD rats when given for up to 90 days. Mice do exhibit kidney lesions, but only at much higher exposure concentrations. Nephrotoxicity was observed in rabbits following intravascular administration of purified fumonisin B1 (Gumprecht et al., 1995). Positive carcinogenicity results have been obtained in several chronic (2 years or longer) bioassays of fumonisin B1 in rodents (Gelderblom et al., 1991; Howard et al., 2001). In each case, tumors were induced when fumonisin B1 in the diet was 50 ppm, even though the target organ differed by species, strain, and sex of the animals. Liver tumors occurred in mice and, depending on strain of the rats, liver (Gelderblom et al., 1991) or kidney (Howard et al., 2001) tumors, some unusually invasive, in rats. Biodistribution, Metabolism, and Excretion The biodistribution, metabolism and excretion of fumonisins have been reviewed previously

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(Voss et al., 2007; EFSA, 2018a). Fumonisins are poorly absorbed after oral ingestion in animals, including pigs, cattle, laying hens, rats, mice, and monkeys. The bioavailable amount, less than 4% of the dose, is rapidly distributed to the tissues (half-life of less than 4 h) and eliminated by biliary excretion in the feces without biotransformation. Similar low oral bioavailability was found in humans consuming fumonisindcontaminated corn diets (Riley et al., 2012). Absorption and biodistribution patterns of FB1 in different species are similar. After oral, intraperitoneal, or intravenous administration, fumonisin B1 is rapidly cleared from the blood and eliminated in feces, with relatively low amounts excreted in urine. FB1 is excreted in bile with significant enterohepatic recirculation shown to occur in swine, thus increasing the half-life in that species and likely in other species with enterohepatic recirculation. Tissue burdens are low, with the highest concentrations occurring in liver and kidneys. As compared with fumonisin B1, fumonisins B2 and B3 appear to be less bioavailable, accumulate less in liver and kidneys, and with lower excretion in urine (EFSA, 2018a). Metabolites found in mammals can be produced via two pathways. Hydrolysis of the ester groups to remove the two tricarballylic acid moieties is mediated by the colonic microbiome of some species resulting in production of hydrolyzed and partially hydrolyzed fumonisin B1. The second pathway is acylation of the amino group, with formation of small but detectable amounts of several N-acyl fumonisin B1 or N-acyl hydrolyzed fumonisin B1 ceramide analogs, which have been shown in vivo both in the kidney and liver of rats and in vitro. This metabolic pathway has been investigated only in rats and the biological significance, if any, of N-acyl metabolites has not been determined. Mode of Action Proposed molecular mechanisms for fumonisin B1 toxicity include disruption of sphingolipid metabolism, induction of oxidative stress, activation of endoplasmic reticulum stress and MAPKs, modulation of autophagy and alteration of DNA methylation (Liu et al., 2019). While disruption of sphingolipid metabolism occurs early following exposure to fumonisins,

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TABLE 6.8 Relevant Fumonisin Toxicity Data From Ruminant, Pig, Poultry, Horse, Rabbit, and Fish Studies No observed adverse effect levels (NOAELs)

Lowest observed adverse effect level (LOAEL)

Cattle (calves)

31 mg FBs (FB1s þ FB2)/kg feed (corresponding to 600 mg FBs/kg bw per day)

148 mg FBs/kg feed (corresponding to 2.9 mg FBs/kg bw per day)

Elevated serum enzymes and cholesterol, suggesting altered liver function; impaired lymphocyte blastogenesis

Cattle (steers)a

108 mg FBs/kg feed (corresponding to approx. 2.7 mg/kg bw per day for 110 days)

N/A

None

Horses

0.2 mg FB1/kg bw iv per day (corresponding to 8.8 mg/kg feed)

1 mg FB1/kg bw iv per day (corresponding to 44 mg/kg feed)

Neurological abnormalities Cardiovascular effects

Pig

1 mg FB1/kg feed (corresponding to 40 mg/kg bw per day)

5 mg FB1/kg feed Corresponding to 200 mg/ kg bw per day

Mild lung lesions in 1 pig at 1 mg FB1/kg feed (NOAEL) At 5 mg FB1/kg feed, increased lung weight, chronic pulmonary changes in lung and liver (LOAEL)

5 mg FB1/kg feed (corresponding to 130 mg FB1/kg bw per day)

Decreased performance and biochemical alteration (serum protein, enzymes) Altered blood formula

Species

Rabbits

Adverse effects observed

Chicken

20 mg FB1/kg feed (corresponding to 2.6 mg/kg bw per day)

40 mg FB1/kg feed (4.7 mg/kg bw per day)

Decreased liver lipids (from 40 mg/kg) Increased ratio GOT:AST (from 80 mg/kg)

Ducks

8 mg FB1/kg feed

32 mg FB1/kg feed

Serum biochemistry, indicative of liver damage

Turkeys

20 mg FBs (FB1sþFB2)/kg feed (corresponding to 0.9 mg FBs/ kg bw per day)

Fish (Carp)

Other fish: Nile tilapia a

None

10 mg FB1/kg feed Reduced weight gain, (corresponding to 0.5 mg/ neuronal apoptosis in brain kg bw per day) 10 mg FB1/kg feed 40 mg FB1/kg feed (corresponding to 0.4 mg FB1/ kg bw per day)

Reduced weight gain

Additional data from Jennings JS, Ensley SM, Smith WN, Husz TC, Lawrence TE: Impact of increasing levels of fumonisin on performance, liver toxicity, and tissue histopathology of finishing beef steers. J Anim Sci 98, skaa 390, 2020. https://doi.org/10.1093/jas/ skaa390. AST, aspartate aminotransferase; bw, body weight; FB, fumonisin B; GOT, glutamic-oxaloacetic transaminase; iv, intravenous; LOAEL, lowest-observed-adverse-effect level; N/A, not applicable; NOAEL, no-observed-adverse-effect level. Table modified from EFSA: Appropriateness to set a group health-based guidance value for fumonisins and their modified forms, EFSA J 16: 5172, 2018a., Table 9, p 74–75 with permission. Note that alteration in Sa/So ratio was not by itself considered an adverse effect. Data stated as “corresponding to” are gross calculations.

7. FUMONISINS

a recent EFSA review concluded that there is not enough evidence to support the hypothesis that this disruption in itself is an adverse effect or that it plays a significant role in some of the observed critical adverse effects (EFSA, 2018c). This conclusion is debatable given previous research which is summarized by Riley and Merrill (2019). Fumonisin B1 and other fumonisins bear a striking structural similarity to the sphingoid bases, sphinganine and sphingosine (Figure 6.13). Fumonisins bind to and are potent inhibitors of ceramide synthases (Merrill et al., 2001). These enzymes (six, designated CerS1–CerS6, have been characterized) catalyze ceramide formation from sphinganine (or sphingosine) and palmitate or other long chain fatty acids. Inhibition of ceramide synthase thus disrupts overall sphingolipid metabolism and leads to, among other changes, increases in cellular sphinganine and sphingosine concentrations, increases in the 1phosphate metabolites of these sphingoid bases, and decreases in cell ceramide and complex sphingolipids. Sphingosine 1-phosphate is a particularly important signaling molecule that exerts diverse physiological effects by acting as a ligand for a family of membrane bound G proteincoupled sphingosine 1-phosphate receptors. Together, ceramide, the sphingoid bases, and sphingosine-1-phosphate regulate a variety of cell functions, including apoptosis and cell replication. Their physiological actions interact with those induced by some cytokines, such as tumor necrosis factor alpha (TNF-a) or FAS ligand (CD95-ligand), that also induce apoptosis and otherwise influence cell survival and replication. Apoptosis is the earliest histopathological finding in liver and kidney following fumonisin exposure in many species. Rodent studies have shown that tissue sphinganine and sphinganine plus sphinganine-1-phosphate concentrations, indicative of ceramide synthase inhibition, increase at doses that are too low to cause apoptosis, while at higher doses increased sphinganine concentrations are positively correlated with increased apoptosis in target organs. As tissue injury and/or dose increases, cell necrosis (as opposed to apoptosis) is followed by mitosis and regenerative cell proliferation, as well as other manifestations of fully developed fumonisin toxicosis. Increased cell proliferation, in this case compensatory following necrosis, is one mechanism proposed for

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fumonisin B1 induced carcinogenesis in mouse liver and rat kidney (Dragan et al., 2001). Hepatocyte megalocytosis, indicative of mitotic disruption and often seen following exposure to hepatocarcinogens, was also observed in rodent studies. Support for the importance of the sphingolipid pathway in fumonisin B1 induced toxicity and carcinogenesis comes from studies with knockout (KO) mice for CerS2 (Pewzner-Jung et al., 2010). The mice are unable to synthesize very long acyl chain (C22–C24) ceramides and have elevated C16-ceramide and Sa levels. Increased rates of hepatocyte apoptosis and proliferation were observed and followed by regenerative hepatocellular hyperplasia that progressed to hepatomegaly and hepatocellular carcinoma. Digital gene expression indicated pathways related to cell-cell and cell-matrix interactions were up-regulated. Disruption of sphingolipid metabolism activates various downstream signaling pathways, including the activation of endoplasmic reticulum stress, PKC, and MAPKs, and alters membrane composition and influences lipid metabolism (reviewed by Riley and Merrill, 2019). These effects in turn lead to a wide variety of biological consequences, such as altered cell growth and differentiation, ER stress, changes in mitochondrial function, cell death, autophagy and lipid peroxidation. Oxidative stress induction by fumonisin B1 is thought to be due to impairment of both mitochondrial and cytosolic redox homeostasis (Liu et al., 2019). Accumulation of sphingoid bases, especially sphinganine, and the accompanying increase in the sphinganine:sphingosine (Sa:So) ratio in tissues, serum and urine, has been demonstrated in a variety of species and can be used as a biomarker of exposure both experimentally and diagnostically in naturally occurring disease in horses and pigs (Haschek et al., 2001; Hsiao et al., 2007; Merrill et al., 2001), These fumonisin induced sphingoid base changes are reversible, so they may not be evident after exposure ceases (Wang et al., 1992). Neural tube defects (NTDs) have been observed in LM/Bc and CD1 mice treated with fumonisin B1 (Voss and Riley, 2013). Fumonisins are considered a potential risk factor contributing to NTD in humans eating a diet high in fumonisin contaminated corn, further discussed below under Human Risk and Disease. The mechanism(s) of NTD induction in mice by

446

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fumonisin B1 has not been established but likely involves one or more interrelated cellular processes (Gelineau-van Waes et al., 2009). There is some evidence that depletion of complex sphingolipids associated with the folate binding protein reduces folate uptake and utilization, which in turn increases the risk of NTDs (Marasas et al., 2004). Other possibilities include cytotoxicity resulting from oxidative stress and signaling by cytokines or modulation of sphingosine 1-phosphate receptor-linked pathways by elevated levels of circulating sphingosine or sphinganine 1-phosphate. Supporting evidence for the latter includes induction of NTDs in LM/Bc mice by the sphingosine 1-phosphate receptor modulator FTY720. Pathophysiological evidence indicates that cardiac failure due to decreased cardiac contractility is the cause of pulmonary edema in swine (Haschek et al., 2001). Sphingoid bases such as sphingosine or sphingosine-1-phosphate are recognized as compounds that influence cardiovascular physiology, theoretically providing a biochemical mechanism for fumonisin cardiotoxicity. Inhibition of L-type calcium channels in the myocardium and in the vasculature by increased cardiac or serum sphingosine is the proposed mechanism of action (Constable et al., 2000; Smith et al., 2000). Advances in the field of sphingolipid-mediated pathophysiology and calcium signaling may further elucidate the mechanism of fumonisin-induced toxicity. In horses with ELEM, sphingolipid alterations in brain tissue were not evident, suggesting that the nervous system is affected secondarily (Haschek et al., unpublished). The primary site of action may be the cardiovascular system, since cardiovascular changes in the horse are similar to those observed in swine. In addition, L-type calcium channels occur in high concentrations in the cerebral arterioles, which are responsible for autoregulation of blood flow to the brain, which is of special importance when the horse lowers its head to eat and drink (Smith, 2018). Other molecular events resulting from fumonisin exposure are poorly characterized. It is generally accepted that fumonisins do not directly interact with DNA, and fumonisin–DNA adducts have not been found. Evidence of genotoxicity was not found upon evaluation of FB1 by various means, including the Salmonella typhimurium assay, unscheduled DNA synthesis assays in primary hepatocyte cultures and in rat liver after oral exposure to fumonisin B1, SOS chromotest

genotoxicity assays, and differential DNA repair and transforming activity studies in mouse embryo cells in vitro. Support for a nongenotoxic mechanism for fumonisin B1 carcinogenesis comes from a 26 week study in p53 heterozygous and p53 homozygous transgenic mice that showed no difference in cancer incidence after exposure to fumonisin B1, with hepatic adenomas and cholangiomas occurring after exposure to 150 mg/kg fumonisin B1 (Bondy et al., 2012). Evidence of DNA damage secondary to reactive oxygen species (ROS) or other undefined mechanism(s) has been demonstrated by Comet assay in target tissues of rats fed fumonisins. Fumonisin B1 induced lipid peroxidation in liver in vivo, in hepatocytes in vitro, and in rat liver nuclei in vitro. a-Tocopherol was protective in fumonisin B1-treated hepatocytes, a finding that suggests oxidative damage to DNA and other cellular macromolecules may be important from a mechanistic standpoint. More recent studies suggest that fumonisin B1 can cause significant DNA damage via epigenetic mechanisms (Arumugam et al., 2020). Therefore, while it is accepted that fumonisin B1 causes renal and hepatic neoplasia in rodents, the mode of action of carcinogenesis is uncertain and is likely a complex process involving more than one mechanism (see Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8; and Riley and Merrill, 2019).

7.3. Manifestations of Toxicity in Animals Overview The toxicity of fumonisins in laboratory and farm animals has been extensively reviewed, with Table 6.8 summarizing NOAEL and LOAEL data (Bolger et al., 2001; Bulder et al., 2012; EFSA, 2018a,c). While the liver is a target organ of toxicity in all species, other organs are affected in a species and sometimes strain and sex specific manner (Table 6.8). In addition to liver and kidney, the nervous, cardiovascular, respiratory and immune systems can be adversely affected. The kidney is a target organ in the rat and renal injury has been reported in a number of other species. Pulmonary edema in pigs and leukoencephalomalacia in horses is believed to occur secondarily to cardiovascular toxicity, which has also been reported in nonhuman primates. Adverse effects on the immune system, where studied, have been

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reported in all species and can result in greater susceptibility to infection. Equidae The effects of fumonisins on horses and swine have been summarized in detail by Smith (2018). Onset of naturally occurring disease in horses may occur as early as 7 days after exposure to a contaminated diet, but signs usually are first seen after 14–21 days; occasionally onset may be delayed 90 days or more. Outbreaks that affect several horses on the same farm are common. In 1901–02, over 2000 horses died in the USA as a result of ELEM; in 1934–35, over 5000 horses died in Illinois alone. In a given outbreak, the overall morbidity is generally low, less than 25%, but mortality usually approaches 100% in affected animals. This disease also occurs in donkeys. Two syndromes have been described in horses with naturally occurring disease: the neurotoxic and hepatotoxic forms, with these occurring independently or concurrently. However, this may be related to the concentration of fumonisins in the diet or amount ingested (contaminated feed is often unpalatable and fumonisin distribution uneven) and the duration of exposure as well as individual animal susceptibility (Smith, 2018). In the field, high-dose exposure is thought to increase the likelihood of the hepatotoxic form, with the more frequently encountered lower doses favoring the neurotoxic form, ELEM. The clinical course of the neurologic disease is generally short with an acute onset of signs followed by death within hours or days. Decreased feed intake, depression, ataxia, blindness, and hysteria are reported. Anorexia occurs due to glossopharyngeal paralysis, and paralysis of the lips and tongue, with loss of ability to grasp and chew food. Incoordination, circling, ataxia, head pressing, marked stupor, and hyperesthesia are common, as are hyperexcitability, profuse sweating, mania, and convulsions. Acutely affected animals often progress through the manic and depressive stages of the syndrome within 4– 12 h of onset and become recumbent and moribund. Death may occur without clinical signs. If hepatotoxicity is the presenting disease, it often takes 5–10 days from time of onset of clinical signs to death. Icterus is usually prominent, and there

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may be edema of the head and submandibular space, as well as oral petechiae. Elevated serum bilirubin concentration and liver enzyme activities are typically present. Terminal neurologic signs may be noted, possibly due to secondary hepatoencephalopathy. The liver is often small and firm, with an increased lobular pattern. Centrilobular necrosis and moderate to marked periportal fibrosis can be observed histologically. Classical neurologic lesions consist of focal or multifocal areas of liquefactive necrosis of the white matter, primarily in the cerebrum (Figure 6.14A). The liquefactive necrosis, most commonly located in the subcortical white matter, is often evident grossly as cavitation or discoloration. Histologically, necrosis with influx of gitter cells, edema, and hemorrhage are primary lesions (Figure 6.14B). Some cases may only exhibit histologic lesions consisting of perivascular edema and hemorrhage, with infiltration of mononuclear and plasma cells and occasionally eosinophils. Experimental administration of purified fumonisin B1, either per os or iv, can induce both neurologic and hepatic disease in horses, with these generally occurring concurrently. The clinical signs and time course of the neurologic disease are similar to those in naturally occurring disease. Horses with neurologic disease had cardiac dysfunction, increased protein in the cerebrospinal fluid and other changes consistent with vasogenic cerebral edema (Foreman et al., 2004; Smith, 2018). Histologic lesions characterized by perivascular edema and hemorrhage, primarily of the white matter, were found in both brain and spinal cord. Serum biochemical evidence of hepatic injury was present in horses with and without neurologic disease (Haschek et al., unpublished data). The LOAEL dose was 0.01 mg/kg iv for 28 days and approximated oral ingestion of fumonisin B1 at 8 ppm. These findings contradict the clinical literature, which suggests that the neurologic form occurs at lower exposure levels while the hepatic form occurs at higher exposure levels. Hepatic lesions were characterized by hepatocellular apoptosis and necrosis. Sphingosine and sphinganine levels were elevated in serum and tissues such as heart, liver, and kidney, but not in brain. An increase in serum cholesterol concentration was also present.

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Cardiovascular abnormalities in horses with neurologic disease were similar to those described for swine below, and included decreased cardiac contractility, heart rate, arterial pulse pressure, and increased systemic vascular resistance (Smith et al., 2002).

FIGURE 6.14 (A) Leukoencephalomalacia in the left cerebrum of a horse whose diet was contaminated with fumonisins. Focal malacia (necrosis, arrows) is confined to the subcortical white matter. (B) Leukoencephalomalacia in the cerebrum of a horse. At low magnification, the white matter is severely disrupted (coagulated [liquefactive necrosis]) and there is accumulation of proteinaceous fluid, scattered neutrophils, and abundant macrophages. The interface between the gray (G) and affected white matter contains diffuse edema, perivascular hemorrhage (not shown here), and blood vessels with small leukocytic cuffs. Blood vessel walls are degenerate or necrotic, and some are infiltrated with neutrophils, plasma cells, and eosinophils. Inset: Note the influx of blood monocytes that mature into tissue macrophages and become “gitter” cells as they phagocytose necrotic debris. H&E stain. Figures reproduced from Fundamentals of Toxicologic Pathology, second Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), Figureure 13.20 and 21, p. 403, with permission.

Swine Outbreaks of a fatal disease in swine fed Fusarium verticillioides-contaminated corn screenings from the 1989 corn crop in the Midwest and Southeast regions of the United States led to the identification of fumonisin B1 as the causative agent of porcine pulmonary edema (PPE). Thousands of pigs died in these outbreaks. In Hungary, outbreaks of this disease have been reported since the 1950s. A decline in feed consumption is usually the first sign following exposure to fumonisin contaminated feed. If fumonisin consumption is high, acute pulmonary edema and death generally follows within 4–7 days after onset of feeding the contaminated diet. Typically, deaths cease within 48 h after animal withdrawal from contaminated food. PPE has been reproduced experimentally in swine by feeding naturally contaminated feed, fumonisin-containing culture material, and purified fumonisin B1 (Haschek et al., 2001). Pulmonary edema is not a finding in any other species. Severe pulmonary edema and hydrothorax occur after several days of treatment in the acute form of fumonisin intoxication (Figure 6.15A). Edema appears to originate in the interstitium, with perivascular edema and markedly dilated lymphatics a prominent feature early in the disease (Figure 6.15B). Liver injury is similar to that found in other species, and is characterized by scattered hepatocellular apoptosis, necrosis, and proliferation. Pancreatic necrosis has been reported in some studies. Alterations in clinical pathology reflect hepatic injury and total serum cholesterol concentration is elevated. Progressive and marked elevations in sphinganine and sphingosine are found in serum and in major organs such as kidney, liver, lung, and heart, indicating a major disruption in sphingolipid biosynthesis. Although altered myocardial morphology has not been documented in studies using purified toxin, fumonisin B1 was shown to decrease cardiac contractility, mean systemic arterial pressure, heart rate, and cardiac output, and increase

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mean pulmonary artery pressure and pulmonary artery wedge pressure. These changes are compatible with the inhibition of L-type calcium channels due to increased sphingosine and/or sphinganine. Ultimately, acute left-sided heart failure occurs and results in pulmonary edema. At lower doses, slowly progressive liver disease has been demonstrated in pigs. Subacute hepatic injury was characterized by hepatocellular cytomegaly, disorganized hepatic cords, and early perilobular fibrosis, while chronic injury resulted in icterus with severe hepatic fibrosis and nodular hyperplasia. Additional findings reported in some studies include esophageal plaques and right ventricular hypertrophy due to pulmonary hypertension (Haschek et al., 2001). In more recent studies, low-dose, longer term feeding studies have shown mild pulmonary edema that progressed to fibrosis localized primarily around lymphatics (ZomborszkyKovacs et al., 2002). As with other mycotoxins, fumonisins affect the immune system. Impairment of innate and acquired immune responses, as well as increased susceptibility to enteric and pulmonary infections have been reported. In swine, fumonisin B1 decreased phagocytosis and inhibited sphingolipid biosynthesis in pulmonary macrophages, and decreased clearance of particulates and bacteria from the pulmonary circulation (Haschek, unpublished data).

FIGURE 6.15 Lung from a field case of fumonisin toxicoses, pig. (A) Pulmonary edema: interlobular septa are widely distended by edema. (B) Prominent proteinaceous interstitial edema is present in the pleura (top) and interlobular septum. Note the widely dilated lymphatics (L) present in the septum and pleura (H&E, 4X) (Images courtesy of Sandeep Akare). Figure 6.15A reproduced from Fundamentals of Toxicologic Pathology, second Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), Figure 6.18A, p. 121, with permission. Figure 6.15B reproduced from Handbook of Toxicologic Pathology, second Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 14A, p. 679, with permission.

Laboratory Animals Similarly to horses and pigs, one of the main target organs in rodents and rabbits is liver. The kidney is also an important target organ in these species. Early pathology studies in animals given purified fumonisin B1, culture material (corn that is infected with a single fungal isolate and then molded under controlled laboratory conditions), or corn naturally contaminated with fumonisins variously described the lesions as “hepatopathy” or “hepatosis,” and “nephropathy” or “nephrosis” because of its noninflammatory nature. Many studies have been performed but the most extensive are the long term feeding studies with F344 rats and B6C3F1 mice conducted by the National Toxicology Program, of the US NIEHS (NTP, 2001). In the liver, the earliest finding was hepatocellular apoptosis. Affected cells were scattered throughout the hepatic lobules. Inflammation was usually absent. With

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increasing hepatocellular injury, apoptotic cells become more numerous, and cytoplasmic vacuolation, mitotic figures, and necrosis were increasingly found. Cytomegaly, anisocytosis, and anisokaryosis occurred as injury progressed (Figure 6.16A). Chronic nonneoplastic lesions (NTP, 2001) consisted of bile duct and oval cell proliferation, fibrosis, nodular regeneration (regenerative hyperplasia), foci of cellular alteration, and cholangiomatous lesions (Figure 6.16B). There was loss of parenchyma around and between central veins. In mice, Kupffer cells or macrophages containing pigment were variably present in the centrilobular zone. Inflammation remained minimal and, when present, was usually associated with focal necrosis. g-Glutamyl transferase (GGT) and glutathione-S-transferase p (GST-P) positive foci were readily demonstrated in rats. Serum biochemical profiles supported the microscopic findings. Alanine and aspartate aminotransferase, alkaline phosphatase, and GGT activities, as well as bile acids and bilirubin concentrations, were increased. Hypercholesterolemia occurred readily. Leukocytosis and altered differential leukocyte counts, thrombocytopenia, changes in serum immunoglobulin levels, all suggested that fumonisins exert immunological and hematological effects. Neoplastic hepatic lesions have been found only in male BD IX rats and female B6C3F1 mice (Gelderblom et al., 1991; NTP, 2001). They have been variously classified as neoplastic nodules, hepatocellular adenomas, or carcinomas. Proliferative biliary lesions, such as cholangiofibrosis, angiofibrosis, and cholangiocarcinoma, occurred only in rats. Hepatocellular carcinomas in rats ranged from well-differentiated to more anaplastic, poorly differentiated types of neoplasms. In female B6C3F1 mice, hepatic tumors ranged from discrete adenomas containing welldifferentiated cells to hepatocellular carcinomas with poorly differentiated, anaplastic cells organized in the trabecular or adenoidal patterns commonly found in murine liver carcinomas. The kidney is the other major target organ in rodents. In Sprague–Dawley and F344 rats, males were more sensitive to nephrotoxic effects than females. In contrast, mice are relatively resistant to fumonisin-induced nephrotoxicity. Lesions, when found, generally consisted of a few scattered apoptotic tubular epithelial cells.

FIGURE 6.16 Effect of fumonisin B1 on liver. H&E stain. (A) Early changes, rat. Hepatocellular apoptosis (arrow) characterized by individualization and cytoplasmic condensation of cells, with margination of chromatin in eccentrically located nuclei. (B) and (C) Chronic dietary exposure, mouse. (B) Polyploid hepatocytes (arrows) are characterized by an enlarged hyperchromatic nucleus.

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FIGURE 6.17 Kidney, subchronic dietary exposure to fumonisin B1 and other fumonisins provided by corn culture material, male rat. Characteristic basophilia of tubules in the outer stripe of the outer medulla (OSOM) and detached apoptotic or preapoptotic cells (arrows). H&E stain. Figure reproduced from Handbook of Toxicologic Pathology, third Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Figure 39.16, p. 1236, with permission.

As in liver, apoptosis is the earliest finding in the kidney (Figure 6.17). Apoptotic epithelial cells were scattered throughout the proximal convoluted tubules of the outer stripe of the outer medulla. Apoptotic cells detached from adjacent cells and sloughed into the tubular lumen. Accelerated apoptosis has been defined as a pivotal feature in the renal toxicity of fumonisin (see Kidney, Vol 5, Chap 2). More advanced renal lesions extended into the adjacent inner cortex and had both regenerative and atrophic components. Regenerative tubules were lined by epithelium that is basophilic and hyperplastic, often cuboidal rather than columnar, and with occasional mitoses. Mitoses were found more readily in advanced cases. Other tubules

= Megalocytosis (arrowhead) with nuclear membrane invagination (pseudoinclusion) and oval cell proliferation are also present. (C) Large numbers of oval cells, small basophilic cells (arrowheads), extend out from the periportal region separating hepatocytes. An occasional apoptotic cell is present (arrow). Figures reproduced from Fundamentals of Toxicologic Pathology, second Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), A from Figure 9.9 (p214), B from Figure 9.17 (p. 221), and C from Figure 9.21 (p. 223), with permission. Figs B and C from slide courtesy of Dr D. Caldwell, Health Canada.

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were atrophic and had flattened, squamous epithelia that made the lumina appear distended, and the basement membrane of affected tubules was amorphously thickened. Interstitial fibrosis was present in the most advanced cases. Inflammation was not a prominent feature. Decreased kidney weight and clinical signs of tubular dysfunction accompanied the renal lesions. The latter included increased serum creatinine and decreased serum total carbon dioxide. Urine output and water consumption were transiently increased. Other urinary findings included decreased osmolality; increased enzyme activities of GGT, N-acetyl-b-glucosaminidase, and lactate dehydrogenase; inhibition of r-aminohippurate and tetraethylammonium transport; and proteinuria. Glomerular involvement was negligible. Renal tubular adenomas and carcinomas have been found in F344 male rats fed high doses of fumonisins over an extended period (50 ppm for 2 years (Hard et al., 2001; Howard et al., 2001; NTP, 2001). Tumor morphology ranged from a clear cell type to a sarcomatous variant composed of spindle-shaped cells. Many of the carcinomas displayed a high degree of anaplasia, numerous mitoses, aggressive infiltration of the surrounding tissue, and metastases to the lung and lymph nodes. The neoplasms arose in kidneys that displayed varying degrees of apoptosis, tubular basophilia, regeneration and hyperplasia, and focal tubular atrophy, suggesting that an imbalance between cell loss and replication played a role in tumor induction. NONHUMAN PRIMATES

There are no reports on the pathological effects of pure fumonisins in nonhuman primates. However, two of three baboons fed a diet contaminated with a strain of Fusarium verticillioides, now known to produce fumonisins, died of acute congestive heart failure after 143–248 days of exposure. After 720 days of exposure, the third baboon was found to have cirrhosis. Other findings in nonhuman primates (vervet monkeys) suggest that fumonisins may have cardiovascular, in this case atherogenic, effects based on serum chemical findings including elevated plasma cholesterol, low density lipoprotein C, and apoprotein B, as well as fibrinogen and coagulation factor VIII activation (Gelderblom et al., 2001; Kriek et al., 1981). South African scientists conducted a 13.5-year study on the effects of fumonisin-containing

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diets (varying concentrations) in vervet monkeys. Clinical findings suggestive of liver dysfunction included increased alanine and aspartate aminotransaminase, GGT, and lactate dehydrogenase activities. As in other species, serum cholesterol concentrations were also elevated. Serum creatinine concentrations were increased and creatinine clearance was decreased, suggesting that fumonisins affected renal function. Descriptions of microscopic lesions were confined to the liver, which was clearly a target organ. Lesions were consistent with those seen in other species, and included apoptosis, bile duct proliferation, nodular hyperplasia, and perilobular fibrosis. Mitotic figures and apoptotic cells were present in the nodular structures (Gelderblom et al., 2001).

7.4. Human Risk and Disease The risks to humans posed by fumonisins are undetermined at present; however, the potential risk to populations that consume large amounts of corn on a regular basis include cancer, NTDs, and growth impairment in children. The International Agency for Research on Cancer considers fumonisin B1 as possibly carcinogenic to humans based on inadequate evidence in humans for carcinogenicity but sufficient evidence in experimental animals (Group 2B) (IARC, 2002). Correlations between consumption of moldy, “home-grown” corn as a dietary staple and high rates of esophageal cancer have been reported. The main types of esophageal cancer are squamous cell carcinoma and adenocarcinoma. Adenocarcinoma is the primary type in developed countries and squamous cell carcinoma in developing countries such as China. Esophageal cancer in developing countries occurs in both men and women, most often at age 50–60 years. It is usually detected late in the disease and therefore prognosis is poor. Retrosternal pain and dysphagia are common clinical signs due to the mass causing esophageal stricture. The tumors are located most frequently in the middle third of the esophagus, followed by the lower third and then the upper third. Ulceration is common. The studies of correlations between fumonisin exposure and esophageal cancer in the Transkei, southern Africa, and Linxiang, north central China, were observational studies with fumonisin concentrations measured in corn. The few studies where sphingolipid biomarkers were examined did not show

a significant association with esophageal cancer (Abnet et al., 2001). However, a recent study performed in the Huaian area, in which serum aflatoxin B1-lysine adduct and urinary fumonisin B1 were measured, showed an association between aflatoxin and fumonisin exposure and risk of esophageal squamous cell carcinoma; there was a greater than additive interaction between these two mycotoxin coexposures (Xue et al., 2019). Similar coexposure to aflatoxin B1 and fumonisin B1 has been shown in the Linziang region. It has been speculated that fumonisins also may be an etiological factor for liver cancer in China, especially in regions where fumonisins and aflatoxin occur together. Liver cancer rates are also very high in Guatemala in both men and women (as mentioned in the section on aflatoxins) and significant exposure to fumonisins has been demonstrated in this population (Smith et al., 2017; Torres et al., 2014). Liver cancer is also one of the more frequently encountered cancers in men from the Transkei region of South Africa. These observations do not conclusively show a link between fumonisins and cancer in man, as a number of other factors have been cited as being the cause of, or contributing to, esophageal and other cancers that are found in these regions. Among these are nitrosamines from food and tobacco, mineral deficiencies in the soil, and poor nutrition. Furthermore, it remains a possibility that other mycotoxins, acting alone or in concert with fumonisins, are the cause of the esophageal cancer that has been associated with Fusarium verticillioides. Support for this possibility comes from the lack of data from chronic experimental studies in rodents and nonhuman primates that would implicate the esophagus as a target organ of fumonisins (Gelderblom et al., 2001; NTP, 2001). Perhaps of concern for humans is the link between cardiovascular disease and fumonisins documented in swine, horses, and nonhuman primates, and the consistent finding of elevated cholesterol levels in all species exposed to fumonisins as discussed above. High level exposure to fumonisin in utero is thought to be a risk factor for NTDs (Gelineauvan Waes et al., 2009). NTDs are anomalies resulting from failure of the neural tube to fully close during early pregnancy (see Embryo, Fetus and Placenta, Vol 5, Chap 11). NTDs present in multiple forms, including spina bifida, exencephaly, anencephaly, and craniorachischisis (externalization of the brain and spinal cord):

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severe forms are incompatible with life. NTDs appear to be multifactorial in origin involving complex gene-nutrient- environment interaction, with folic acid supplementation known to decrease the occurrence. Fumonisins were implicated in an NTD outbreak that occurred within the Mexican-American population of South Texas during 1990–91. Associated factors included higher than average fumonisin concentrations in the 1989 corn crop, a simultaneous increase in Texas ELEM cases (see above), and reports that NTD rates are high in areas of northern China, Guatemala, and southern Africa, where the local population is heavily dependent on corn as a food source. Retrospective case-controlled investigations conducted in the affected area (1995–2000) indicated that the risk of an NTD-affected pregnancy increased with increasing tortilla consumption during the first trimester of pregnancy up to a critical amount and decreased thereafter. The same “inverted U”-shaped response was found when NTD outcome and maternal postpartum serum sphinganine/sphingosine ratios, an indicator of fumonisin exposure, were considered. These results, while not definitive, provide the basis for further investigations on fumonisins as potential risk factors for NTDs in humans. Growth impairment in children is a serious global public health risk, with highest prevalence in sub-Saharan Africa, South Asia, and Central America. Interest in the contribution of dietary mycotoxins, such as fumonisins, to this problem has increased since many animal studies have shown reduced weight gain and feed efficiency. However, this has been observed mainly in studies using partially purified fumonisin mixtures and fungal culture materials. Several epidemiological studies from Tanzania have shown an association between ingestion of fumonisin contaminated food and stunting in children, with one study using fumonisin B1 concentration in urine samples as an indicator of exposure (Chen et al., 2018). Human exposure is dietary, with the toxins found in a variety of commercial foods, including corn meal, corn flour, grits, masa, polenta, snack foods, and beer. In general, corn meal and baking mixes have higher levels than more highly processed products. Sweetcorn and popcorn have low fumonisin concentrations, and residues in

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meat, milk, and eggs are negligible. Milling and distilling do not appreciably degrade these mycotoxins. Fumonisins distribute unevenly in milling fractions, with higher amounts found in bran, gluten, distiller’s grains, steep liquids, flour, meal, and grits. Cornstarch, an important commercial product used in sweeteners, contains negligible amounts of fumonisins. Fumonisins are found in corn-based food products because they are relatively heat stable under many cooking and processing conditions. However, alkaline cooking (known as nixtamalization) effectively reduces fumonisins in tortillas and other masa products (Voss et al., 2017). Reduction is achieved by a combination of extraction into the (discarded) cooking liquid, conversion of fumonisins to the less toxic hydrolyzed forms, and perhaps formation of other reaction products. Depending on the specific conditions, extrusion cooking, which combines high heat and pressure, can significantly reduce fumonisins in the cooked corn products. The amount of reduction is enhanced if glucose or another reducing sugar is added before processing. Extruded corn products and the fumonisin glucose reaction products N-(1-deoxy-D-fructos-1-yl) fumonisin B1 and N-carboxymethyl fumonisin B1 exhibited reduced toxicity in bioassay experiments. Efforts to develop robust and reliable methods for routinely assessing fumonisin exposures in humans continue. The increase in serum, whole blood, or tissue sphinganine or sphinganine 1-phosphate concentrations or sphinganine:sphingosine ratios that arise as a result of ceramide synthase inhibition have proven to be useful exposure biomarkers in laboratory animals. Their use in epidemiological surveys has, however, been limited, and is problematic due in part to the rapid reversibility of these effects. Methods for measuring fumonisins in urine, feces and tissues have been developed. While the use of urinary fumonisin concentration as an exposure biomarker has been limited, this approach shows promise, as illustrated by the results of a study of South African subsistence farmers in which a positive correlation was found between urinary fumonisin B1 excretion (normalized to creatinine) and dietary exposure. A study in Guatemala showed that high levels of fumonisin B1 intake were correlated with changes in sphinganine 1-phosphate and the sphinganine 1-phosphate/

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sphingosine 1-phosphate ratio in human blood in a manner consistent with FB1 inhibition of ceramide synthase (Riley et al., 2015).

7.5. Diagnosis, Treatment, and Prevention In horses and pigs, diagnosis of ELEM or PPE is based on a history of ingestion of corn, particularly corn screenings or unscreened corn, together with characteristic clinical signs and lesions. Detection of approximately 10 ppm fumonisin in horse feed or >50 ppm in swine feed is highly suggestive of toxicosis. However, lower levels or lack of detectable fumonisin does not eliminate the possibility of fumonisin toxicosis, since feed originally present may no longer be available for testing or false negative results are obtained because mycotoxin distribution in the suspect feed lot is heterogenous. Assays (HPLC derivatization) are now available for fumonisins at some veterinary diagnostic laboratories, and ELISAbased screening tests for fumonisins in feed are commercially available. Recovery rates from food and feed may be poor because of the presence of hidden or matrix-bound fumonisins. Elevated sphinganine in serum and/or tissues (frozen or formalin fixed) is an excellent biomarker of exposure, although this assay is not routinely available in diagnostic laboratories. Research on the human population has shown that there is a good correlation between fumonisin B1 in urine and that in recently consumed corn (Riley et al., 2012; Torres et al., 2014). There is no known effective therapy. Removal of animals from contaminated feed may be effective in exposed but nonaffected animals. Fumonisin reduction in crops can include preharvest and postharvest interventions. Good agricultural practices and development of resistant corn varieties is a start. GMO technology has been used to develop corn more resistant to the European corn borer and therefore to fungal infection. Fungicides or biological control methods can be used to further mitigate fungal infection. Postharvest interventions largely focus on following the regulation and guidance provided below. Food processing, such as nixtamalization (alkaline cooking with lime) which is extensively used in central and south America, as well as by the Hispanic population in the US, in the preparation of tortillas, can significantly reduce the amount of fumonisin B1. For animal

feed, corn screenings should not be fed to susceptible species. Use of clay adsorbents or ammoniation, as used for some other mycotoxins, has not proved successful. However, in Europe, a commercial fumonisin esterase has been shown to be a safe and effective additive for degrading fumonisin in feed used for pigs and poultry (EFSA, 2018e).

7.6. Regulations and Guidances In 2018, a tolerable daily intake (TDI) of 1.0 mg/kg bw/day was established for fumonisin B1 by the EFSA Panel on Contaminants in the Food Chain (CONTAM) (EFSA, 2018a). This was partly based on a chronic study in mice in which an increased incidence of megalocytic hepatocytes was found (Bondy et al., 2012). Fumonisins B2, B3, and B4, but not modified fumonisins, were included with fumonisin B1 in a group TDI. However, the uncertainty of the assessment was considered high. Previously, a group TDI for fumonisins B1, B2 and B3, alone or in combination, of 2.0 mg/kg bw had been set by JECFA in 2001 and retained in a subsequent reevaluation (Bolger et al., 2001; Bulder et al., 2012). In addition, the US FDA has established advisory levels for total fumonisins B1 þ B2 þ B3 in corn for human consumption, which varies from 2 to 4 ppm, depending on intended use of the corn (FDA, 2001; FDA, 2021). EFSA also evaluated the risks for animal health from fumonisins (B1, B2 and B3) (EFSA, 2018c). The NOAEL or LOAEL was identified from the available literature and an additional factor of 1.6 was applied to the data to account for the possible occurrence of hidden forms of fumonisins. Sphingolipid alterations were considered a biomarker of exposure and not an adverse effect per se. Based on mean exposure estimates from European countries, the risk of adverse health effects of feeds containing fumonisins B1–3 was considered very low for ruminants, low for poultry, horse, rabbits, and fish, and of potential concern for pigs. The US FDA recommends that corn and corn by-products intended for equids and rabbits contain aflatoxin B1) suggest that adduct conformational differences contribute to differences in mutagenic potency between aflatoxin B1 and sterigmatocystin (Baertschi et al., 1989; EFSA, 2013). Sterigmatocystin causes necrosis, blocks mitosis, inhibits DNA and RNA synthesis, and causes cell cycle arrest at the G2 phase in mammalian cells. In vitro studies have identified multiple pathways involved in cellular toxicity and responses to toxic insult by sterigmatocystin, including impaired mitochondrial ATP synthesis that may contribute to cell death, and oxidative stress that may contribute to cellular damage and necrosis. Induction of MAPK and PI3K signaling pathways in response to DNA damage, leading to regulation of cyclins, can lead to cell cycle arrest (EFSA, 2013). MANIFESTATIONS OF TOXICITY IN ANIMALS

Sterigmatocystin is 10–100 times less acutely toxic than aflatoxin B1. Oral LD50s for aflatoxin B1 in rodents range from 1 to 18 mg/kg bw (EFSA et al., 2020a). In comparison, the acute oral LD50 of sterigmatocystin in rats was 166 and 120 mg/kg in male and female rats, respectively. The LD50 for 5-day old chicken embryos exposed to sterigmatocystin was 14.9 mg/egg (EFSA, 2013). Acute and subacute oral exposure caused dose-dependent necrosis and hemorrhage in the liver and kidneys of rats (Purchase and van der Watt, 1969, 1970). Hepatic changes present in liver biopsies from vervet monkeys exposed intragastrically to 20 mg/kg bw sterigmatocystin every 14 days for 12 months included single-cell necrosis of hepatocytes, and progressive expansion of the fibrous septa with disruption of the lobular architecture. After 12 months, large hyperplastic nodules containing hepatocytes with pleomorphic nuclei were evident. The liver is also the major target organ in pigs and poultry. Long term exposure to sterigmatocystin is carcinogenic to rats, mice, Mongolian gerbils, monkeys and fish, resulting in hepatocellular carcinomas, hemangiosarcomas in the liver, angiosarcomas in brown fat, lung adenomas, and other lesions (EFSA, 2013).

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Sterigmatocystin has not been definitively linked to human illness or disease, although higher incidence and content of sterigmatocystin in grains were detected in high versus low incidence areas for stomach and liver cancers in China. Several studies of Chinese patients explored potential associations between sterigmatocystin exposure and cancer. Sterigmatocystin–DNA adducts were found in 14 out of 28 tissue samples of tumors collected from 12 Chinese patients with liver or stomach cancer. In another study, there was a statistically significant higher prevalence of sterigmatocytins in blood from patients with liver cirrhosis or liver cancer (EFSA, 2013; WHO, 2018). Since sterigmatocystin has been demonstrated to be genotoxic and carcinogenic in animal studies, the lack of conclusive data on associations between human cancers and sterigmatocystin exposure does not obviate concerns about health risks due to human exposure. However, EFSA concluded that a sterigmatocystin exposure of low human health concern (0.016 mg/kg b.w. per day) would range from 1.5 to 8 mg sterigmatocystin/kg in grain and grain-based foods, and recommended that further occurrence data are needed for both food and feed (EFSA, 2013).

9.4. Tremorgenic Mycotoxins Source/Occurrence The tremorgenic mycotoxins are a large group of indole-diterpene secondary metabolites notable for their production by taxonomically discontinuous fungi. Members of this group are neurotoxins that cause intermittent or sustained tremors in vertebrates. The producing organisms grow on, or are associated with, a variety of foods, plants, and other substrates. A partial list of tremorgenic toxins includes the Penicillium-associated tremorgens penitrem A (Figure 6.26) and roquefortines, found in spoiled meat, cheese, eggs, cereals, nuts, processed foods, and compost. These toxins are associated with intoxications caused by ingesting moldy food or refuse, often by dogs due to their scavenging behaviors. Other toxins, like verruculogen, tryptoquivaline tremorgens, territrems A and B, and aflatrem, are produced by Penicillium and Aspergillus sp. that grow in soil, on cereal grains and other cereal-

HO

O

Cl

N H

OH O

OH O

FIGURE 6.26 Chemical structure of penitrem A (www.chemispider.com).

based substrates. Endophytic fungi in the genera Neotyphodium and Claviceps are associated with an intoxication known as “ryegrass staggers” in grazing animals. Ingestion of perennial ryegrasses contaminated with lolitrems A-D, paxilline and lolitriol produced by Neotyphodium sp., or the Claviceps-produced paspalitrems and paspalinine are the causative agents. Janthetrims A, B and C are also produced by Penicillium sp. that grow on pasture ryegrasses (Evans and Gupta, 2018; Saikia et al., 2008). Toxicology, Toxicokinetics and Mechanism of Action Tremorgens are lipophilic. Once ingested, uptake from the GI tract is rapid and maximum plasma concentrations are reached within 30 min in most species, with rapid distribution to tissues. They easily cross the blood –brain barrier. Signs of toxicity may be apparent within hours, as in dogs ingesting moldy food containing penitrem A, or days, as in ruminants consuming tremorgen-contaminated grasses. Tremorgenic toxins are not thought to accumulate in tissues, and are excreted primarily via biliary excretion into the feces (Evans and Gupta, 2018). Penitrem A undergoes Phase I biotransformation in intoxicated dogs, leading to the presence of mono- and di-oxygenated isomers, as well as hydrated products, in plasma (Uhlig et al., 2020).

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The cellular targets of tremorgen toxins are not fully understood but, in general, tremorgens interfere with inhibitory neuroreceptors and enhance excitatory amino acid neurotransmitter release mechanisms. It is uncertain that the mode of action is the same for all tremorgenic toxins, and some structure-function differences have been noted (Evans and Gupta, 2018; Reddy et al., 2019). In experimental systems, tremorgens inhibit gamma aminobutyric acid (GABA) receptors and antagonize large conductance calcium-activated potassium ion channels (Big Potassium or BK channels) (Imlach et al., 2008; Moldes-Anaya et al., 2011). Reactive oxygen species production, disruption of calcium homeostasis and activation of the JNK pathway may also play a role in penitrem A-induced neurotoxicity (Berntsen et al., 2013). Manifestations of Toxicity in Animals Upon consuming refuse or spoiled food contaminated with tremorgenic toxins, dogs suffer rapid onset of continuous, generalized tremors that may be severe enough to resemble seizures. Further symptoms include excessive salivation, panting, pyrexia, dilated pupils, nystagmus, hypersensitivity to noise and touch, vomiting, diarrhea, flatulence, and tachycardia. In mild cases, presumably due to the ingestions of relatively low doses of tremorigenic toxins, dogs may recover within 24–96 h. Severely affected dogs may have persistent symptoms cerebellar injury, ataxia and weakness for months or years (Barker et al., 2013; Hocking et al., 1988). Massive liver necrosis, convulsions and death occurs in dogs acutely exposed intraperitoneally to high doses (up to 5 mg/kg) of penitrem A (Hayes et al., 1976). Similar symptoms occur in laboratory rodents under experimental conditions. Rats injected ip with 3 mg/kg penitrem A developed a whole-body tremor within 10 min, with half developing episodes of extensor spasms; facial myoclonus and nystagmus were also observed. Tremor intensity peaked at 6 h after dosing and was no longer observed after 72 h. Rats appeared to be normal 8 days posttreatment, except for a hopping gait likened to that of sheep afflicted with ryegrass staggers. Pathological changes were seen only in the cerebellum and consisted of widespread Purkinje cell degeneration and necrotic foci in cerebellar granule cell

layers (Cavanagh et al., 1998). Mice injected ip with lolitrem B or aflatrem displayed whole body tremors, uncoordinated gait, hyperactivity and hypersensitivity to sound and touch, with lolitrem B eliciting more severe responses (Gallagher and Hawkes, 1986). Ryegrass stagers have been reported in grazing animals such as horses, cattle, sheep, and deer. Intoxications are more prevalent in the summer and early fall, with drought stress and over grazing contributing to increased incidence. Head tremors and muscle fasciculations in the neck and legs are followed by head nodding, swaying, hypersensitivity, and transient ataxia aggravated by stimulation, evident as the disease progresses. Death does not normally occur, unless by misadventure while symptomatic (Plumlee and Galey, 1994; Reddy et al., 2019). Human Exposure and Disease Descriptions of human intoxication associated with tremorgenic mycotoxins indicate that symptoms are similar to those seen in other animals. Human illness suggestive of tremorgenic mycotoxicosis has been documented after consumption of beer or soup contaminated with Penicillium crustosum (Cole et al., 1983; Lewis et al., 2005).

9.5. Fusarium Toxins Beauvericin and Enniatins SOURCE/OCCURRENCE

Beauvericin and enniatins are structurally related cyclic hexadepsipeptide mycotoxins (Figure 6.27). They are most often found in cereal grains as a result of preharvest colonization by various species of Fusarium plant pathogens (Tolosa et al., 2019). Of the almost 30 enniatin analogues identified so far, enniatins A, A1, B and B1 are most frequently detected in foods and feeds (EFSA, 2014). TOXICOLOGY, TOXICOKINETICS AND MECHANISM OF ACTION

Partial information on beauvericin and enniatin toxicokinetics is available for rodents, pigs, and poultry. Beauvericin, enniatin A and enniatin B are detectable in rat plasma after oral administration in feed or by gavage. In a single

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N

O O

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respectively, in mice. The genotoxic potential of enniatin B, but not beauvericin, was demonstrated in Comet assays performed on tissues from mice after acute oral exposure. Repeated exposure to beauvericin or enniatin B by oral gavage to male and female mice caused a spectrum of mild changes in thyroid, adrenals, kidneys, spleen, thymus, duodenum, and reproductive organs, with NOAELs ranging from 0.1 to 1.8 mg/kg bw/d. Taken together, mouse studies indicate a potential for endocrine and immune disrupting effects of beauvericin or enniatins. Rodent reproductive and developmental toxicity assays were largely negative.

O O

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N

O

O

FIGURE 6.27 Chemical structure of beauvericin A (www.chemispider.com).

pig, a bolus intragastric dose containing 0.5 mg/ kg bw of each toxin resulted in relative maximum plasma concentrations of enniatin B > B1 > A1 > A, with beauvericin levels near or below detection limits. High oral bioavailability and rapid elimination were observed in pigs given a single oral dose of 0.5 mg enniatin B/kg bw. Relatively low concentrations of beauvericin and enniatins B and B1 reach the liver, muscle and eggs of poultry exposed through feed. Hydroxylated or carboxylated biotransformation products of enniatin B have been detected in chicken liver, serum or eggs (EFSA, 2014). In addition to antibiotic, insecticidal and cytotoxic activities, beauvericin and enniatins are known for their ionophoric properties. Due to their lipophilicity, the enniatins and beauvericin have membrane channel-forming properties and can also act as carriers, facilitating the transport of monovalent ions across membranes and disrupting ion balance, pH and mitochondrial functions. Inhibition of enzymes, such as acyl-CoA:cholesterol acyltransferase, 30 ,50 cyclo-nucleotide phosphodiesterase, topoisomerases, and various transport proteins has been demonstrated in vitro (EFSA, 2014; Tonshin et al., 2010). MANIFESTATIONS OF TOXICITY IN ANIMALS

Poultry are relatively insensitive to beauvericin and/or enniatins in contaminated feeds. Oral LD50s for beauvericin or an enniatin mixture were 100 and 350 mg/kg bw,

HUMAN EXPOSURE AND DISEASE

There are no reports of human illness or disease associated with exposure to beauvericin or enniatins in food. A comprehensive review by the EFSA concluded that no human health concern was indicated for acute exposure to beauvericin and enniatins, but that, based on gaps in the in vivo toxicology data, no firm conclusions could be drawn on human health concerns related to chronic exposure (EFSA, 2014). A mixture of enniatins, in the form of the drug fusafungine, was prescribed to humans for its antimicrobial and antiinflammatory properties. Administered by inhalation, it was used in some European countries to treat upper respiratory airway diseases, including sinusitis, rhinitis, rhinopharyngitis, laryngitis and tracheitis, as well as angina, Studies in guinea pig and rabbit models were conducted to confirm its antiinflammatory effects. It was withdrawn from the European market in 2016 due, in part, to an increase in the reporting rate of serious allergic reactions (Hedenmalm et al., 2019). Oral exposure to beauvericin and enniatins has unclear consequences with respect to immunotoxicity. There are no in vivo studies on the immunotoxic effects of beauvericin after oral exposure. Oral exposure to 465 mg enniatin A/kg diet for 28 days increased the percentage of CD4þ and decreased the percentage of CD8þ T lymphocytes in Wistar rats, with no clear functional implications (Maranghi et al., 2018).

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Moniliformin Moniliformin was isolated in 1973 from a Fusarium verticillioides (previously F. moniliforme) strain that was toxic to 1-day old chicks (Burmeister et al., 1980). It is a low molecular weight, water soluble mycotoxin produced by multiple species of Fusarium and at least one species of Penicillium (Figure 6.28. Like other Fusarium toxins, it is found on cereal grains and in cereal-based products. Limited toxicokinetics data from rats indicate that 20%–30% of moniliformin is excreted in urine, with less than 2% appearing in feces and the remainder unaccounted for. It tested negative in bacterial mutagenicity assays, but induced chromosomal aberrations, sister chromatid exchanges and micronuclei in mammalian cells, indicating that moniliformin exposure has the potential to be directly or indirectly genotoxic. A primary mode of action appears to be interference with the tricarboxylic acid cycle by cofactor-targeted inhibition of thiamin pyrophosphatase dependent enzymes (EFSA, 2018d; Gruber-Dorninger et al., 2016; Pirrung et al., 1996). Oral LD50s for acute moniliformin exposure range from about 50 mg/kg/d in mice, 19– 25 mg/kg bw/d in rats, and selenomethionine > selenate >selenite. As the chemical forms of selenium associated with selenium accumulator plants (A. bisulcatus and S. pinnata) are primarily selenate and methylselenocysteine, these plants should be considered highly toxic (Davis et al., 2011; Raisbeck, 2000). Signs of acute poisoning include dyspnea, pulmonary edema, and sudden death. Lower doses over longer duration may produce heart failure with subsequent edema, thoracic and abdominal transudates, and vascular distension that is seen as a jugular pulse. Histologic lesions include acute swelling and necrosis of cardiomyocytes (Figure 7.21) with pulmonary edema and vascular congestion. Animals that survive longer develop more extensive lesions with focally extensive myocardial necrosis, and fibrosis with adjacent edema and minimal infiltrates of lymphocytes and macrophages (Figure 7.22). The myocardial Purkinje cells may also be swollen and vacuolated with peripheral edema and infiltrates of lymphocytes and macrophages. The livers from these animals were congested with dilation and congestion of many of the central veins. Pigs are uniquely sensitive to Se poisoning and develop characteristic poliomyeloencephalomalacia in addition to the characteristic Se-related hoof lesions and lameness. Chronic Se poisoning in other livestock can be more variable. Some animals have minimal change with altered hair and hoof growth (Figure 7.23). Severely poisoned animals have more extensive lesions, including sloughing and loss of hooves, extreme lameness, and marked alopecia, especially of the mane and tail. Anecdotal reports suggest chronic poisoning alters fertility and reproduction; however, this has not been documented experimentally. Sheep may be more tolerant regarding the reproductive effects of Se poisoning. Ewes fed A. bisculcatus and alfalfa pellets to obtain Se doses of nearly 30 ppm had normal estrus cycles, estrus behavior, and conception rates. In a larger study, ewes dosed for 72 weeks with feed containing up to 20 ppm Se (as sodium selenite) did not develop clinical or histologic changes suggestive of toxicity and did not have detectable alterations in gestation or lactation. More work is needed to determine the chronic reproductive effects of high Se exposures

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FIGURE 7.21 Heart of a sheep acutely poisoned with a seleniferous plant. Notice the massive myocyte swelling and hypereosinophilia. H&E stain. Bar ¼ 200 mm. Inset is higher magnification of myocyte necrosis with adjacent inflammatory infiltrates. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.20. p. 1289, with permission.

FIGURE 7.22 Heart of a cow that survived nearly 2 weeks after ingesting highly seleniferous plants. Notice the extensive necrosis mixed with areas of fibrosis and nuclear proliferation. H&E stain. Bar ¼ 100 mm. Inset is higher magnification of degenerative myocytes (arrow) entrapped in the fibrous connective tissue. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.21. p. 1289, with permission.

FIGURE 7.23 Hoof of a cow with chronic selenosis. Notice the prominent growth ridges and abnormal growth. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.22. p. 1289, with permission.

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in other species. The mechanism of Se toxicity has not yet been clearly defined. It has been speculated that in acute poisonings intermediate substrates, such as glutathione and Sadenosylmethionine, are depleted, leaving cells susceptible to oxidative damage. Alternatively, free radicals and additional oxidative damage may be produced when selenium reacts with various thiols. Additionally, Se may be incorporated in place of sulfur or seleno-amino acids in proteins, resulting in altered and dysfunctional proteins that lack key disulfide structures. It is more likely that all these mechanisms contribute to toxicity, though their individual extent may be dependent on individual chemical forms or doses of selenium (Kolbert et al., 2016). Se toxicokinetics has been studied in cattle and sheep using serum, whole blood, liver, and skeletal muscle. In classical cases of Se poisoning in cattle, the elimination rate or half-life was 40.5 (8.2) days in serum, 115.6 (25.1) days in whole blood, 38.2 (5.0) days in liver, and 98.5 (19.1) days in skeletal muscle. Though these clearances are quite slow, the tissue concentrations were low (0.562  0.107 ppm [wet weight] at 30 days postexposure), suggesting that in most cases the risk of secondary poisoning is low.

5. SELECTED TERATOGENIC PLANTS (For more information, see Embryo, Fetus, and Placenta, Vol 5, Chap 11.)

5.1. Lupine In North America, the Lupinus genus includes more than 150 species whose similarities are such that identification is challenging. The lupines are generally highly nutritious legumes that under many conditions are an important component of livestock and wildlife nutrition. However, they are also rich in many different toxic and teratogenic alkaloids. As mentioned previously, lupines are neurotoxic, and poisoning has been reported in sheep, cattle, horses, and goats. However, the most common condition relating to lupine ingestion is “crooked calf syndrome/disease.” This is a congenital condition in calves resulting in skeletal contracture-type malformations and cleft palate after their mothers have grazed lupines

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during sensitive periods of pregnancy. Though the disease is most often seen in cattle it has been reproduced in various other rodent and livestock species, suggesting that all species are susceptible. In North America, most lupine species are found in and west of the Rocky Mountains. Generally, they are classified as an increaser species, as their populations expand in disturbed areas. In some conditions they may dominate plant communities, especially in times of poor precipitation and drought. Though lupine species may appear similar, there are large population differences. Many similar plant populations are often chemically unique, with alkaloid profiles that directly relate to their potential for toxicoses and teratogenicity. For example, Lupinus sulphureus, a yellow lupine species that is found in different geographical regions of Oregon, Washington, and British Columbia, has seven distinct populations as identified by unique alkaloid profiles. These profiles correlate directly with the incidence of disease and the relative risk that lupine will poison livestock in those locations. This illustrates that taxonomic classification of these lupines without verifying their chemical profiles is of little value in predicting risk of lupine poisoning or of crooked calf disease. The lupine-induced “crooked calf syndrome” was first reported in 1959 and later experimentally reproduced in 1967. Crooked calf syndrome includes various skeletal contracture-type birth defects and, occasionally, cleft palate. Subsequent studies identified the quinolizidine alkaloid, anagyrine, as the lupine teratogen. Later, a second teratogen, the piperidine alkaloid, ammodendrine, was identified in Lupinus formosus. Most lupine species contain quinolizidine alkaloids, a few contain piperidine alkaloids, and some contain both. Teratogenic risk can be based on chemical profile, and presence and concentrations of these teratogenic alkaloids. Plant alkaloid concentration is highest in the new early growth; the alkaloids are often diluted as the plant biomass increases and they peak at the pod stage, concentrating in the pods themselves. Following seed shatter, both concentration and pools of all alkaloids decline precipitously, leaving the senescent plant relatively nontoxic. The most sensitive period for teratogenicity in the cow is gestation days 40–70. Though the morbidity is lower, birth

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defects can be produced through gestation day 100. Crooked calf disease has been reproduced using dried ground lupine containing anagyrine, ground lupine containing piperidine, and with purified anagyrine in cattle. Ultrasonic monitoring of poisoned fetuses suggests that the mechanism of action is due to alkaloid-induced reduction in fetal movement during the critical stages of gestation. The inhibition of fetal movement is due to stimulation followed by desensitization of skeletal muscle type nicotinic acetylcholine receptors (nAChRs). This mechanism is common for other plant alkaloids that produce similar terata, including poison hemlock (C. maculatum) and wild tree tobacco (Nicotiana glauca) (Panter et al., 1999a). The molecular mechanism has been confirmed in TE-671 cells that express human fetal-muscle type nAChR, and SH-SY5Y cells which express human autonomic-type nAChR (Green et al., 2010, 2017). Some alkaloids have speciesspecific effects. For example, anagyrinecontaining lupines only cause birth defects in cattle, and not in sheep or goats. Conversely, the piperidine-containing lupine L. formosus induced birth defects in cattle and goats. More work is needed to identify the mechanism of these differences. The incidence of lupineinduced crooked calf disease is variable geographically and from year to year within a given herd. When lupine populations expand under certain environmental conditions and when susceptible animals are exposed with few other forage alternatives, the incidence of disease can nearly be 100% of a given calf crop. As dose and duration of exposure varies, the lesions in affected fetuses and neonates also vary. Most affected calves are born alive at full term. Dystocia may occur when calves are severely deformed. Arthrogryposis is the most common malformation, and it is often accompanied by one or more of the following: scoliosis, torticollis, kyphosis, and/or cleft palate (Figure 7.24). Some articulations may present as fused or immobile; for example, the elbow may be immobile because of malalignment of the ulna with the articular surfaces of the distal humerus. Much of this malalignment results from the lateral rotation of the radius and ulna. Similar rotations and ankylosis can occur in the pelvic limbs, though they are less frequent and always less severe.

FIGURE 7.24 Calf with “crooked calf disease” due to maternal ingestion of Lupinus sulphureus. Notice the angular limb deformities with lateral rotation of the front legs. There is also prominent kyphosis and lordosis. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.23. p. 1291, with permission.

In severe cases there may also be axial rotation and ankylosis of the cervical and thoracic vertebrae. The ankylosis and deformation can be so severe that articular motion is reduced to such a degree that the calf cannot stretch and pass through the pelvis at parturition. Many such calves have to be delivered via a cesarean. Others cause severe dystocia resulting in extensive maternal damage and subsequent endometritis with reduced reproductive capability. Mild angular deformities may resolve if the calf can walk and nurse. More severe lesions with gross articular misalignments are permanent, and as the calf grows, the additional weight and stresses result in progressive osteoarthritis. Various simple management solutions have been used to prevent crooked calves. Most limit exposure to lupine-infested pastures during the susceptible period of gestation (40–70 days). Elimination of the lupine alkaloids in the urine is quite rapid (t1/2 ¼ 6.32  6.88 h), suggesting that intermittent grazing with short clearance

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times in lupine-free pastures may prevent the disease (Panter et al., 1999a). There is no treatment for the malformations, and euthanasia is recommended for serious skeletal defects and cleft palate.

5.2. Veratrum californicum Of the five species of Veratrum in North America, Veratrum californicum has been shown to cause birth defects in various livestock and rodent species. Poisoning was first recognized in the mid-20th century, when in some areas nearly 25% of pregnant ewes that grazed on pastures containing V. californicum gave birth to lambs with a variety of craniofacial malformations. These malformations ranged from mild brachygnathism to severe cyclopia with anencephaly (Figure 7.25). The lesions were often so spectacular that the Basque shepherds called affected lambs “chatto” which translates as “monkey-faced” lamb, and the syndrome became known as monkey-faced disease. V. californicum grows primarily in the high mountain ranges of the western United States. Most Veratrum species are found in moist, open, high alpine meadows or in open woodlands, marshes, along waterways, in swamps or bogs, and along lake edges in high

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mountain ranges. They are coarse, erect plants about 1–2.5 m tall with short perennial rootstalks. The leaves are smooth, alternate, parallel-veined, broadly oval to lanceolate, up to 30 cm long and 15 cm wide, in three ranks and sheathed at the base. The inflorescence has panicle flowers; the lower ones are often staminate and the upper ones are perfect. Over 50 complex steroidal Veratrum alkaloids have been identified and divided into five classes: veratrines, cevanines, jervanines, solanidines, and cholestanes. The veratrines and cevanines are of considerable toxicologic interest as they are neurotoxins and hypotensive agents that bind to sodium channels, delaying closure and causing cardiotoxic and respiratory effects. The jervanines are most significant for their teratogenic effects; the most notable alkaloids were named cyclopamine and jervine, both potent inducers of the congenital cyclopia or monkey faced lamb disease. This cyclopic defect is induced in the sheep embryo during the blastocyst stage of development when the pregnant mother ingests the plant during the 14th day of gestation. The lesion is extremely dependent on the time of fetal exposure, as early studies suggest that durations as short as an hour may produce the defect. Later exposure may produce other terata. For example, early embryonic death may occur if the fetus is exposed during other

FIGURE 7.25 Series of lambs with “monkey-faced lamb disease” due to maternal ingestion of Veratrum californicum. Notice the spectrum of craniofacial defects that range from mild superior brachygnathism to anencephaly. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.24. p. 1292, with permission.

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periods before the 19th day of gestation, and phocomelia and tracheal stenosis may occur when maternal ingestion occurs on gestation Days 28–33. The mechanism of teratogenesis has been identified as jervanine alkaloid inhibition of the sonic hedgehog signaling pathway. A key player in craniofacial development during embryogenesis, the sonic hedgehog gene pathway and the related genes that it regulates have also been implicated in the development of numerous cancers, birth defects, and other anomalies. As a result, cyclopamine has become a significant tool in the study of this complex pathway and its involvement in such diverse physiologic processes and diseases. Clinical signs of direct poisoning are most likely caused by the neurotoxic cevanine alkaloids present in most species of Veratrum. Typical signs begin with excess salivation with froth around the mouth, slobbering, and vomiting, progressing to ataxia, collapse, and death. The elimination half-life of cyclopamine in sheep is approximately 1.1 h, and it is assumed that the elimination rates of the other similar Veratrum alkaloids are similar. This suggests a withdrawal period of 8 h for >99% of the Veratrum toxins (Welch et al., 2009). Control of Veratrum is relatively easy with broad-leaf herbicides that have been shown to be effective for decades. The teratogenic effects of Veratrum can be avoided by keeping sheep and other livestock species off pastures containing the plants during the first trimester of pregnancy.

5.3. Poison Hemlock Plant material and seeds from C. maculatum (poison hemlock) cause contracture-type skeletal defects and cleft palate similar to those caused by lupine. Cases of teratogenesis have been reported in cattle and swine in the field, and experimentally induced in cattle, swine, sheep, and goats. The birth defects include angular limb deformities, arthrogryposis, scoliosis, torticollis, and cleft palate. The teratogenic effects are undoubtedly related to the neuromuscular effects on the fetus and have been shown to be related to reduction in fetal movement. Likewise, cleft palate is caused by the tongue

interfering in palate closure during the reduced fetal movement and occurs during the 30- to 40-day gestation period in swine, the 32- to 41day period in goats, and the 40- to 50-day period in cattle. Fetal muscle AChR seems to be the alkaloid target in teratogenesis, as the autonomic AChR activation is much less sensitive. In cattle, the defects, susceptible period of pregnancy, and probable mechanism of action of teratogenesis are the same as in “crooked calf syndrome” induced by lupines. In swine, sheep, and goats, the susceptible period of gestation is from 30 to 60 days. Cleft palate has been induced in fetal goats when pregnant goats were treated with plant or purified toxins on Days 35–41. In some goats, the incidence was nearly 100% when treatment began on gestation Day 32. As many other genetic and toxic insults can produce similar terata, identifying plantinduced arthrogryposis or other terata is generally based on clinical history of exposure during susceptible gestation times. In acute poisoning, plant alkaloids can be detected in rumen contents, liver, urine, or blood from clinical cases; if poisoning is fatal, the presence of plant material in the stomach and a characteristic pungent odor of the gastrointestinal content with chemical confirmation of the alkaloids are diagnostic (Lee et al., 2020). Prevention of poisoning is based on recognizing the plant and its toxicity, and avoidance of exposure to livestock when they are hungry. If a lethal dose has not been ingested, the clinical signs will pass and a full recovery can be expected. Avoidance of stressing animals poisoned on Conium is recommended. However, if lethal doses have been ingested, supporting respiration, gastric lavage, and activated charcoal are recommended. Control of plants is easily accomplished using broadleaf herbicides; however, persistent control measures are recommended as seed reserves in the soil will quickly reestablish a population.

6. SELECTED NEPHROTOXIC PLANTS (For more information on nephrotoxicity, see Kidney, Vol 5, Chap 2.)

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6. SELECTED NEPHROTOXIC PLANTS

6.1. Oak Oaks (Quercus spp.) are woody plants that have worldwide distribution and encompass hundreds of species. Different species range from smaller shrubs to large trees. All oak species should be viewed as potentially toxic. Oak poisoning is most commonly associated with seedlings, early bud growth, and acorns. Thus, poisoning frequently occurs in early spring or fall. This is due both to greater toxicity in the acorns’ early growth, and to the fact that these are the times of year when other available forages are often limited. Toxicity does occur during other seasons when alternative forages are limited due to drought or overgrazing. All forageable parts of oak are potentially toxic, but new sprouts, new bud growth, and acorns are more toxic than mature leaves. Tannins have historically been identified as the cause of oak toxicity. However, purified tannins are only toxic at relatively high doses with extended exposure periods, and they do not produce all of the components of clinical oak toxicity. This suggests there must be some other toxin or synergistic factor in oak that has yet to be identified. Tannins, named for their ability to tan leather, are a complex family of polyphenolic compounds that are often subdivided into hydrolyzable and condensed tannins. Toxicity has been attributed to tannin’s free hydroxyl groups that cross-link proteins and other macromolecules, denaturing proteins and altering protein interactions. It has been suggested that hydrolyzable tannins are more likely involved in toxicoses, as they are more concentrated in plants associated with clinical poisoning. Poisoning occurs when large doses are ingested over several days to weeks. Calves appear to be more susceptible than adult cattle. Goats are less susceptible to poisoning, and this resistance has been attributed to rapid binding of tannins with goat salivary proteins which are enzymatically metabolized in the rumen. Although predominantly affecting the gastrointestinal tract and kidney in ruminants, high acute doses or nonoral administration of tannins can be hepatotoxic. Tannins can also be hepatotoxic in nonruminants. At low doses, most tannins are bound to salivary and microbial macromolecules that may be degraded in the rumen, or, when they are absorbed, are quickly detoxified in the liver.

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With higher doses these protective mechanisms are overwhelmed, resulting in gastrointestinal mucosal damage and nephrosis. Mucosal damage results in greater tannin absorption and more extensive damage. Early clinical signs of poisoning include lethargy, constipation, tenesmus, polydipsia, polyuria, and a brown discoloration to the urine. Initially these signs may be subtle, and they quickly progress in subsequent days into hemorrhagic diarrhea, abdominal pain, rumen atony, and anorexia. Severely poisoned animals often die. As animals develop renal disease, urinalysis shows isosthenuria, glucosuria, proteinuria, and hematuria. Biochemical changes are consistent with renal insufficiency, and include increased serum potassium, phosphorus, blood urea nitrogen (BUN), and creatinine. Transient increases in SDH and AST activities may also occur. Gross lesions of acute poisoning include the presence of oak material/acorns in the digesta, subcutaneous edema, perirenal edema, edema of the mesenteric lymph nodes, ascites, hydrothorax, pale kidneys with petechial hemorrhage, and multifocal ulcerative and hemorrhagic rumenitis, gastritis, and enteritis. Histologic findings include diffuse cortical renal tubular degeneration and necrosis characterized by loss of tubular epithelium and formation of hyaline, granular, and cellular casts. In some areas the remaining tubular epithelial cells form a flattened epithelium. The medullary tubules are generally spared or only mildly affected. In cases with liver disease, the hepatocellular degeneration and necrosis is characterized by swelling, degeneration, and necrosis of hepatocytes with focally extensive hemorrhage. In monogastric animals or in ruminant studies when tannins are administered via routes that bypass the rumen, the predominant lesions are hemorrhagic gastroenteritis and hepatic necrosis. The risk of oak poisoning in humans is low as acorns used for food are typically processed, which removes most of the tannins. Diagnosis of poisoning is generally made using the clinical presentation, pathologic findings, and evidence of ingestion. Detection of tannin metabolites such as pyrogallol is of limited use diagnostically, as they are readily eliminated and often undetectable in tissues of poisoned animals at the time of death. Poisoning can be prevented by limiting intake of oak materials to less than

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50% of the diet. Treatment of poisoned animals is generally symptomatic and supportive for the gastric and renal damage. There is some evidence that supplementing animals with hydroxide at 5%–15% in the diet will reduce the incidence of toxicosis.

6.2. Lily and Grapes Cats are uniquely sensitive to the nephrotoxic effects of several Lilium and Hemerocallis genera plants. First reported in 1990, most poisonings are due to Easter lily (Lilium longiflorum). This common ornamental houseplant is brought indoors during holidays, where it can be eaten by house cats. As no cases from natural, outdoor exposures have been documented, this suggests that outdoor cats probably do not eat lilies. However, many outdoor lilies are toxic, and there is the potential for poisoning. Nephrotoxic effects have not occurred when dogs have ingested lilies; also, they could not be reproduced in rabbits fed the plant material, nor in rats nor mice that were gavaged with plant material. Nephrotoxicity has been documented in cats after ingestion of as few as two to three leaves. The causative toxin has not been identified, but it is water soluble and the clinical presentation suggests it is rapidly absorbed. Urinary elimination of the toxin is also likely as the renal effects may be avoided with aggressive fluid dieresis. Clinical signs often occur by 1-h postingestion, and include increased salivation, vomiting, lethargy, and anorexia. Plant leaf material is often found in the vomitus. The vomiting and salivation generally stop 8–18 h postingestion, when animals develop polyuria and dehydration. The polyuric renal failure state is followed by anuric renal failure usually within 36–72 h. When anuric, poisoned cats are severely lethargic, dehydrated, anorexic, and inactive, they may begin vomiting again. Glucose, protein, and cellular casts can be observed in the urine as early as 12 h postingestion, whereas increased serum BUN, creatinine, phosphorus, and potassium are not typically increased until 18–24 h postingestion. Early cellular casts have distinct cellular structural detail, suggesting relatively intact sloughing of tubular epithelium. Gross lesions may be subtle and may include renal swelling and perirenal edema. Histologically, there is severe renal tubular necrosis

characterized by sloughing and necrosis of the proximal convoluted tubular epithelium (Figure 7.26). If the lesion is extensive, it may spread into the loops and collecting ducts. Affected tubules are often devoid of lining tubular epithelial cells and filled with cellular debris mixed with cellular and granular casts. The tubule basement membranes usually remain intact, and crystals are rarely observed. In animals that survive past 3–4 days, remaining tubular epithelial cells flatten to cover the basement membrane and may begin to regenerate, observed as occasional mitotic figures. Diagnosis is based on the clinical history, evidence of exposure, and confirmatory histologic findings. Cases that have been identified early, prior to anuric renal failure, have responded well with extensive fluid diuresis for 24–48 h. Chemical diuretics are generally not effective at reestablishing urine production, and severely poisoned cats may require dialysis for 7–10 days before the tubules heal sufficiently to produce urine. Prevention is easily accomplished by teaching owners to exclude plants and floral arrangements containing lilies from feline environments. A second species-specific plant poisoning is canine nephrosis associated with ingestion of

FIGURE 7.26 Kidney of a cat poisoned with lily. Notice the extensive tubular necrosis (arrows) with sloughing of tubular epithelium into the tubules and cast formation. H&E stain. Bar ¼ 100 mm. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.25. p. 1295, with permission.

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grapes or raisins. Although grapes are among the largest fruit crops in the world, poisoning is relatively rare. Reports of renal failure associated with grape ingestion have been reported in North America, Europe, and Korea. Initial comparisons of these cases suggest there are no breed, age, or gender differences in susceptibility. Grape color or preparation (cooking, drying, crushing, or fermenting) does not seem to alter toxicity. Clinical signs of poisoning include vomiting, ataxia, oliguria, or anuria, with less frequent changes of hypertension, dehydration, edema, hypothermia, trembling, polydipsia, polyuria, ptyalism, and seizures. Clinicopathologic changes include increased serum calcium and phosphorus, azotemia (increased BUN and serum creatinine), acidosis, isosthenuria, proteinuria, glucosuria, crystalluria, and both granular and hyaline casts (Eubig et al., 2005; Yoon et al., 2011). Microscopic changes include renal tubular degeneration and necrosis that is most severe in the proximal tubules. Tubular epithelial regeneration is common as are intratubular granular and cellular casts. Mineralization can be present in older animals that occasional may affect tubular and glomerular basement membranes. Other extrarenal changes were inconsistent but included hepatocellular swelling and degeneration, splenic lymphoid depletion, lymph node and intestinal submucosal lymphoid atrophy, and minimal metastatic mineralization in various tissues (Morrow et al., 2005). Treatment includes careful fluid therapy and diuresis with furosemide, dopamine, or mannitol. Peritoneal dialysis may also be helpful. Though several toxins have been suggested, none has been definitively identified as the cause. More work is needed to identify the cause and potential biomarkers that might better identify poisoned animals. The accessibility of grapes, raisins, and other grape products suggests that the incidence of poisoning is probably underestimated.

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on the species poisoned, and the dose, duration, and chemical form. This section relates to plantassociated oxalate poisoning. Plant oxalates have been divided into soluble and insoluble fractions. Insoluble plant oxalates include calcium oxalate, are found in crystalline form in plants, and are very irritating to mucosal membranes. When animals eat these plants, the crystals are immediately irritating, causing mechanical damage to the oral cavity and gastrointestinal tract. Consequently, most animals avoid eating them. However, when other forage is not available and animals are forced to eat them, buccal irritation and lesions develop that are seen clinically as mucosal hyperemia, swelling, and marked hypersalivation (Figure 7.27). With discontinuation of exposure, these lesions rapidly heal and the clinical disease resolves. The soluble plant oxalates include sodium and potassium oxalate and oxalic acid. These are more easily absorbed and generally cause systemic poisoning. Numerous plant species contain soluble oxalates at low concentrations, but only a few contain concentrations high enough to be systemically toxic. These high oxalate-containing plants occur worldwide, and include various species of the Agave, Beta, Bassia, Chenopodium, Halogeton, Oxalis, Rhuem,

6.3. Oxalate-Containing Plants Oxalate poisoning occurs from ingesting oxalate-containing plants; from ingesting feed that is contaminated with oxalic acid, oxalate salts, or fungi that produce oxalates; or from ingesting ethylene glycol. The clinical disease and subsequent lesions appear to be less dependent on the oxalate source and more dependent

FIGURE 7.27 Horse with severe gingivitis caused by plants containing insoluble calcium oxalate crystals. Notice the extensive salivation and slobbering. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.26. p. 1296, with permission.

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Rumex, Sarcobatus, and Setaria genera. Leaves contain the highest soluble oxalate content, seeds contain less, and stems contain the least. As oxalates are soluble, most of them leach from plants as they weather and age during senescence; however, they can remain if plants are preserved in hay or other prepared feeds. Soluble oxalate toxicity is species dependent, with monogastric animals responding very differently than ruminants. Ruminants are less susceptible to chronic poisoning but are commonly poisoned when they are exposed to and ingest plants containing relatively high oxalate concentrations without time for the body to adapt. Horses and other monogastric animals are infrequently acutely poisoned, but more commonly chronically poisoned when they are exposed to low oxalate doses for extended periods. The type of poisoning is largely determined by plant oxalate concentration. Plants with 10% soluble oxalates or greater generally produce acute disease that is usually nephrotoxic. Plants with lower oxalate concentrations, around 0.5%, usually produce chronic disease that may present as altered calcium (Ca) metabolism which may produce osseous lesions and secondary hyperparathyroidism. The toxic mechanism of action for soluble oxalates is probably multifactorial. Absorbed soluble oxalates effectively bind and sequester calcium and magnesium, resulting in functional deficiencies. Certainly, the loss of free intracellular calcium and magnesium affects multiple enzymatic and cellular functions (Rahman et al., 2013). More recent studies, using ethylene glycol as a model of oxalate poisoning, suggest that calcium oxalate monohydrate (COM), not oxalate ion, damages mitochondria, inducing mitochondrial permeability transition. Others also have shown that COM crystals alter membrane structure and function and increase reactive oxygen species that cause mitochondrial dysfunction. Intoxication results in decreased tricarboxylic acid enzymes (succinate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, and other respiratory enzymes) and increased oxygen stress (decreased antioxidant enzymes and glutathione with increased reactive oxygen species and lipid peroxidation). The impact each of these different mechanisms has in toxicity is unknown, as acute poisoning can produce sudden death prior to the

development of crystalline nephropathy. Oxalate toxicity can be altered but not avoided with calcium supplementation or antioxidant therapy. Certainly, COM crystals are closely linked with oxalate induced nephrosis. Insoluble COM crystals form in areas of highest oxalate content and are often seen in rumen vasculature and renal tubules. Crystals may also be present in other vessels, including cerebral vessels, and may contribute to the neurologic changes seen in some poisonings. In acute poisoning, soluble oxalates are readily absorbed from the gastrointestinal tract and clinical onset of toxicosis often occurs as early as 1–2 h postingestion. In monogastric animals, the acid pH of the stomach more readily favors absorption of the oxalic acid. The alkaline rumen promotes calcium oxalate formation which decreases the potential absorption due to precipitation. Once absorbed, soluble oxalates readily undergo glomerular filtration, but they are concentrated in the renal tubules where they can bind calcium and precipitate as COM crystals. The overall toxicokinetics can also be affected by rumen adaptation, as some rumen flora can break down oxalates into nontoxic by-products. Thus, repeated low-dose exposure can result in some degree of adaptation and protection from sudden higher exposures. Chronic low dose poisoning by soluble oxalates primarily affects monogastrics and presents as nutritional hyperparathyroidism in horses. Chronic poisoning in ruminants is less well understood and has been linked to a wasting syndrome in sheep. Acute oxalate poisoning is characterized by hypocalcemia, lethargy, anorexia, muscle tremors, weakness, stiffness, diarrhea, ataxia, tachypnea, dyspnea, tetany, recumbency, rumen atony, coma, and death. Animals that are forced to move may develop hyperesthesia and seizures. If these animals survive, they develop azotemia; if severely affected, they will develop clinical renal failure and, potentially, encephalopathy (Naude and Naidoo, 2007). Gross lesions in most animals are minimal and characterized by gastritis or rumenitis with serosal edema and hemorrhage. Secondary accumulation of pleural fluid and ascites, as well as pulmonary edema, may also occur. The kidneys are often pale and swollen. If the animal survives, the kidneys often become pale, firm, and shrunken. Histologic lesions are associated

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with deposition of birefringent COM crystals in the renal tubules, abomasal mucosa, and gastric vasculature. Renal tubules are typically distended with crystalline material and are lined by flattened and degenerating epithelium in acute cases (Figure 7.28). With more chronic cases there can be a thinning of the renal cortex, with discoloration between the cortex and medulla that is associated with accumulation of crystalline materials in the tubules. Fibrotic change in chronic cases can be encountered (Rood et al., 2014). Chronically poisoned horses show weakness, stiffness, intermittent lameness, inability to work, roughened hair coat, weight loss, and swelling of the osseous structures of the head. Grossly and histologically, these horses may develop fibrous osteodystrophy, with swelling of the nasal bones, maxilla, and mandible (“bighead”). Histologic changes include increased osteoclast activity with proliferation of fibrous connective tissue and poorly mineralized bone. During World War I, oxalate poisoning in humans was attributed to the use of rhubarb leaves instead of spinach. Poisoning has also occurred when curly dock (Rumex spp.) was included in soup. Though oxalate poisoning is uncommon now, the potential for

FIGURE 7.28 Kidney from a sheep poisoned with halogeton. Notice the intracellular and tubular calcium oxalate monohydrate crystals with associated crystal associated nephrosis and cellular tubular casts. H&E stain with polarizing filters. Bar ¼ 100 mm. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.27. p. 1297, with permission.

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human poisoning still exists as many oxalatecontaining plants are used as herbs or medicinals. Oxalate intoxication is easily diagnosed via identification of COM crystals histologically, consistent clinical signs, and evidence of consumption of oxalate-containing plants. To better predict risk and the type of potential poisoning, plant material can be analyzed by gas chromatography for the presence and quantification of oxalates. Treatment of acutely poisoned animals is symptomatic, using parenteral calcium to correct hypocalcemia if indicated. Oral calcium therapy may also be useful in binding soluble oxalate in the rumen to prevent absorption. Allowing ruminants to adapt with low-dose exposure to soluble oxalates for 8– 25 days will decrease the risk of poisoning. As with most toxic plants, most poisoning is easily avoided by recognizing the potential for poisoning and managing exposure so that animals do not ingest too much too fast.

6.4. Amaranthus spp. The Amaranthus genus includes over 60 species and many more hybridized species that can be found throughout the world. Commonly referred to as “pigweeds,” they are annual weeds with prolific seed production that allows them to easily colonize disturbed areas in paddocks, on field edges, and along fences, ditches, and roads. Historically they were used as food by native populations, and some species have again gained favor for use as a natural food source. However, Amaranthus retroflexus (red pigweed) and several additional species of this genus are toxic to cattle, sheep, goats, pigs, and, rarely, horses. Several potential toxins, including nitrates, oxalates, and several unknown nephrotoxic and myocardiotoxic factors, have been associated with Amaranthus poisoning in livestock. Most recently, toxicity has been attributed to nitrates (see Section 7.3). Nitrate poisoning in ruminants is characterized by rumen nitrate conversion to nitrite, rapid absorption with associated methemoglobin formation, and sudden onset of clinical signs. If the dose is nonlethal, animals quickly recover. The myocardial and renal toxins and mechanisms of toxicity are unknown, but both appear to require plant ingestion for several days to

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several weeks. As animals can develop clinical signs 5–10 days after exposure, it is likely that elimination of these toxins is slow. The clinical signs of nitrate-associated poisoning include sudden onset of tachypnea, weakness, and recumbency. The syndrome progresses rapidly to death or to a full recovery by 24 h or less. Lesions are minimal or absent, but can consist of darkened, brownish blood in tissues. In pigs, the cardiotoxic effects include sudden death after eating Amaranthus caudatus contaminated grain for 5–7 weeks. Histologically, these animals often have localized areas of myocardial hemorrhage and necrosis with subsequent fibrosis and scarring. Many other tissues may be subsequently congested and edematous with occasional effusions. Renal disease also requires extended ingestion of Amaranthus spp. and poisoning clinically progresses over 1–2 days from weakness, muscle tremors, ataxia, knuckling of pasterns to more severe recumbence, paralysis, hemorrhagic diarrhea, hemorrhages, coma, and death. Serum chemistry changes include increases in serum potassium, phosphorus, BUN, and creatinine. Gross histologic lesions are predominantly fluid accumulation with straw-colored fluid in the abdominal and thoracic cavity with pale, potentially swollen kidneys, and prominent perirenal edema. Microscopically, there is marked necrosis and regeneration of the convoluted renal tubular epithelium with interstitial edema. Many renal tubules are dilated and contain proteinaceous debris and casts. The disease in surviving animals often progresses to interstitial renal fibrosis. Despite relatively common exposure, there are no indications of human risk from the cardiotoxic or nephrotoxic Amaranthus syndromes. Some antinutritive effects have been identified in Amaranthus seed diets in a rodent model. Identification of poisoning is made by linking exposure and ingestion with the clinical signs and lesions. Nitrate-induced methemoglobinemia can also be evaluated and forages can be analyzed to confirm toxic nitrate concentrations. Animal serum or ocular fluid can be evaluated for nitrate or nitrite content postmortem. The diagnosis of nephrotoxic or cardiotoxic diseases is based on known or verifiable ingestion, clinical presentation, and compatible pathologic lesions (Alegbejo, 2013). Prevention and treatment are aimed at limiting

exposure, and supportive care, respectively. Treatment with electrolyte solutions of calcium and magnesium has provided minimally beneficial effects early in the syndrome but has no overall effect on survival. Animals that survive may take several weeks to recover, and they are at high risk of having diminished renal function (Casteel et al., 1994).

6.5. Calcinogenic Glycoside-Containing Plants Solanum malacoxylon, Solanum verbascifolim, Solanum torvum, Nierembergia veitchii, Trisetum flavescens, and Cestrum diurnum contain glycosides of 1,25-dihydroxycholecalciferol (calcitriol) or physiologically similar compounds that act as active vitamin D (cholecalciferol) resulting in hypercalcemia and calcification of many tissues and organs. Cholecalciferol increases Ca absorption in the intestinal tract, increases Ca resorption from bone, and decreases renal Ca excretion, resulting in marked hypercalcemia and hyperphosphatemia. This results in osteopetrosis and, eventually, metastatic calcification. On nearly every continent, cattle, horses, sheep, goats, and pigs have been poisoned by calcinogenic plants (Table 7.3). Plant toxicity and the mechanism of poisoning have also been confirmed and studied using a variety of laboratory animals. Poisoning is characterized by a progressive disease that is first seen as depression, weakness, weight loss, infertility, anorexia, cardiac arrhythmias, and impaired stilted gait (Figure 7.29). Poisoned animals become lame and then recumbent, and death is often attributed to emaciation in addition to cardiac and pulmonary insufficiency. Clinically, animals have hypercalcemia and hyperphosphatemia. This often resolves when animals are removed from the plant, despite extensive tissue mineralization. With extended disease most animals develop renal disease, with subsequent increases in BUN, creatinine, and phosphorus concentrations. Radiologic exams often identify mineralization of many organs and tissues, including mineralization of vessels in the legs (Dirksen et al., 1970). Grossly, mineralization is seen as gritty white deposits in kidneys, intestines, stomach, heart, lungs, arteries, bones, tendons, and ligaments (Figure 7.30). The renal tubular

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TABLE 7.3

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Country, Plant, and Common Names of the Disease Produced by Calcinogenic Plants

Country

Plant

Disease

Argentina

Solanum malacoxylon

Enteque seco

Brazil

S. malacoxylon Nierembergia veitchii

Espichamento Calcinosis

Uruguay

S. malacoxylon

Calcinosis

United States

Cestrum diurnum Solanum torvum

Enzootic calcinosis Naalehu disease

Jamaica

C. diurnum S. torvum

Manchester wasting disease

Cuba

C. diurnum

Calcinosis

New Guinea

S. torvum

Calcinosis

Australia

Solanum esuriale

Humpy back

South Africa

Solanum verbascifolium

Calcinosis

Austria

Trisetum flavescens

Weidekrankheit Kalzinose

Germany

T. flavescens

Enzootische kalzinose

Switzerland

T. flavescens

Enzootische kalzinose

FIGURE 7.29 Cow with enzootic calcinosis due to Solanum malacoxylon poisoning. Notice the erect, stiltlike stance especially in the hocks. Photograph courtesy of Dr. Alan A. Seawright. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.28. p. 1300, with permission.

Table reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Table 40.3. p. 1299, with permission

basement membranes, glomerular tufts, and Bowman’s capsule may be mineralized. Histologic mineralization may also be seen in bronchioles, alveoli, endocardium, vessel walls, and walls of the intestine and stomach. In response to chronic hypercalcemia, thyroid follicular cells often become hyperplastic and the parathyroid becomes atrophic. Poisoned animals with extensive mineralization rarely recover completely, and lesions have been shown to persist for years (Hanichen and Hermanns, 1990). Mineralization in the walls of the aorta and in tendons was found to be especially persistent and seemed unlikely to resolve in either experimental or natural poisoning (Hanichen et al., 1970). Consequently, prevention and control of toxicoses are recommended. Removing these weeds or using alternative forage crops may be useful. Most

FIGURE 7.30 Aorta from a cow with enzootic calcinosis. There is extensive metastatic mineralization of the aorta. Photograph courtesy of Dr. Alan A. Seawright. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.29. p. 1300, with permission.

remain toxic when included in green forage, but the calcinogenic potential decreases when they are stored for extended periods in dried feeds (Mello, 2003).

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7. OTHER TOXIC PLANTS 7.1. Pine Needles Pregnant cattle that are allowed to eat needles from ponderosa pine (Pinus ponderosa) and several other related pine trees may abort, resulting in dead or premature calves, retained placentas, and uterine infections. The risk of pine-needle consumption and subsequent abortion is greater during severe winters and during cold snowstorms. Other pine trees found to induce abortions in cattle include lodgepole pine (Pinus contorta) and Monterey cypress (Cupressus macrocarpa). Several other trees contain many of the pine needle toxins, but they have not been associated with clinical abortion and are not included in this discussion. In pine needle abortion both the fresh green needles and old dry needles can be abortifacients. Consequently, any pine needles, even dry duff

around the tree, trees that might have fallen during a storm or trimmings from trees should be considered risky. Cattle and bison are uniquely susceptible to abortion, as other species have not been shown to abort when exposed to or treated with pine needles or their toxins. Though the exact physiologic mechanism and active metabolite that produces pine-needle abortion has not been identified, the pineneedle compounds that produce the abortion have been isolated and identified as labdane acids, of which isocupressic acid is the main toxin in ponderosa pine needles (Figure 7.31). Other labdane acids, including agathic acid, imbricatoloic acid, and dihydroagathic acid, are also abortifacients (Figure 7.31). Most of these labdane acids are quickly metabolized in the rumen and liver, and they are rapidly eliminated from the serum and tissues. However, one metabolite, tetrahydroagathic acid, can be detected in the serum of poisoned animals and

FIGURE 7.31 Chemical structures of labdane acids found in pine needles that cause abortions in cattle: (1) isocupressic acid, (2) imbricatoloic acid, (3) agathic acid, (4) dihydroagathic acid, and (5) tetrahydroagathic acid Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Fig. 40.30. p. 1301, with permission.

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fetal fluids for longer than 72 h. It is speculated that pine needles affect the fetal/placental unit by reducing blood flow to the fetus and subsequently initiating parturition. Regulation of caruncular arterial blood flow is complex and includes short-term (phasic) and long-term (tonic) contractile mechanisms that are balanced by numerous adrenergic receptors, potentialsensitive calcium þ channels, and hormonesensitive receptors. As isocupressic acid does not appear to act directly at the site of the uterine artery muscle, it is likely that some metabolite or other second messenger, such as tetrahydroagathic acid, directly reduces caruncular blood flow. More research is needed to identify the exact mechanism of abortion and develop antidotes or methods to reduce abortion. Factors including the gestation stage, dose, duration, animal condition, environmental stress, and nutritional status all influence abortion incidence. There is huge animal variation in response to poisoning. Cows may abort within a couple of days to as long as several weeks after exposure to a single dose of pine needles. Other cows require daily doses of nearly 2 weeks’ duration to produce abortions. Cows in late gestation are most susceptible. Most abortions occur during the late winter or early spring, when storms force near-term pregnant cattle into the pine trees for shelter. The signs of pending abortion include weak uterine contractions, incomplete cervical dilation, and excessive mucous discharge. Dystocia is common and most require assistance. If aborted, fetuses are often stillborn and if they are born alive, they are small and weak, requiring assistance to stand and nurse. Cows that have aborted will have retained fetal membranes, and further complications, including endometritis, agalactia, rumen stasis, and death, are common. The death of the calf and/or the cow from the abortion is believed to be the result of the prematurity and complications, and not necessarily related to any toxic effects from pine needles (Gardner et al., 1999). At high doses, or when other diterpene acids that are found in ponderosa pine needles at relative lower concentrations were isolated and dosed, pine needles may be directly toxic. Signs of this intoxication included anorexia, rumen stasis and acidosis, dyspnea, and hepatic and muscular disease. Histologically, affected animals had nephrosis,

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vacuolation of basal ganglia neuropil with patchy perivascular and myelinic edema, and skeletal myonecrosis. Under normal grazing conditions, intoxication from these other diterpene acids rarely occurs (Stegelmeier et al., 1996). No notable human risks are known from livestock consumption of pine needles. There are no known methods to prevent abortions after pregnant cattle have consumed pine needles. Currently, the best method to prevent abortion is to deny cows access to pine needles during the late periods of pregnancy (third trimester) by either removing pregnant cows from the pines or eliminating the pine trees by burning or clear cutting. Providing adequate food and shelter can help reduce losses from pine-needle abortion. If cows abort from grazing pine needles, professional care is essential to prevent postpartum complications. The survival of the calf is usually dependent on the stage of gestation (fetal maturity) and the neonatal care received. Diagnosis of pine needle abortion can in some cases be confirmed by analysis of sera and fetal fluid samples for metabolites of isocupressic acid.

7.2. Cyanogenic Plants More than 2500 plants throughout the world contain cyanogenic glycosides. Many of these are common plants that are used as foods and forage crops. Some cyanogenic glycoside containing species include many types of cherries (Prunus spp.), elderberry (Sambucus spp.), service berry (Amelanchier alnifolia), various sorghum, Johnson and Sudan grasses (Sorghum spp.), corn (Zea spp.), vetches (Vacia sativa), white clover (Trifolium repens), birdsfoot trefoil (Lotus spp.), arrowgrass (Triglochin spp.), and others. Although under certain conditions all these plants are potentially toxic, only a handful of them have been associated with poisoning in livestock. In plants, toxic cyanide is sequestered as glycosides such as amygdalin, prunasin, lucumin, and others that are composed of an alphahydroxynitrile aglycone with a unique sugar moiety. To become toxic, cyanogenic glycosides must be hydrolyzed to cyanide or prussic acid. This conversion is facilitated when the plant is damaged as by crushing, chewing, freezing, or wilting. Drying or ensiling the plants decreases the cyanogenic potential as the cyanide is slowly

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degraded and released over time. The concentration of cyanogenic glycosides in plants varies and can be higher in young plants growing rapidly in cold moist weather; when plants are heavily fertilized, frost- or drought-stressed; or when plants are treated with certain herbicides. Plant cyanogenic potential can be measured chemically, and forages with concentrations of greater than 200 ppm are considered to be potentially toxic, especially if the plant is wilted or otherwise damaged, facilitating hydrogen cyanide release (Cressey and Reeve, 2019). Cyanide is highly toxic to all animals as it avidly binds with iron in cellular cytochrome oxidase, inhibiting cellular respiration. As this step is a key player in transferring electrons to oxygen as the terminal electron acceptor in oxidative phosphorylation, oxygen is not used, and it accumulates as oxyhemoglobin. Affected animals accumulate supersaturated oxyhemoglobin in erythrocytes and other tissues, causing them to appear “cherry red.” Affected tissues quickly deoxygenate postmortem, causing this discoloration to persist only temporarily after death. Low, nonlethal doses of cyanide have been associated with lathyrism-like disease, goiter, birth defects such as arthrogryposis, and spinal cord degeneration and cystitis. The mechanism of many of these changes has been suggested to be due to neurologic myelin damage. Identifying clinical poisoning in livestock can be difficult, as most animals die quickly. Acute poisoning can cause hyperventilation, dyspnea, anxious and excited mental state, hypotension, and staggering that may be followed by convulsions, paralysis, and death. The blood and tissues may be a bright cherry-red immediately after death. This color darkens and may not be apparent after 2–6 h postmortem, depending on temperature. Petechial hemorrhages may also be found in the abomasum, endocardium, and epicardium. These probably relate to stress and agonal struggling. Some report that cyanogenic plants produce a “bitter almond” odor postmortem. This finding is also sporadic, as it quickly dissipates postmortem. Diagnosis of poisoning is usually based on linking the clinical signs and lesions with evidence of plant consumption. As cyanide quickly dissipates from tissues, tissues such as liver and muscle, and rumen contents must be collected within a couple of hours of death, frozen in sealed,

airtight containers, and quickly analyzed. Poisoned animals should be treated with intravenous sodium nitrite (22 mg/kg) and sodium thiosulfate (600 mg/kg). These oxidize hemoglobin, forming methemoglobin, which binds cyanide and protects the cytochrome oxidase system from its effects. The best treatment is preventiondavoiding harvesting and feeding these plants when they are likely to be toxic. As some plants are only sporadically toxic, potentially toxic feeds can be tested for their cyanogenic potential (Cressey and Reeve, 2019; Vetter, 2000). Human exposure to cyanogenic glycosides is associated with consumption of cassava, cycad, lima beans, sorghum, and flaxseed. Apricot, peach, cherry, and apple kernels, as well as some stems, leaves, and roots, may contain glycosides, such as amygdalin in concentration of up to 6%. In fruit pulp from these same plants, concentrations are usually less than 0.01%. Cassava (Manihot esculenta C.) roots contain linamarin and lotaustralin, which are mostly removed with processing (sundrying, soaking, and boiling). Cassava is used to produce various foods (gari, lafun, and fufu) that are important for over 500 million people in Africa and South America. Inefficient processing of cassava root results in cyanohydrincontaminated cassava meal, which can result in poisoning. Poisoning is generally associated with drought or changes in processing techniques, or when plant parts are included in teas or porridges. Cycad seeds (Cycas revoluta T.) contain cycasin and neocycasin with several different methylations. Cycad seeds are commonly used as food and medicinals. As with cassava, most preparations are relatively safe as most of the cyanogenic potential is removed during processing. The other lesstoxic cyanogenic glycoside contaminated food stuffs are not as well studied, and seemingly less toxic. Acute poisoning is characterized by sudden onset of vomiting and crying followed by fainting, lethargy, and coma. Other less common signs include colic, headache, dizziness, weakness, tachycardia, and diarrhea. Most patients recover within 24 h. Chronic poisoning has been linked to several sporadic and endemic diseases. Epidemiological studies have linked these diseases with cassava ingestion, but definitive evidence or causality is lacking. Tropical ataxic polyneuropathy with ataxia,

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sensory polyneuropathy, optic nerve atrophy, and neurosensory deafness may be related to cassava ingestion in Nigeria. Konzo or “tired legs” is a spastic upper motor neuron disorder of Mozambique, Tanzania, the Central African Republic, Cameroon, and the Democratic Republic of Congo. This is characterized by spastic paraparesis or tetraparesis and it has also been associated by several studies with cassava ingestion. Western Pacific Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia complex is a neurodegenerative disorder with symptoms similar to amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease. Though initially linked with cyanogenic compounds, this disease of Guam and several other Pacific populations may be due to beta-methylamino-L-alanine toxicity. More work is needed to better understand the effect of chronic sublethal cyanide exposure, and whether such exposure plays in these syndromes (Barceloux, 2009; Spencer, 2020; Kashala-Abotnes et al., 2019).

7.3. Nitrate-Accumulating Plants Another relatively common toxicity occurs when certain pasture and cultivated forages accumulate nitrates. In the rumen, and possibly in other portions of the gastrointestinal tract, nitrates are reduced to nitrite, which is absorbed and oxidizes hemoglobin, producing methemoglobin. Methemoglobin is nonfunctional, as it will not bind oxygen. Monogastric animals, including horses, are less susceptible to nitrate poisoning, and it has been suggested that this is because nitrates are not as easily reduced in the GI tract of monogastrics as they are in the rumen. However, there are reports of poisoning in horses, cats, and dogs (Hintz and Thompson, 1998; Worth et al., 1997). Sheep and goats are less susceptible to poisoning which is probably due to their relatively quick elimination of nitrate in the urine (t1/2 in sheep is 4.2 h, and in cattle is 9 h). The elimination rate in bovine fetuses is even longer (t1/2 > 24 h), which may explain some of the effect of nitrate poisoning on pregnant cows. Poisoning is cumulative, and all other potential sources, such as water or feed additives, should be considered. Nitrates accumulate in all plant parts, but they may be especially high in stalks and leaves. Seeds or

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grains are generally safe. Plants that commonly accumulate toxic nitrate concentrations (>0.5%) are listed in Table 7.4. Many of these are weeds that commonly invade fields where they can contaminate forages. For example, fireweed (K. scoparia) and pigweed (Amaranthus spp.) can accumulate nitrates, and they often contaminate and are harvested in new hay fields. Dried and stored forages retain their toxicity. Nitrate accumulation is also provoked or enhanced by nitrogen fertilization, drought or frost stress, and some herbicide treatments. Water may be directly toxic if it is contaminated with nitrates from fertilizer, silage-pit or feedlot runoff, or when included in irrigation water as fertilizer. Ruminants are most often poisoned by nitrates, and early clinical signs reflect tissue oxygen deprivation. Initial signs include exercise intolerance, weakness, trembling, brown or cyanotic mucous membranes, dyspnea, brown discolored blood, abortion, and death. Abortion is frequently associated with sublethal nitrate poisoning, and most evidence suggests that abortions usually occur 3–7 days after exposure. Some clinical nitrate-associated abortion storms have been attributed to plants with nitrate concentrations as low as 0.5%, a concentration that may be considered safe for nonpregnant animals. Reproducing nitrate-associated abortion has been difficult. Sodium nitrite given intravenously to pregnant cattle is fetotoxic. Both clinical and nitrite poisoned fetuses have elevated ocular fluid nitrates and nitrate concentrations. It is generally considered that concentrations greater than 20 mg NO3/mL (20 ppm) are indicative of maternal nitrate poisoning. Maternal blood, serum, and ocular nitrate concentrations can also be diagnostic. The lesions of nitrate poisoning can be minimal and overlooked, especially if postmortem examination is delayed. Acutely poisoned animals often have chocolate-colored blood, and many tissues may appear brown. As methemoglobin is reduced, this change diminishes postmortem. If promptly measured, blood methemoglobin concentrations will be elevated. Other nonspecific changes include congestion of the mucosa of the rumen and abomasum. Poisoned animals may be treated with intravenous methylene blue (8 mg/kg in cattle). As methylene blue is rapidly cleared, treatment may need to be repeated every 2 h. In most cases poisoned animals die

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Select Plants that Accumulate Toxic Nitrate and Have Been Associated with Livestock Poisoning

CROPS

TABLE 7.4

Select Plants that Accumulate Toxic Nitrate and Have Been Associated with Livestock Poisoningdcont’d

Milk thistle

Silybum marianum

Oats

Avena sativa

Nightshades

Solanum spp.

Beets

Beta vulgaris

Golden-eyes

Viguiera spp.

Rape, turnips

Brassica spp.

Soybeans

Glycine max

Table reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Table 40.4. p. 1304, with permission.

Barley

Hordeum spp.

Flax

Linum usitatissimum

Alfalfa

Medicago sativa

Pearl millet

Pennisetum typhoides

Rye

Secale cereal

Sorghum, Sudan, and Johnson grass

Sorghum spp.

Wheat

Triticum aestivum

Corn

Zea mays

WEEDS

quickly, precluding treatment. Nitrate poisoning can be prevented by recognizing crops, weeds, and forages that are likely to accumulate nitrates and avoid contact with susceptible species. Forage nitrate concentrations of >0.5% and water concentrations >200 ppm should be considered dangerous. Contaminated forages can still be used if they are diluted with good feed or fed to less susceptible species (Lee and Beauchemin, 2014; Casteel and Evans, 2003).

7.4. Photosensitizing Plants

Ragweed

Ambrosia spp.

Pigweed

Amaranthus spp.

Wild oat grass

Avena fatua

Musk thistle

Carduus nutans

Lambsquarters

Chenopodium spp.

Canadian thistle

Cirsium arvense

Field bindweed

Convolvulus arvense

Jimson weed

Datura stramonium

Barnyard grass

Echinochloa spp.

Cudweeds

Gnaphalium spp.

Sunflowers

Helianthus spp.

Fireweed

Kochia scoparia

Skeleton plant

Lygodesmia spp.

Cheeseweed

Malva parviflora

Sweetclover

Melilotus officinalis

Smartweed

Polygonum spp.

Docks

Rumex spp.

Russian thistle

Salsola pestifer (Continued)

Photosensitization is light-induced dermatitis caused by heightened sensitivity of the skin to sunlight. Increased sensitivity is often due to the presence of photodynamic agents or chromophores in the circulation and skin. These chromophores absorb light energy, transform into an excited, high-energy state, and transfer energy to proteins, nucleic acids, and receptor molecules. This energy can directly damage tissues, generate reactive molecules, or initiate chemical reactions in dermal components. The clinical signs of photosensitization dermatitis develop within hours after exposure to strong sunlight. Hair and dermal pigments are protective as they absorb light energy before it activates chromophores and damages dermal tissues. Thus, lightly pigmented areas with little hair protectiondespecially the muzzle, ears, eyelids, face, tail, vulva, and coronary bandsdthat are exposed to the sun are most severely affected. However, with severe disease even black, heavily haired animals can develop photosensitivity. Clinical signs include photophobia and discomfort, seen as scratching and rubbing the ears, eyelids, and muzzle. Affected areas develop erythema followed by edema, serous exudation, scab formation, and skin necrosis. The resulting

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inflammation may be extensive, resulting in epidermal necrosis, sloughing of necrotic layers, and extensive suppurative exudate (Figure 7.32). Photosensitization differs from sunburn, in which lightly pigmented skin slowly becomes inflamed following prolonged exposure to rays in the ultraviolet range. In human and animal photosensitivity, the dermal photodynamic chromophores are most often plant or fungal products, drugs, chlorophyll metabolites, or other chemicals. In man, cattle, swine, and cats, there is also a congenital photosensitivity associated with porphyria. This disease, referred to as osteohemochromatosis or pink tooth, is caused by a meta-bolic defect in uroporphyrinogen III cosynthetase, an enzyme of hemoglobin synthesis. Except for photosensitization, this disorder is clinically harmless. Historically, photosensitization has been divided into primary photosensitization and secondary or hepatogenous photosensitization. Primary photosensitization is caused directly by chromophores from plants or drugs. Secondary photosensitization is caused by defective liver function with subsequent accumulation of photodynamic phylloerythrin, a chlorophyll metabolite from plants.

FIGURE 7.32 Skin from a horse with secondary photosensitization due to pyrrolizidine alkaloid induced secondary or hepatogenous photosensitization. Inset is histologic section that demonstrates typical necrotizing dermatitis. H&E stain. Bar ¼ 100 mm. Figure reproduced from Handbook of Toxicologic Pathology, 3rd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2013) Academic Press, Vol. 2, Figure 40.31. p. 1305, with permission.

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Primary Photosensitization Primary photosensitization occurs when plant, drug, or other chemical photodynamic agents enter the skin and are activated by sunlight. By definition, primary photosensitization does not include hepatic damage. However, some toxins, especially those from several grasses, are both directly phototoxic and hepatotoxic, making the distinction less obvious. Most agents are ingested, but some may induce lesions by contact with the skin. Several of these are weedy plants that can contaminate pastures and feed. Additionally, as some of these plants are more commonly used as herbals and medicinal plants, the incidence of photosensitization is increasing (Stegelmeier et al., 2020). Hypericism Hypericum perforatum is commonly known as St. John’s wort, goat weed, Tipton weed, amber, or Klamath weed. It is a perennial that grows along roadsides and in meadows, pastures, rangelands, and waste places in the western United States, Europe, Australia, New Zealand, and South America. It prefers dry, gravelly, or sandy soils in full sunshine. It is considered a noxious weed in most countries. St. John’s wort is a smooth-branched, erect plant that may reach a height of 2 m. The leaves are covered with clear, small dots that reportedly contain high concentrations of hypericin. Hypericin is the photodynamic toxin that recent work suggests is produced by a plant-associated endophyte. It has five-petaled flowers growing in clusters; they are orange-yellow with occasional black dots along the edges. St. John’s wort is dangerous at all stages of growth. Young tender shoots may attract animals in the spring. Normally, livestock will not eat mature St. John’s wort if alternative forages are available. However, if included in stored feeds it is readily eaten, and contaminated hay can cause poisoning in the winter. Signs of clinical poisoning usually appear 2–21 days after animals have access to St. John’s wort. St. John’s wort has traditionally been used as an herbal medicine, and recently purified or synthesized hypericin has been used and tested extensively as an antiviral, antitumor, and antidepressant pharmaceutical (Burrows and Tyrl, 2013). Most of its pharmaceutical activities have been shown to be light dependent, and it has been speculated

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that this restriction may result in little clinical application; however, its use in medical photochemistry has generated much interest. With specific binding affinities to some neoplasms or antibody-directed binding to others, hypericin shows promise in allowing specific targeting of light therapy in specific and controlled locations including neoplasms. St John’s wort is a potent inducer of cytochrome P450, and consequently can alter the metabolism of other pharmaceuticals. Though some interactions may be beneficial (decreased irinotecan toxicity), others may not. For example, H. perforatum use alters the bioavailability of oral contraceptives resulting in decreased effectiveness (Jendzelovska et al., 2016; Kusari et al., 2008). Fagopyrism Buckwheat speciesdFagopyrum esculentum, Fagopyrum tataricum, and other Fagopyrum spp.dare upright perennial subshrubs or vines with simple, alternate leaves and perfect flowers. Found in many parts of the world, they are up 0.6 m tall and commonly grow and expand in disturbed soils along field margins and fences. Native to Asia, they were initially used as a summer cover crop. In most countries, buckwheat use has been replaced by better, less toxic plants, making poisoning of historical interest. The toxins fagopyrin, photofagopyrin, and pseudohypericin have structures and toxicity similar to hypericin, though little is known of their synthesis or origin. Poisoning is most common in sheep and cattle. Furocoumarins Furocoumarins contain a furan ring fused with a coumarin nucleus (psoralen derivatives). They are thought to be synthesized as both primary plant compounds and phytoalexins (fungal pathogens). More work is needed to verify their origin. Poisoning has been reported in sheep, cattle, and horses, and contact dermatitis in various animals, including humans, is relatively common. Plants commonly associated with furocoumarin-induced photosensitivity include spring parsley (Cymopterus watsonii) and several other related Cymopterus spp., bishop’s weed (Ammi majus), dutchman’s breeches (Thamnosma texana and Thamnosma montana), and celery and parsnip. Various species of these fungal

phytoalexins have been identified (xanthotoxin and tripsoralen) and monitored in plants as they are infected (Stegelmeier et al., 2019a). Drugs and Other Toxicants Several pharmaceuticals have been shown to induce photosensitivity. In ruminants, phenothiazine can be metabolized to phenothiazine sulfoxide, a photoactive molecule. Other drugs or treatments that have been associated with primary photosensitivity include thiazides, acriflavins, sulfonamides, tetracyclines, methylene blue, coal-tar derivatives, furosemide, promazine, chlorpromazine, quinidine, and some antimicrobial soaps. Hepatogenous Photosensitization (Secondary Photosensitization) Photosensitization caused by increased circulating phylloerythrin concentrations has been identified as hepatogenous or secondary photosensitization. Phylloerythrin is a chlorophyll metabolite that is formed by enteric microorganisms. Phylloerythrin is absorbed from the gastrointestinal tract and carried to the liver via the portal circulation. Within the normal liver, the hepatocytes conjugate phylloerythrin and excrete it in the bile. However, if the liver is damaged or bile excretion is impaired, phylloerythrin accumulates in the liver, blood, and subsequently in the skin, causing photosensitivity. This is probably the most common cause of photosensitization in livestock, and it is a common clinical result of the hepatotoxic plants discussed in other sections. Photosensitization Sequelae Photosensitization of livestock in most cases is related directly to the presence of photodynamic compounds in the blood and cutaneous tissues. These molecules are generally not directly toxic but require irradiated energy to transform and subsequently damage adjacent tissues. Most affected animals quickly recover if they are protected from additional solar irradiation and the dermal lesions are treated and allowed to heal. However, the economic cost of such poisoning can be large, as photosensitized animals may prematurely wean their babies (the teats and udders are often severely affected) and working animals are not able to perform for weeks and

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sometimes months. With proper treatment, most animals recover without permanent sequelae (Stegelmeier et al., 2020).

7.5. Bracken Fern Bracken fern (Pteridium aquilinum) subspecies and varieties are found throughout the world and are the fifth most distributed and common weed. Though it commonly grows in semishaded, well-drained open woodlands, it can grow and spread in a variety of soils and environmental conditions. It is a perennial, erect plant with deciduous fronds that range from 0.5 to 4.5 m long. It is a prolific spore producer but spreads primarily through a dense rhizome network. Under certain conditions, such as in disturbed locations that have been burned or disturbed, bracken fern populations can be highly expansive so that they dominate local plant communities. Although it has been used historically as both food and a medicinal, in most locations it is classified as a weed problem. Animals will eat bracken fern, which remains toxic in stored and prepared feed. Livestock poisoning has been reported on nearly every continent. Several bracken fern toxins have been identified and characterized. Many others are suspected, partially described, or have been identified, but have not been associated with clinical poisoning. Many bracken fern parts often contain cyanogen glycosides, thiaminases, thiamine-inhibiting compounds, several steroid-like compounds, and several other radiomimetic toxins and carcinogens. Of these, the best described is ptaquiloside, a norsequiterpene glucoside. Ptaquiloside and its metabolites have been shown to be mutagenic, clastogenic, and carcinogenic, and it is thought that they initially damage rapidly dividing cells in the bone marrow and gastrointestinal tract. At longer dose durations and depending on the conditions in which they are hydrolyzed, they have also been shown to be potent carcinogens as they produce bovine esophageal, gastric, and urinary tract neoplasms. Additional bracken toxins that have carcinogenic potential include isoptaquiloside and caudatoside. Variable and often remarkably high ptaquiloside concentrations are found in the vegetative plant parts, but the rhizomes, roots, and spores contain very little. Nevertheless, bracken spores have been shown

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to be carcinogentic suggesting there may yet be an unidentified bracken carcinogen. More work is needed to identify these unknown carcinogens and determine their role in bracken fern-related carcinogenesis. Several thiaminases have also been isolated from bracken fern. Thiaminase concentrations are highest in the rhizomes, which often is remarkable. Additionally, several other antithiamine compounds, caffeic acid, astragalin, and isoquircetin have also been identified and probably contribute to bracken fern– induced thiamine deficiency. Prunasin, a cyanogenic glycoside, has also been identified in bracken fern; however, it has not been directly connected with clinical poisoning or with the related syndromes. The role prunasin and chronic cyanide poisoning plays in bracken fern poisoning especially in horses needs to be better defined. This is interesting, as many clinical changes of chronic cyanide poisoning in humans are very similar to the neurologic disease that develops in bracken fern–poisoned horses. As suggested by the variety of toxins, various syndromes have been associated with bracken fern poisoning. These syndromes include acute hemorrhagic disease, bovine enzootic hematuria, bright blindness, upper alimentary carcinomas, and thiamine deficiency. Current evidence suggests these different toxicologic presentations are dose- and species-specific (Hintz, 1990; Smith and Seawright, 1995). Acute Hemorrhagic Disease and Enzootic Hematuria These syndromes primarily affect ruminants, and they are probably a continuum of the same disease process that is separated by dose and duration. As the names suggest, these syndromes are characterized clinically by hemorrhagic disease or chronic intermittent hematuria and anemia. Cattle and occasionally sheep are most often poisoned. Hemorrhagic disease occurs most often in late summer when other feed is scarce. It may also occur when animals are fed hay containing bracken fern. Poisoning requires prolonged exposures, as affected livestock must ingest bracken fern for several weeks to years before disease develops. Affected cattle are weak and rapidly lose weight. They become febrile and dyspneic with pale mucosal membranes. Hemorrhages vary from minor mucosal petechia to effusive bleeding at times. Severe cases have

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massive hemorrhage seen as large blood clots which may be passed in the feces. Coagulation is prolonged and bleeding pronounced and excessive, so that even small wounds such as insect bites or other minor scratches bleed profusely. Once animals develop clinical disease, poisoning is almost always fatal. Postmortem examination usually reveals multiple serosal and soft tissue hemorrhages and bruises. There may also be intestinal hemorrhage with ulceration. The urinary bladder mucosa is nearly always hemorrhagic with vascular dilation and congestion. The uroepithelium of the urinary bladder and urethra may also contain numerous vascular, fibrous, or epithelial neoplasms. Though less common, neoplasms in the upper gastrointestinal tract have also been reported. Enzootic hematuria is clinically characterized by intermittent urinary hemorrhage that is due to extensive epithelial and mesenchymal neoplastic transformation of the urinary tract. The neoplastic lesions are of mixed origin as they include vascular, mesenchymal, or epithelial differentiation ranging from papillomas to invasive carcinomas and sarcomas. The adjacent urinary epithelium is often ulcerated, and there are large submucosal hemorrhages. In most cases mixtures of hemorrhagic and neoplastic lesions are present, making separation of these syndromes artificial. Both the hemorrhagic syndrome and uroepithelial neoplasms have been reproduced experimentally with both bracken fern and purified ptaquiloside. The clinical presentation is dose related, as high doses of short duration produce acute poisonings seen as hemorrhagic disease. This is due to ptaquiloside’s cytotoxic effects on proliferating bone marrow stem cells. Microscopically, there is depletion of bone marrow megakaryocytes followed by panhypoplasia. The leukogram often has a mixed response. In the initial phase there is a pronounced monocytosis, which is followed by granulocytopenia and thrombocytopenia. Final phases include marked thrombocytopenia with anemia, leukopenia, and hypergammaglobulinemia. Urinalysis generally includes hematuria and proteinuria. Affected animals have both an increased susceptibility to infection and a tendency for spontaneous hemorrhage. Bracken fern exposures of lower doses with longer duration are more likely to be carcinogenic. The effects are probably cumulative, as animals may be exposed repeatedly for years before disease onset.

Often the onset of clinical disease can be delayed for weeks, or even months, after animals have been removed from bracken fern–infested ranges and pastures. In addition to livestock, the carcinogenic potential of bracken fern and ptaquiloside has been confirmed in rats, mice, guinea pigs, quail, and Egyptian toads. Ptaquiloside is excreted in the urine and milk of poisoned animals. Contaminated milk retains toxicity, as it has been shown to produce gastrointestinal neoplasms in rats. Several investigators have suggested that ptaquiloside neoplastic transformation may be promoted or enhanced by bovine papilloma virus infection. However, this is probably secondary, as bracken fern–associated myelodysplasia and subsequent immunosuppression may promote papilloma virus infection (Prasad and George, 1986). Bright Blindness Another bracken fern syndrome that may be a less common component of ptaquiloside toxicity has been called bright blindness or progressive retinal degeneration of sheep. It is characterized clinically as marked degeneration and thinning of the retina that is seen clinically as tapetal hyperreflectivity. It is most commonly reported in England and Wales but is also described with lower frequency in New Zealand and Australia. Affected sheep may be partially visual, and in efforts to see, they adopt a characteristic wide-eyed or alert attitude. The pupils respond poorly to light. Ophthalmoscopic findings are those of retinal degeneration characterized by a hyperreflective fundus with narrowing of arteries and veins and pale tapetum nigrum with patchy gray spots. Histologically, the lesion consists of severe atrophy of the retinal rods, cones, and outer nuclear layer that is most pronounced in the tapetal portion of the retina. Affected animals may also have many of the other bracken fern–associated lesions, including bone marrow suppression, hemorrhage, immunosuppression, and urinary tract neoplasia. Retinal degeneration has also been experimentally reproduced in sheep, using both powdered bracken fern and purified ptaquiloside (Hirono et al., 1993). Bracken Staggers Bracken fern staggers or neurologic disease is the bracken fern poisoning syndrome that is

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described in monogastric animals. It was first recognized as a neurologic disease when horses consumed contaminated hay. Subsequent studies showed that horses fed 20%–25% bracken fern for 3 or more months developed the characteristic staggers. The clinical presentation of bracken staggers in horses includes anorexia, weight loss, incoordination, and a crouching stance while arching the back and neck with splayed feet. When forced to move, affected animals tremble and, if they are severely poisoned, horses develop tachycardia and arrhythmias. These signs may progress to convulsions and opisthotonos, and such severely affected animals generally die. As this disease is similar to vitamin B1 (thiamine) deficiency and most animals respond with thiamine therapy, the pathogenesis of these changes has been attributed to bracken fern thiaminases and other potential antithiamine factors. Perhaps partially due to availability and feeding practices, horses seem to be particularly susceptible to bracken fern–induced neurologic disease. The disease is rare in pigs and the signs are less distinct, including anorexia, weight loss, and sudden onset of dyspnea, recumbency, and death. In ruminants, thiamine deficiency is generally associated with polioencephalomalacia, which is not a common finding in ruminant bracken fern poisoning; however, impaired thiamine metabolism in sheep has been associated with consumption of bracken fern and rock or mulga fern (Cheilanthes sieberi) in Australia (Burrows and Tyrl, 2013). Human Poisoning Epidemiologic studies suggest that consumption of milk from cattle with access to bracken fern produces increased risk of human esophageal or gastric cancer. Certainly, the greater risk to humans is greater in the case of direct consumption of bracken fern. Bracken rhizomes have been used to make flour. The young shoots or croziers are considered a delicacy in many parts of the world. Though preparing and cooking lessen toxicity, ptaquiloside has been identified in all these foods. Additionally, ptaquiloside has been found as an environmental contaminate in soil and water, where further exposure is possible. Exposure through all these means should be of concern, as ptaquiloside is a proven carcinogen (Smith and Seawright, 1995).

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Treatment and Prevention As bracken fern poisoning, apart from thiamine deficiency, is essentially untreatable, it should be controlled by preventing exposure. Bracken fern is usually grazed for want of alternative forages. Avoiding exposure by improving pasture management and increasing the production of alternative forage is essential. Recent work has found that some bracken fern populations contain very little or no ptaquiloside. More work is needed to identify these populations, determine why they are not toxic, and use this information to predict or reduce toxicity. As with most toxic plants, the initial step should be to remove poisoned animals from bracken fern–containing pastures. Treatment of the induced thiamine deficiency in horses is effective if the diagnosis is made early. Thiamine should also be administered to animals similarly exposed but not yet showing signs, as they may develop disease days or weeks after removal from the source of bracken. Antibiotics may be useful to prevent secondary infections. Blood or even platelet transfusions may be appropriate. Most animals that develop hemorrhagic and neoplastic disease do not recover.

7.6. Ricinus spp. Castor beans (Ricinus communis) were named after wood ticks, Ixodes ricinus, as the mottled seeds resemble a blood-gorged tick. Originally from Africa or Asia, castor beans are now distributed throughout the world and seeds and evidence of cultivation have been discovered in many cultures as early as 7000 BCE. Today, many populations have escaped cultivation and are considered a weed, although in some situations they are still used as an ornamental and for oil production. The oil has been used medicinally as a purgative and it has also been used as an industrial lubricant and coolant. The seeds have been used ornamentally in jewelry. Poisoning occurs in all species, including humans, livestock, and wildlife. For example, entire flocks of ducks have been fatally poisoned after feeding on remnants of castor bean fields. Though the seed coat is thick and impairs toxin absorption, human poisoning is generally related to ingestion of the beans and medicinal use of castor oil. It has been reported

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that poisoning has occurred due to the ingestion of as few as a couple of beans. The leaves and pericarps are also toxic and seem more likely to produce neurologic disease (Akande et al., 2016). Three toxins have been identified in castor bean. As previously mentioned, ricin is a glycoprotein composed of an enzymatic A chain and a lectin B chain. These chains have been described as facilitator and effector chains. The lectin binds epithelial cells, altering absorption and causing mucosal necrosis. The effector chain has been described as a class 2 ribosomal inhibitor protein. Detailed studies have been made of the molecular and physiologic effects of both of these toxins. Ricin is extremely toxic, and as little as 500 mg can be lethal for an adult human. Toxic both by inhalation and ingestion, ricin has been developed and used nefariously, and its possible use as a weapon (Lord et al., 2003). The second toxin is ricinin, an alkaloid that is thought to cause neurologic signs by binding to GABA receptors and antagonizing nicotinic receptors in neuromuscular junctions. The third toxin is the lectin, agglutinin. It agglutinates erythrocytes, but its toxicologic effect has not been defined. It may be immunogenic and contribute to the allergic response that castor bean elicits in many individuals. In most species, signs of poisoning include severe diarrhea, which may be watery or bloody and is often accompanied by severe colic, abdominal straining, nausea, sweating, vomiting, and sudden collapse and death. Some cases develop neurologic signs of weakness, seizures, and coma. Affected animals may also develop liver and kidney disease. The mucosa of rumen, stomach, and small intestine is severely congested and edematous. The mesenteric lymph nodes are often swollen and edematous, and there may be petechial and ecchymotic hemorrhages on the intestinal and cardiac serosal surfaces. Some cases may have degeneration and necrosis of hepatocytes and renal tubular epithelia. Most of these changes develop within 6 h of ingestion, and the duration is generally less than 24 h. Treatment is generally supportive, with fluids, electrolytes, glucose, and dieresis. Activated charcoal may be useful early, but when mucosal damage is extensive the prognosis for survival is poor.

8. ADDITIONAL RESOURCES There are many additional references that describe toxic plants and the diseases and lesions that relate to their toxicity. The Internet expands daily with globally available information. The Poisonous Plant Research Laboratory of the United States Department of Agriculture provides a web page (https://www.ars.usda.gov/pacificwest-area/logan-ut/poisonous-plant-research/docs/ main/) with information of toxic plants, current research progress, and contacts for specific problems and questions. Additionally, there are numerous texts that are excellent material for many toxic plants found in North America, and in other parts of the world (Burrows and Tyrl, 2013; Kingsbury, 1964; Botha and Penrith, 2008; Knight and Walter, 2001; Riet-Correa et al., 2009a). For the past 45 years toxic plant researchers have held an international symposium on toxic plants. The proceedings of this International Symposium on Poisonous Plants are published in book form. These chapters often contain unique information of lesser-known plants and disease syndromes that are not available through most indexing services (Acamovic et al., 2001; Colegate et al., 1994; Garland et al., 1998; James et al., 1992; Keeler et al., 1978; Panter et al., 2007; Riet-Correa et al., 2009b; Seawright et al., 1985).

REFERENCES Acamovic T, Stewart CS, Pennycott TW: Poisonous plants and related toxins, Wallingford Oxon, 2001, CABI Publishing. Akande TO, Odunsi AA, Akinfala EO: A review of nutritional and toxicological implications of castor bean (Ricinus communis L.) meal in animal feeding systems, J Anim Physiol Anim Nutr 100:201–210, 2016. Alegbejo JO: Nutritional value and utilization of Amaranthus (Amaranthus spp.)- A review, Bayero J Pure Appl Sci 6:136– 143, 2013. Allen JG: The toxicity of phomopsin in a crude extract of Phomopsis leptostromiformis to pigs and sheep. In Seawright AA, Hegarty MP, James L, Keenan RW, editors: Plant toxicology, Melbourne, 1985, Dominion Press- Hedges and Bell. Anderson RC, Majak W, Rassmussen MA, et al.: Toxicity and metabolism of the conjugates of 3-nitropropanol and 3nitropropionic acid in forages poisonous to livestock, J Agric Food Chem 53:2344–2350, 2005.

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REFERENCES

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Stegelmeier BL, Molyneux RJ, Asano N, et al.: The comparative pathology of the glycosidase inhibitors swainsonine, castanospermine, and calystegines A3, B2, and C1 in mice, Toxicol Pathol 36:651–659, 2008. Stuart BP, Cole RJ, Gosser HS: Cocklebur (Xanthium strumarium, L. var. strumarium) intoxication in swine: review and redefinition of the toxic principle, Vet Pathol 18:368–383, 1981. Vetter J: Plant cyanogenic glycosides, Toxicon 38:11–36, 2000. Welch KD, Green BT, Panter KE, et al.: If one plant toxin is harmful to livestock, what about two? J Agric Food Chem 62: 7363–7369, 2014. Welch KD, Panter KE, Gardner DR, et al.: The acute toxicity of the death camas (Zigadenus species) alkaloid zygacine in mice, including the effect of methyllycaconitine coadministration on zygacine toxicity, J Anim Sci 89:1650–1657, 2011. Welch KD, Panter KE, Lee ST, et al.: Cyclopamine-induced synophthalmia in sheep: defining a critical window and toxicokinetic evaluation, J Appl Toxicol 29:414–421, 2009.

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8 Animal Toxins Brad Bolon1, Kathleen Heinz-Taheny2, Kara A. Yeung3, Justin Oguni4, Timothy B. Erickson5, Peter R. Chai5, Charlotte E. Goldfine5 1

GEMpath, Inc., Longmont, CO, United States, 2Eli Lilly and Company, Indianapolis, IN, United States, 3Brigham and Women’s Hospital, Massachusetts General Hospital, Boston, MA, United States, 4The Veterinary Clinic West, Marietta, GA, United States, 5Brigham and Women’s Hospital, Boston, MA, United States

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3. Zootoxin Classification 3.1. Zootoxin Classification 3.2. Zootoxin Classification 3.3. Zootoxin Classification 3.4. Zootoxin Classification

by by by by

Source Molecular Structure Function Mechanism of Action

4. Clinical Presentations and Pathologic Manifestations of Zootoxin-Mediated Diseases 4.1. Blood Vessels and Blood Components 4.2. Epithelium (Cutaneous and Mucosal Surfaces) 4.3. Kidney 4.4. Liver 4.5. Lung 4.6. Muscle (Cardiac and Skeletal)

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1. INTRODUCTION Humans have been fascinated for centuries by the powerful properties of animal toxins. This allure is attested through history by numerous legends, facts, and flash-in-the-pan trends. The legend of Queen Cleopatra of Egypt choosing Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00008-3

4.7. Neuromuscular Junction and Other Peripheral Synapses 4.8. Systemic (Multi-Organ) Failure

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5. Diagnosis and Treatment of Zootoxin-Mediated Diseases 5.1. Diagnosis 5.2. Treatment

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6. Regulatory Guidance Regarding Zootoxins 6.1. Sources and Major Indications of Medicinal Zootoxins 6.2. Practices for Developing Zootoxin-Based Medical Products 6.3. Practices for Developing Antivenom Products

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the asp (Egyptian cobra [Naja haje]) as an instrument of suicide. The invoking of animal parts in concocting the witches’ “hell-broth” in a theatrical masterpiece (“Eye of newt, and toe of frog,/Wool of bat, and tongue of dog,/Adder’s fork, and blind-worm’s sting,/Lizard’s leg, and owlet’s wing.” Macbeth, Act IV, Scene I, by

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William Shakespeare)deven though these ingredients are actually pseudonyms for medieval pharmaceutical plants known to apothecaries (Larum, 2019; Tishma, 2019). Acts of murder using animals (a “swamp adder” in the Sherlock Holmes story The Speckled Band, by Arthur Conan Doyle) or their toxic products (e.g., the blister beetle [Epicauta sp.]–derived poison cantharidin as a favored instrument of assassination by some medieval aristocrats) (Karamanou et al., 2018). The snake oil craze that swept the United States near the close of the 19th century thanks to the mesmerizing tales and dubious concoctions of Clark Stanley, the Rattlesnake King (Haynes, 2015; Stanley, 1897) (Figure 8.1). Modern-day exposures to animal-derived toxins are generally less sensational but no less memorable. Millions of animals and people are stung (mainly by jellyfish, insects, or scorpions) or bitten (chiefly by snakes or spiders) each year, resulting in significant morbidity and mortality. Counterbalancing the significant public health issue posed by animal toxins is the growing number of novel therapies made possible due to an expanding arsenal of animal toxin–derived drugs that have redirected the harmful properties of these substances to alleviate various human and animal diseases. The kingdom Animalia brings together more than one million distinct species of creatures equipped with myriad diverse characteristics designed to assist in surviving and thriving in a hostile world. Among these features, the evolution of biochemical and molecular means for defense (warding off predators) and/or offense (food acquisition (Figure 8.2) and sometimes digestion of food, elimination of competitors) are among the most essential and interesting adaptations for meeting environmental challenges (Arbuckle, 2017; Nekaris et al., 2020; Rash and Hodgson, 2002; Rode-Margono and Nekaris, 2015; Walker et al., 2018). Other animals have adapted these potent molecules for alternative purposes such as communication (e.g., laying pheromone trails by army ants on the march), defeating rivals during courtship (e.g., platypus [Ornithorhynchus anatinus] and some slow loris [primate (Nycticebus spp.)] species), and domestic hygiene (e.g., protecting broods from bacteria and parasites by some wasps) (Arbuckle, 2017; Yan and Wang, 2015; Zhang, 2015). Some insects utilize venom zootoxins placed by prey species

as homing signals to find new hosts to parasitize (Sharma and Fadamiro, 2013). Toxins may be passed from individual to individual, presumably for defense; this phenomenon occurs during copulation (male transfer to female) for several beetle species, and from the mother to gametes and/or offspring for some species of marine and terrestrial invertebrates as well as a few bony fishes and poison dart frogs (Nelsen et al., 2014). The ability of such substances to harm cells and tissues

FIGURE 8.1 Label for “Clark Stanley’s Snake Oil Liniment,” a popular 19th century nostrum touted as a remedy for many inflammatory and painful conditions. In modern parlance, this product was an overthe-counter patent medicine having limited efficacy that was sold directly to consumers and apothecaries using sensational advertising gimmicks. This label, designed by the liniment’s inventor, features a virile outdoorsman (a stylized self-portrait) bracketed by twin rattlesnakes (perhaps paying homage to the paired serpents encircling the staff of the caduceus). Myriad conditions cured by this panacea are listed along the borders of the label. Original label credited to Mr. Clark Stanley (c. 1905).

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FIGURE 8.2 Acquisition of food is a key driver for zootoxin evolution, and death of prey represents the most extreme adverse outcome associated with zootoxin exposure. Adult eastern copperhead (Agkistrodon contortrix) engaged in consuming a deer mouse (Peromyscus spp.). Original image by Dr. Justin Oguni.

warrants their classification as toxins (i.e., toxic molecules of biological origin), or more specifically zootoxins (i.e., animal-derived toxins). Production of zootoxins incurs a high metabolic cost that is balanced against the advantages supplied by venom use, especially greater success in food acquisition and defense (Evans et al., 2019; Pintor et al., 2010). Venoms have arisen independently by convergent evolution many times across various phyla of the kingdom Animalia (Fry et al., 2009a). Every one of the invertebrate phyla (Porifera [sponges]; Cnidaria [e.g., corals, jellyfish, sea anemones]; Platyhelminthes [flatworms]; Nematoda [roundworms]; Annelida [segmented worms]; Mollusca [e.g., clams, octopi, squids, snails, slugs]; Arthropoda [e.g., arachnids, centipedes, crustaceans, insects, scorpions]; and Echinodermata [e.g., starfish, sea urchins]) has numerous species that produce one to thousands of zootoxins. Similarly, in vertebrates (phylum Chordata, subphylum Vertebrata), the five major taxonomic divisions of higher chordatesdsuperclass Pisces and superclass Tetrapoda with its classes Amphibia, Reptilia, Aves, and Mammaliadall have multiple species that produce zootoxins. Animals fill most aquatic, airborne, and terrestrial niches, so animals and humans encounter zootoxins with frequency. Zootoxins typically occur as complex cocktails of bioactive materials rather than as single toxic entities (Gwaltney-Brant et al., 2018; Watkins,

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2013). The precise molecular structure of individual zootoxins and the combination of toxins produced by a species have developed under positive selection (Zhang, 2015), often as a consequence of gene duplication (Vonk et al., 2013). Common toxic substances within these zootoxin mixtures include alkaloids, biogenic amines, amino acids, enzymes, lipids, nucleosides, metal ions, peptides, polysaccharides, quinones, and steroids, among others (Chen et al., 2018; Touchard et al., 2016; Watkins, 2013). These materials often act in additive or synergistic fashion in exerting their harmful effects. The combination of zootoxins varies among species, and among individuals of the same species (Arbuckle, 2017; Rash and Hodgson, 2002; Upadhyay, 2018; Yang et al., 2019). Furthermore, zootoxin potency is influenced by many factors including the body size, sex, age, and diet of the individual as well as various environmental influences such as the season and geographic location (Fowler, 1993; Gwaltney-Brant et al., 2018; Rash and Hodgson, 2002). For example, in the wild the neurotoxic alkaloids batrachotoxin in the skin of poison dart frogs and homobatrachotoxin in the feathers of some passerine birds are not synthesized through endogenous metabolic pathways but instead are bioconcentrated by the consumption of zootoxinladen melyrid beetles (Dumbacher et al., 2004). The same frog and bird species are nontoxic when kept in zoological collections and fed instead on nonpoisonous insects (GwaltneyBrant et al., 2018). Slow loris venom is a mixture of saliva with an apocrine gland secretion (from a gland in the axilla [“armpit”]), each of which is toxic but which exhibit synergistic potency when combined during grooming (Nekaris et al., 2013). Zootoxins typically are categorized as either poisons or venoms depending on the manner in which an animal or human is exposed to the toxin (Gwaltney-Brant et al., 2018; Watkins, 2013). In this context, a poison is a mixture of zootoxins generated in nonspecialized tissues through the accumulation of toxic metabolic by-products. Exposure to animal-derived poisons usually occurs through oral (or rarely dermal or inhalational) contact because poisonous animals lack any means for actively delivering the zootoxin brew into the victim’s tissues. In contrast, venom is a zootoxin mixture

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produced in a specialized tissue (usually a gland), and venomous animals have evolved one or more specific apparatuses (e.g., grooved or hollow teeth, stingers, stinging cells) to actively introduce the zootoxin cocktail into tissues of a hostile or prey animal. The process of venom delivery, termed envenomation or envenoming, involves mechanical injury. The term toxungen has been proposed as an intermediate category of zootoxins based on distinct mechanisms for delivery not relevant to poisons and venoms (Nelsen et al., 2014). Toxungens are toxic substances actively administered onto the external surface of another organism via a delivery method that does not produce mechanical injury (e.g., spitting or spraying). The toxungen concept is not discussed further in this chapter, and any exposures appropriate for this proposed term are grouped under poisons. This chapter provides a brief introduction to the toxinology of animal toxins, emphasizing their comparative toxicology and pathology. Toxinology is the study of the toxins, poisons, and venoms made by living organisms (animals, plants, and microbes), while toxicology is the study of the effects that toxic substances (including but not limited to toxins) have on living organisms. Section 2 will provide a short description of typical exposure scenarios in which animals and humans may encounter zootoxins. Section 3 will consider potential classification schemes for zootoxins. Section 4 will explore important mechanisms of zootoxin action. Section 5 will examine the clinical presentation and major pathologic manifestations resulting from zootoxin exposure. Section 6 will provide current diagnostic and treatment methods employed for zootoxicoses (i.e., a toxicant-induced disease state [“toxicosis”] caused by exposure to a zootoxin). Finally, Section 7 will cover regulatory guidance related to use of zootoxins in the production of therapeutic products.

estimated 100,000 deaths (Feola et al., 2020; Kasturiratne et al., 2008; Rojnuckarin et al., 2012). Snakebite is among the top 10 causes of death in many Asian countries. Moreover, an estimated 400,000 cases of chronic disability occur in snakebite survivors annually due to persistent pain and swelling, joint contractures, necrosis and loss of extremities (digits or entire limbs), or psychological disorders (Feola et al., 2020; Tasoulis and Isbister, 2017). Some poisonous and venomous animals use warning behaviors (e.g., coiling and rearing by snakes), have evolved visual cues (e.g., brilliant colors of poison dart frogs [Figure 8.3] and cone snails), or deliver audible signals (e.g., tail shaking in rattlesnakes) to minimize encounters (Maan and Cummings, 2012; Sunagar et al., 2014a). In contrast, other species are not detected at the time of zootoxin exposure but are inferred to have been present later based on the clinical and pathologic effects in their victims (e.g., severe, locally extensive dermal necrosis induced by bites of brown recluse spiders [Loxosceles reclusa]). Humans and animals encounter zootoxins in several situations. The nature of the exposure depends on such factors as the setting (e.g.,

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FIGURE 8.3 Photograph of a strawberry poison dart frog (Oophaga pumilio) demonstrating the brilliant hues that provide visual cues to deter potential predators. Image reproduced from https://commons.wikimedia.org/wiki/ File:Pumilio_010_800_cristobal_to.jpg under a Creative Commons license (CC BY-SA 3.0).

Zootoxin encounters are a global public health priority. The incidence of envenomation due to snakebite alone is estimated at 4 million cases worldwide annually in humans, resulting in an

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human-modified or wild environment), geographic region (i.e., the species within that biosphere), and whether exposures occur inadvertently as poisoning or envenomation or deliberately via therapeutic interventions in which zootoxins comprise an active component of a medication or weapon.

2.1. Poisoning As noted previously, poisoning with zootoxins requires direct contact between the victim and the toxic mixture because poisonous animals lack an active mechanism for delivering their toxins. The usual routes for poison exposures in both medical and veterinary medical practices are by ingestion, less frequently direct contact with the skin (or mucosa), and uncommonly by inhalation. Ingestion Zootoxin exposure by the oral route commonly involves ingestion of the poisonous animal (or pieces thereof). Encounters may be accidental (e.g., consumption of blister beetles [Epicauta spp.] buried inside hay bales), adventurous (e.g., paresis and paralysis after eating the Japanese delicacy fugu [pufferfish, Fugu spp.]), ill-informed (e.g., attacks on cane toads or rattlesnakes by inexperienced dogs), unexpected (e.g., amnesic and paralytic shellfish poisonings), or in some cases deliberate (cantharidin from blister beetles is a key component in the aphrodisiac “Spanish fly” and also is a reputed ingredient in cantarella, a Renaissance poison made by the Borgias as a tool for eliminating personal and political rivals (Emsley, 2017; Karamanou et al., 2018)). Zootoxins may be widely distributed within the animal (e.g., the entire skin of many true toads from the family Bufonidae (Mailho-Fontana et al., 2018) [Figure 8.4] and most organs of ciguatoxincontaminated reef fish (Friedman et al., 2017)) or confined to particular organs (e.g., the ovaries, liver, and intestines of pufferfish (Lago et al., 2015)). Thus, the likelihood of experiencing a zootoxic episode depends on the ingested species and the particular organs that are consumed. Toxic effects following poison consumption may be localized to the digestive tract. Some zootoxins have poor oral bioavailability (Yang et al., 2019),

FIGURE 8.4 Photograph of a Sonoran Desert (or Colorado River) toad (Incilius alavarius) showing the cutaneous distribution of poison-filled glands. A parotid macrogland (arrows) appears as an elongated postaural bulge while common poison glands are located at the cores of the many upraised dorsal warts. Original image by Dr. Justin Oguni.

at least in part due to their large molecular size and complexity and/or their inability to withstand the harsh chemical environment within the digestive tract (e.g., peptides and proteins). Nonetheless, such nonabsorbed zootoxins may impact digestive tract function through direct contact with the mucosal lining. Therefore, their key toxic effects reflect local irritation and tissue destruction to the mucosa of the digestive tract. For instance, dogs that have mouthed toads (e.g., Rhinella marina [formerly Bufo marinus], commonly known as the cane toad, or Incilius alvarius [formerly Bufo alvarius], commonly known as the Colorado River toad or Sonoran Desert toad) or toad larvae (“tadpoles”) exhibit hypersalivation with reddening of the oral mucosa (Eubig, 2001; Reeves, 2004), while livestock (primarily cattle and horses) that have consumed blister beetles develop “blisters” (raised, fluid-filled vesicles), hemorrhage, inflammation, and ultimately ulcers (i.e., local sloughing of necrotic mucosa) anywhere from the oral cavity to the intestine (Krinsky, 2009). Such effects may be accompanied by numbness of the oral mucosa and tongue as well as nausea and vomiting. Other ingested zootoxins are well absorbed orally due to their molecular structure (e.g., alkaloids), especially if present at high levels (e.g., cantharidin comprises up to 5% of the dry weight

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of blister beetles) (Krinsky, 2009; Reeves, 2004). Small molecule zootoxins induce a number of systemic effects following absorption. The nature of the clinical signs depends on the biodistribution and molecular action of the toxin. For example, ciguatoxin and tetrodotoxin are dispersed widely upon absorption, ultimately exerting potent neurotoxic effects by blocking the function of voltage-gated ion channels that are critical for nerve impulse transmission at the neuromuscular junction. Cantharidin is distributed in the blood and is excreted unchanged through the kidney, thus producing hemorrhage, inflammation, and necrosis affecting renal tubular epithelium and the urinary bladder mucosa (Krinsky, 2009). The urinary tract irritation can lead to priapism (long-lasting erection) and genital warmth in men and women, which explains the use of cantharidin historically as a main ingredient in the oral aphrodisiac “Spanish fly.” Other traditional aphrodisiacs (used to enhance male virility) and homeopathic remedies in many East Asian countries include numerous bioactive molecules that induce severe and even lethal clinical disease if an overdose is consumed. For instance, cane toad extracts used as an aphrodisiac contain many cardiac glycosides with digitalis-like activity, which may induce lethal disturbances including severe cardiac electrophysiological (bradycardia) and metabolic (acidosis and hyperkalemia) effects in extreme cases (Gowda et al., 2003). Similarly, “snake wines” (a rice-derived alcoholic libation infused with herbs and spices and containing one or several intact venomous snakes and sometimes scorpions) used in several East Asian countries to treat chronic inflammatory conditions may be associated with clinically significant coagulopathy if the coagulotoxic zootoxins do not become denatured over time by exposure to ethanol (Moon and Chun, 2016). Common zootoxicoses resulting from ingestion vary depending on the toxin and its molecular mechanism of action as well as the geographic location and local culture of the susceptible population. The various seafoodrelated illnesses effectively illustrate this principle. Ciguatera fish poisoning (or simply “ciguatera”) is the most common food-borne zootoxicosis in the United States. This condition results from consumption of fillets from ray-

finned fish (barracuda, grouper, mahi-mahi, red snapper, sea bass, tuna, etc.). Flesh from these apex predators bioconcentrate ciguatoxin and maitotoxin, two lipophilic polycyclic neurotoxins produced by the unicellular dinoflagellate Gambierdiscus toxicus (see Phycotoxins [Vol 3, Chap 5]). These toxins are heat-stable and thus remain active after cooking. Neurological signs and symptoms develop within 0.5–48 h depending on the dose and typically begin with tingling of the lips, tongue and throat (Friedman et al., 2017). These initial signs are followed by a spectrum of effects ranging from abdominal cramping, nausea, vomiting, and impaired vision to life-threatening convulsions, muscle paralysis, coma, and sometimes death (in 12% of the approximately 50,000 annual cases) (Friedman et al., 2017). Clinically, pufferfish (fugu) poisoning in Japan resembles ciguatera, including a similar constellation of human neurological disturbances, the potential for death, and the lack of an effective antitoxin. However, the toxic agent in fugu (tetrodotoxin) is a bioconcentrated bacterial product and not a zootoxin per se (Lago et al., 2015; Magarlamov et al., 2017; Wu et al., 2005) (see Bacterial Toxins [Vol 3, Chap 9]). Paralytic shellfish poisoning is caused by consumption of bivalve mollusks (clams and mussels) that have ingested unicellular dinoflagellates of the genera Alexandrium, Gonyaulax, and Pyrodinium. This condition usually occurs in coastal dwellers or tourists at coastal resorts, but it also has been implicated in deaths of sea otters (which consume shellfish) (DeGange and Vacca, 1989) and humpback whales (which eat predatory fish) (Geraci et al., 1989). The active molecules are neurotoxic alkaloids (e.g., saxitoxin) that reversibly block voltage-gated ion channels on nerve terminals. Initial effects of saxitoxin consist of local burning, tingling, and numbness of the lips, tongue, and face, but effects in severe cases may expand to include muscle weakness, difficulty in swallowing, muscle paralysis, and eventually death due to failure of skeletal muscles essential in respiration (Etheridge, 2010). Chelonitoxism is a food poisoning syndrome in subtropical and tropical Asia resulting from consumption of turtle meat, especially of various marine species. Concentration of chelonitoxins in milk may prove lethal to nursing infants (Rasamimanana et al., 2017). Signs of digestive

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tract injury (e.g., abdominal pain, diarrhea, nausea, vomiting) are accompanied by systemic pathological findings that include gastrointestinal tract edema and hemorrhage as well as renal and splenic congestion. An algal neurotoxin is postulated as the cause due to substantial neurological signs (e.g., increased salivation, lethargy, reduced reflex activity, vertigo) that rarely may produce death by respiratory failure. Finally, scombroid fish poisoning is a pseudoallergy resulting from ingestion of inadequately preserved and/or refrigerated albacore, bonito, mackerel, mahi-mahi, swordfish, tuna, or related species. Unlike genuine food allergies, which occur year-round in sensitive individuals, this condition may affect many people simultaneously and often occurs in the summer (Stratta and Badino, 2012). The organs of scombroid fish naturally contain high levels of histidine, which is converted to histamine by symbiotic bacteria that have colonized improperly stored fillets. This vasoactive amine is resistant to chemical preservation (curing and smoking) as well as pressure (canning) and temperature (cooking and freezing) extremes. Initial signs include facial flushing, burning and/or peppery-tasting sensations of the mouth or throat, headache, nausea, and sometimes hypotension (due to vasodilation) and tachycardia. A facial rash (often with pruritus) or sometimes torso rash accompanied by widespread edema may develop over time, while severe cases lead to substantial tongue swelling and respiratory distress. Signs are more severe in individuals with histamine intolerance (Maintz and Novak, 2007). The rash often recedes first (within 2–5 h), and full recovery typically occurs in 36 h with or without administration of antihistamine drugs. Dermal or Mucosal Contact Zootoxin poisoning by contact with an external body surface occurs in many settings. The poison usually is applied when the victim accidentally contacts the poisonous animal. Individuals who slap a blister beetle crawling on their skin will develop burning pain and cutaneous vesicles (Selander and Fasulo, 2000). Recreational divers who brush against fronds of red beard sponge (Microciona prolifera) will experience redness, swelling, and stiffness of the skin and digits. Indigenous hunters who

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roughly manipulate poison dart frogs (Dendrobates spp., colloquially known as “poison arrow frogs”) by hand rather than using waxy leaves typically will develop cutaneous burning, muscle cramping, and pain. Wildlife biologists (or tourists) who stroke the breast and belly feathers of the hooded pitohui (“rubbish bird” [Pitohui dichrous] of New Guinea) without using gloves may experience burning, tingling, or numbness of the skin (or eyes or lips if they touch these body parts before washing their hands). Other birds that accumulate zootoxins in their feathers and skin include the bluecapped ifrit (Ifrita kowaldi) and several variants of shrikethrush (Colluricincla spp.). Surface effects of zootoxins are potentiated if breaks in the cutaneous or mucosal barrier permit entry of the toxins into deep tissues. For instance, fire ants (Solenopsis spp.) use their mandibles to pierce the epidermis and then spray venom from abdominal glands into the resulting wounds. The venom acts as a poison in this context since the concentrated mixture of formic acid and other zootoxins does irritate the nearby skin surface; the burning pain occurs when the deposited venom is applied directly to any exposed free sensory nerve endings within the traumatized epidermis. Blister beetles concentrate cantharidin in their hemolymph (“blood”). When disturbed, the beetle reflexively expels hemolymph through leg joints to apply cantharidin as a caustic agent onto the cutaneous or mucosal surface (Nelsen et al., 2014). Inadvertent entry of poison dart frog secretions into cuts on the skin increases the speed and severity with which pain and cramping can develop. Finally, purposeful application of poison to wounded skin may cause powerful systemic effects. An example of this latter situation is “kamboˆ” (also called “vacina do sapo” [frog vaccine]), a ritual poisoning in which indigenous hunters deliberately burn their skin and then smear skin secretions from the Amazonian giant leaf frog (Phyllomedusa bicolor) over the fresh wounds (den Brave et al., 2014). The rich peptidome in the secretions leads to diarrhea, nausea, and vomiting and often may induce facial edema, hypotension, and palpitations as well. Some individuals describe pleasant psychoactive effects (Schmidt et al., 2020), possibly through affinity of the zootoxic peptides for opiate receptors (Junior and Martins, 2020). Kamboˆ has been

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adapted as an alternative medicine cleansing practice in Europe and the United States in recent decades. Zootoxin exposure via mucosal membranes may induce both local and systemic effects. Corneal inflammation potentially leading to ulcers, persistent conjunctivitis, and leukoma (white discoloration of the normally translucent cornea) are frequent sequelae of ocular contact with venom streams expelled by spitting cobras (Naja spp.) in Africa and Asia. As noted above, oral contact with cane toad secretions causes mucosal irritation (indicated by hypersalivation and reddening of the oral mucosa) even if the toad is not eaten (Reeves, 2004). The practice of “toad licking” has been used for centuries as a ritual hallucinogen (Davis and Weil, 1992). The skin secretions of some species (e.g., the Colorado River toad [Incilius alvarius]) contain large quantities of psychoactive alkaloids, principally 5-methoxy-N,N-dimethyltryptamine (5-MeODMT). This molecule is metabolized by cytochromes P450 to bufotenine (5-HO-DMT), which chemically resembles the neurotransmitter serotonin (chemical name: 5-hydroxytryptamine [5HT]) (Shen et al., 2010) and the hallucinogens lysergic acid diethylamide (LSD) and psilocybin (the neuroactive molecule in “magic mushrooms”). Bufotenine is absorbed and reaches the brain as attested by such neurological effects as transient hallucinations (fading in about an hour) and sometimes confusion and centrally driven nausea (that lasts from hours to days). Inhalation Inhalation is an uncommon route for zootoxin exposure, but in certain instances poisoning does take place across epithelial surfaces dedicated to gas exchange. In aquatic settings, mucosal surfaces of the gills (the lung equivalent in aquatic invertebrates and fish) and respiratory tract (in air-breathing animals) may be exposed, respectively, to water currents or wind-blown mists containing high quantities of dinoflagellate zootoxins. Massive overgrowth of these unicellular eukaryotes results in “red tides” (Figure 8.5), leading to death of many fish exposed via their gills; deaths in terrestrial animals occur from ingestion of contaminated water or zootoxin-laden fish (see Phycotoxins [Vol 3, Chap 5]). Humans exposed to aerosolized zootoxins while playing at the beach under “red

FIGURE 8.5 A “red tide” in a temperate harbor due to massive overgrowth of dinoflagellate algae. Arrows indicate a dead bony fish (lower) and a ray (upper). Image reproduced from https://commons.wikimedia.org/wiki/ File:Red_tide%EF%BC%BF2017.jpg under a Creative Commons license (CC BY-SA 4.0).

tide” conditions may experience transient, dosedependent lower respiratory symptoms such as wheezing (Backer et al., 2003). Deliberate intake of toad venom–based alkaloids for religious rituals has shifted in recent decades from oral exposure (toad licking) to inhalation exposure by combustion of semidry pastes or dried powders made from toad skin secretions (Weil and Davis, 1994). In toads, fluids from the common poison glands in cutaneous warts have low toxin amounts while maintaining high levels of psychoactive molecules as compared to the secretions from the large parotid macroglands (Figure 8.4). Therefore, exposure to dehydrated secretions from dorsal skin by the inhalation route retains the potent activity of the original fluid encountered during licking.

2.2. Envenomation As stated above, envenomation with zootoxins involves active introduction of venom into superficial and deep subcutaneous tissues of the victim. The usual routes for venom delivery in both medical and veterinary medical practices are by inadvertent mechanical injection via bites or stings. However, deliberate administration of animal venoms by humans may occur in both nontherapeutic and therapeutic settings.

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Injection of venom by mechanical disruption of cutaneous (or rarely mucosal) barriers is the most common means of zootoxin exposure (Nelsen et al., 2014). Most encounters involve human–animal interactions in rural environments, such as densely populated agricultural regions (mainly in African, Asian, and South American countries) or recreational wilderness (aquatic or terrestrial). Victims include humans (especially agricultural workers) and animals (both livestock and pets, primarily dogs). Affected tissues in envenomation cases depend on the thickness of the skin as well as the length of the injection apparatus (i.e., fang or stinger) or depth of the wound inflicted by teeth. Bites Venomous animals that bite are equipped with sharp apparatuses that both break the skin barrier and introduce the venom. The quantity of venom that can be delivered is a function of body size of the venomous animal. Bites typically are inflicted on body parts of the victim that are closest to the habitat of the venomous animal. For example, animals that encounter spiders or snakes are often bitten on the face (muzzle or near the eye) or tongue (Gwaltney-Brant et al., 2018). Extensive zootoxin-induced edema of the affected soft tissues may lead to closure of the larynx and/or proximal trachea, leading to death. In contrast, humans are often bitten on the distal leg, ankle, or foot when walking or on the distal arm, hand, or fingers if reaching into foliage or rocky wilderness terrain. Such bites may be rapidly lethal if delivered by species with potent neurotoxic venoms (e.g., elapid snakes such as brown snakes, cobras, coral snakes, kraits, and mambas). Species that introduce necrotoxic venoms, such as crotalid snakes (New World [or “pit”] vipers) and viperid snakes (Old World vipers), may engender a sequence of cytotoxic and hemotoxic events leading to transient or permanent dysfunction or even loss of the limb. In most cases, human deaths due to envenomation are associated with either respiratory paralysis (driven by neurotoxins), renal failure due to acute tubular necrosis, or hemodynamic events like shock (Feola et al., 2020). The means of venom delivery depends on the species. As mentioned above, fire ants use their mandibles to incise the superficial epidermis

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surface and then spray venom stored in abdominal glands into the wounds. Spiders and many snakes use hollow fangs connected to venom glands to penetrate the thick outer covering of prey, whether that is an exoskeleton (arthropods) or skin (invertebrates and vertebrates). Prototypic fangs have been shaped by evolutionary factors to optimize function while protecting structural integrity; their elongated curvature delivers optimal force to ensure penetration while their conical cross-sectional profile limits deformation and thus minimizes breakage (BarOn et al., 2014; Broeckhoven and du Plessis, 2017). Interestingly, in snakes hollow fangs tend to exhibit more curvature (Figure 8.6) than grooved fangs (where the surface groove serves as an open conduit to channel venom), but both apparatuses experience the same degree of mechanical stress during a bite (Broeckhoven and du Plessis, 2017). Venomous lizards such as Gila monsters (Heloderma suspectum) and Komodo dragons (or Komodo monitors, Varanus komodoensis) seize prey and use their blunt teeth to macerate the flesh of their prey. Venom flows into the wound by capillary tension along grooves in the teeth. The morbidity associated with Komodo dragon bites appears to be driven by venom and not by the long-held belief that transfer of toxigenic oral bacteria into oxygen-poor deep wounds induces sepsis and toxemia (Fry et al., 2009b). The outcome of a bite depends heavily on the quantity of venom injected by the animal. For instance, sea snakes have highly potent venoms but often deliver relatively small volumes (Peterson, 2004). Funnel web spiders (Atrax spp. and Hadronyche spp.) have the ability to modulate the amount of venom they deliver. “Dry bites” involve penetration of the skin, often drawing blood, in the absence of venom injection. “Modulated bites” result from the introduction of a calibrated venom quantity; less venom is injected for small prey (e.g., flies) than for larger prey (e.g., beetles). “Maximal bites” release as much venom as possible, likely as a deterrent against potential attackers. Spiders appear to target venom delivery to the thorax or head of prey to maximize zootoxin efficacy (Wigger et al., 2002). Similarly, snakes can regulate the amount of venom they deliver using compressor muscles associated with the venom glands (Figure 8.6). About 25% of snakebites are dry

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FIGURE 8.6 Anatomy of the venom apparatus in snakes. A: Head of an elapid (forest cobra [Naja melanoleuca]) with the skin removed to demonstrate the proteroglyphous (straight, forward-positioned, solid, unretractable) fang (A) and primary venom gland (B). B: Head of a viperid (common European adder [Vipera berus]) with skin and superficial muscles removed to reveal (from left to right) the solenoglyphous (curved, forward-positioned, hollow, retractable) fang (F), accessory venom gland (Ga), primary venom duct (Dp), and primary venom gland (Gp). The compressor glandulae muscle (Mc) assists in moving venom from the Gp forward into the Dp. Image A reproduced from https:// commons.wikimedia.org/wiki/File:Naja_melanoleuca_head_d issection_A-_fang_B_-_venom_gland.jpg under a Creative Commons license (CC BY-SA 4.0). Image B reproduced from https://commons.wikimedia.org/wiki/File:Vipera_berus_-_Ve nom_delivery_apparatus.JPG under a Creative Commons license (CC BY-SA 4.0).

bites (Peterson, 2004). Venom volumes delivered in an average bite are larger when administered by bigger animals (Peterson, 2004). The amount of protein injected by an eastern diamondback rattlesnake (Crotalus adamanteus) ranges from 200 to 850 mg (Russell, 1980), the brown recluse

spider delivers 30–65 mg of protein (da Silva et al., 2004), and an ant administers between 10 and 300 mg of protein depending on its body size (Touchard et al., 2016). The impact of a bite also depends on the composition of the venom delivered by the animal. For example, venom potency appears to increase with age in snakes (Peterson, 2004). In addition, snake venom is reported to cause greater morbidity but less lethality compared to the venoms of other animals (Sitprija and Suteparak, 2008). In the United States, the likelihood of dying by snakebite is approximately 10fold less than death by Hymenoptera (bee, hornet, and wasp) envenomationdand 3-fold less relative to cattle attack (Ingraham, 2015)! Funnel web spiders from Australia produce many neurotoxic peptides, notably d-hexatoxins (HXTX). Venoms produced by males are at least 6 times more potent than those of females. This discrepancy exists because the male HXTX complement includes zootoxins active against both insect prey and vertebrate predators (e.g., d-HXTX-Ar1, typically designated “d-atracotoxin” or “robustoxin”) while females have HXTX arsenals that are directed primarily against insects (Herzig et al., 2020). The evolutionary driver for this difference is thought to be the need for males, which migrate annually during warm months in search of safely burrow-bound female mates, to mount a defense against likely vertebrate predators encountered along the way. Stings Stings typically are inflicted on exposed body parts. In animals and humans, stings usually occur on glabrous (hairless) skin of the face, limbs, or torso. Envenomation may be lethal if an individual is sensitized to one or more venom components (i.e., hypersensitivity [“allergy”]) or if the victim is stung by large numbers of venomous animals over a short period (e.g., aggressive Africanized honeybees [Apis mellifera scutellata]) (Emsley, 2017). Bee sting incidents are second only to snakebites as a cause of death by envenomation in humans in some countries (Chippaux, 2015). Venomous animals that sting are equipped with barbed, smooth, or whiplike appendages that both pierce the skin (or mucosal) barrier and deliver venom. For example, various species

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of Hymenoptera possess barbed stingers (bees) or smooth stingers (hornets and wasps) (Das et al., 2018). The stinging apparatuses of Hymenoptera are repurposed female reproductive organs: the stinger is a modified ovipositor, and the venom glands are adapted accessory sex glands. The spiraled barbs on bee stingers reduce the force needed to penetrate the skin but also anchor the apparatus so firmly in the wound that removal and reuse are nearly impossible. Instead, the bee’s stinging apparatusdstinger, venom gland, and associated musculaturedoften is detached and remains at the sting site (Pucca et al., 2019). The isolated apparatus continues to pump venom into the wound for 30 s, with approximately 90% of the venom injected within the first 20 s (Schumacher et al., 1994). In contrast, smooth stingers may be withdrawn and reused multiple times, inflicting more mechanical damage even if all venom has been delivered during prior stings. Scorpions possess smooth stingers mounted high on forward-curved tails and are capable of delivering multiple stings. Nematocysts are stinging organelles in cnidocytes (specialized “stinging cells”) wielded by cnidarians (e.g., corals, jellyfish, Portuguese man o’ wars, sea anemones). Fronds or tentacles of these animals are equipped with myriad cnidocytes, each of which is armed with a nematocyst that consists of a tightly coiled thread confined in a cylindrical capsule (Beckmann and Ozbek, 2012). Nematocysts are discharged by mechanical or certain chemical stimuli, at which time the thread is ejected toward the stimulus along with a zootoxic payload of crinotoxin (i.e., a toxin produced in a specialized venom cell or gland that is released passively into the environment). Clinical signs develop from both mechanical damage and zootoxin exposure. Tentacles can deliver stings for hours or even days after becoming detached or after the death of the animal. As with venoms delivered through bites, outcomes associated with envenomation by sting also depend on many factors. For example, scorpions optimize the utility of their stinging apparatus and metabolically expensive venom by using several behavioral and physiological adaptations. Among these, the ability to vary the stinging rate, the venom volume, and the zootoxin spectrum are perhaps the most important

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(Evans et al., 2019). Female scorpions are reported to deploy their stinger earlier, at a faster rate, and deliver more strikes than males (Carlson et al., 2014). Similar to funnel web spider bites, centipedes and scorpions can regulate their venom volume when stinging (Evans et al., 2019; von Reumont et al., 2014). Options used by adults include administering “dry stings” (no venom), “modulated stings” (variable venom), and “maximal stings.” This variation reflects the slow pace of venom regeneration (8 days or more to fully replenish an emptied venom gland); inability to regulate venom release would leave the animal unable to forage or fend off predators for extended periods (Carcamo-Noriega et al., 2019). Small venom volumes are injected for easily subdued and small prey. Young scorpions default to the “maximal sting” option, presumably due to both inexperience and the inability of their small pedipalps (“pincers”) to physically restrain prey or predators. Finally, scorpions are able to adjust the composition of their venom to suit the needs of particular situations. The first portion of venom (“prevenom”) is a peptide- and proteinpoor fluid with 16-fold more potassium (Kþ) compared to venom. This mixture is hypothesized to cause rapid and massive depolarization leading to immediate paralysis of invertebrate prey and severe pain in vertebrate predators (Inceoglu et al., 2003). Furthermore, the chemically simple constituents of prevenom are likely to incur less metabolic cost compared to production of peptide- and protein-rich venom (Evans et al., 2019).

2.3. Deliberate Administration Venoms as whole liquids, dried powders, semidried pastes, or more recently as isolated active zootoxin components have a long history of use by humans on fellow humans. Such exposures have been undertaken for both nontherapeutic purposes and as treatments for various ailments. This section briefly reviews common reasons for deliberately administering venoms to humans. Aggression and Defense Historically, venoms have been administered deliberately to maim or kill. Various animal venoms have been reputed to be among the toxic

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principles applied to poison arrows, which have been used in hunting and warfare for millennia around the world. The most celebrated (albeit mythical) example is found in the Greek legend of Hercules, who slays many foes during the course of his Twelve Labors by using arrows dipped in the blood of the Lernaean Hydra (a water monster vanquished during his Second Labor). In the modern era, whole venom injected directly by a biting snake has been employed in contract homicides (Ambade et al., 2012) and suicides (Knight et al., 1977; Yadlowski et al., 1980). The science of venomicsdan integrated analysis of genomic, transcriptomic, and proteomic profiles to characterize venom composition (Clark et al., 2019; Saez et al., 2010; von Reumont et al., 2014; see also Toxicogenomics: A Primer for Toxicologic Pathologists [Vol 1, Chap 15])dhas been crucial in procuring murder convictions by detecting snake proteins in forensic specimens (Chan et al., 2021). Importantly, venomous animals rarely bite from aggression. Instead, virtually all bites in the wild that do not involve subduing prey are the result of self-defense to counter a perceived threat. Therapeutic Applications Venoms have been used from ancient times to the present in the Far East, Middle East, and some Mediterranean civilizations to treat many ailments in humans. Diseases in which venoms have been used traditionally include chronic conditions characterized by multifocal inflammation (e.g., age-related degenerative arthritis, leprosy, insect stings) or persistent pain. Recent advances in venom biology and synthetic chemistry have expanded the list to include approved zootoxin-based drugs that treat cardiovascular diseases, coagulopathies, and diabetes (Table 8.1). Many additional indications including autoimmune diseases (Chen et al., 2018); multidrugresistant bacterial infections (Fratini et al., 2017; Samy et al., 2017; Yacoub et al., 2020); primary and metastatic neoplasia (Upadhyay, 2018); and even neurodegenerative diseases (Awad et al., 2017; de Souza et al., 2018; Nalivaeva et al., 2012; Silva et al., 2015) may be targeted in the future by zootoxin-derived products currently in development. Venomics has propelled the rapid pace of discovery research seeking to identify natural bioactive molecules that might be

refined into new drugs (Saez et al., 2010; Yan and Wang, 2015). Historically, venoms have been administered as components of homeopathic remedies for ingestion or topical application. Widely recognized examples of such products include snake oils in Europe and North America (de Loeches, 1719; Haynes, 2015; Stanley, 1897) as well as snake wines, scorpion wines, snake and scorpion wines, and similar traditional medicines produced in several Asian countries by bottling venomous animals in alcohol (Moon and Chun, 2016). Quan Xie powder (made by pulverizing the tails of Chinese scorpions [Buthus martensii Karsch]) is ingested for pain control; overdoses have been reported to cause chest pain, diaphoresis (sweating), dizziness, generalized involuntary limb twitching, and hypertonia (abnormally high muscle tone) (Lam et al., 2014). Raw honey has been ingested or applied topically for centuries to reduce respiratory tract inflammation (e.g., asthma) or aid wound healing. The efficacy of honey has been ascribed to antioxidant, antibacterial, and antiviral activities (Samarghandian et al., 2017). Recently, the effectiveness of some honey-based nutraceuticals as treatments for chronic pain has been attributed in part to the presence of small venom quantities (Yaghoobi et al., 2013). The actual contribution of venom in honey to therapeutic efficacy following oral administration is unclear. Many salubrious effects of venom-containing therapies may be due to other components like plantderived flavonoids and polyphenols in raw honey (Samarghandian et al., 2017) or alcohol and added herbs (e.g., garlic) in snake wine. Application of venoms by injection has been used to treat many conditions. Apitherapy, the use of bee venom (either extracts or whole venom) as a therapeutic agent, has been practiced since approximately 500 BCE and still is used in traditional Chinese medicine and homeopathic practices in Western countries to alleviate chronic pain associated with arthritis or neuropathy (von Reumont et al., 2014). Venom may be introduced using a needle (“bee venom acupuncture” or hypodermic injections) or by having the insects sting the patient (“bee sting therapy”) (Lee et al., 2005; Lin and Hsieh, 2020; Yoon et al., 2012) at key anatomic locationsdtermed “acupoints” (Li et al., 2015)d where manipulation of neural pathways and/

II. SELECTED TOXICANT CLASSES

3. ZOOTOXIN CLASSIFICATION

or energy meridians can modulate cell and tissue pathology in diseased local tissues and distant viscera. Scorpion neurotoxins have been used in traditional Chinese medicine as treatments for chronic neuromuscular disorders and neuropathic pain (Lam et al., 2014); the variable thermostability of the potent bioactive peptides responsible for most neurotoxic effects permits efficacy to be retained while toxicity is reduced during production of scorpion powders (Yang et al., 2019). Ziconotide (a synthetic analog of u-conotoxin MVIIA, a neurotoxic peptide produced by a fish-eating marine cone snail [Conus magnus]) is given as an intrathecal infusion to alleviate severe neuropathic pain. This drug is particularly appropriate for chronic pain management because it does not induce addiction or tolerance (McGivern, 2007). Injection of venom is also required for production of antivenoms and toxoids, the sole specific treatments for envenomation. Antivenom (or antivenin [based on the French word “venin” meaning “venom”]) is a serum or serum byproduct containing antibodies to neutralize zootoxin components of venom. Antivenom thus works in a similar manner to antitoxin, an antibody-based product that provides transitory passive immunity to a circulating bacterial zootoxin (see Bacterial Toxins [Vol 3, Chap 9]). Specific antivenoms traditionally have been necessary for each venomous species due to the unique constellation of proteins and peptides responsible for the most severe toxic manifestations of that venom. However, recent work has raised the likelihood that broadspectrum antivenoms raised against a conserved zootoxin (e.g., phospholipase A2 [PLA2]) will exhibit efficacy for many closely related species (Castillo-Beltra´n et al., 2019; Ratanabanangkoon et al., 2020; Xiao et al., 2017). The need to neutralize multiple zootoxins in complex venoms simultaneously means that the most effective antivenoms are polyvalent (i.e., having antibodies directed against many epitopes on one or more zootoxins and thus tend to be effective against venoms from multiple species) rather than monovalent (i.e., having antibodies effective against a single toxin for a single species). Antivenom historically has been produced by injecting nonlethal doses of active whole venom into animals, after which immune serum is harvested and concentrated (Calmette,

559

1896). This technology remains the foundation of current antivenom technology, with most antivenoms being produced in modest amounts at great cost by hyperimmunization of horses or sheep, or occasionally other animals (Liau and Huang, 1997; Theakston et al., 2003; WHO, 2016). In future years, next-generation antivenoms will likely adapt existing highthroughput protein engineering methods to generate appropriate antibodies in bioreactors under controlled conditions (Knudsen et al., 2019; Laustsen et al., 2018). Toxoids are inactivated zootoxins administered prophylactically as vaccines to generate a toxin-specific immune response capable of preventing or moderating clinical disease during future toxin exposures. Zootoxin inactivation typically is performed by chemical (formaldehyde or glutaraldehyde) or physical (heat) modification of the peptide. Such treatments quench the toxic activity but leave the immunogenic potential intact, or even improved (Liau and Huang, 1997; Sadahiro et al., 1984). In general, toxoids are mixed preparations, raising an immune response against multiple venom components and not a single purified zootoxin. Toxoid potency is confirmed in vivo in rodent bioassays (often guinea pigs), and then the ability to neutralize toxins in humans is estimated using nonhuman primates (Sadahiro et al., 1978). Injection of a toxoid does not cause envenomation but instead initiates an acquired immune response directed against toxin antigens. With that said, a toxoid may become reactivated (i.e., zootoxins regain their toxic activities) if stored improperly (Sawai and Fukuyama, 1978). Immunological memory induced by toxoids fades over time, so periodic booster vaccinations are needed to sustain antizootoxin immunity.

3. ZOOTOXIN CLASSIFICATION The census of zootoxins is enormous and constantly expanding. For example, more than 80,000 conotoxins (mainly bioactive peptides) have been identified in various species of cone snails (Yang et al., 2019), and an estimated 100,000 zootoxins (mainly bioactive peptides) have been described among scorpion species (King, 2011).

II. SELECTED TOXICANT CLASSES

TABLE 8.1

Approved Human Biopharmaceuticals Based on Zootoxins in Venoms Approval date (Agency)

Generic name

Trade name

Therapeutic use

Mechanism of action

Species of origin

Production

Batroxobin (1)

Defibrase

Anticoagulant

Serine protease that acts to cleave fibrinogen

Snake

Common lancehead / fer-de-lance (Bothrops atrox)

Purified from venom

1989 (MHLW)

Plateltex-Act

Gel formation for topical application

Cleavage of fibrinogen to produce fibrin that binds platelets into gel

Vivostat

Generation of fibrin as a surgical sealant

Cleavage of fibrinogen to produce autologous fibrin

Bee venom (4)

Apitox

Analgesic for chronic inflammatory diseases

Multiple toxins inhibit proinflammatory signals and signaling pathway

Insect

Western honeybee (Apis mellifera)

Whole venom

Phase II and III clinical trials ongoing

Bivalirudin (2)

Angiomax

Anticoagulant

Thrombin inhibitor (reversible)

Leech

European medicinal leech (Hirudo medicinalis)

Synthetic

2000 (FDA)

Captopril (3)

Capoten

Antihypertensive, congestive heart failure

Angiotensinconverting enzyme (ACE) inhibitor

Snake

Jararaca pit viper (Bothrops jararaca)

Synthetic

1981 (FDA)

Cobratide (cobrotoxin) (2)

Ketongning

Analgesic for chronic inflammatory and pain conditions

a-Neurotoxin that blocks postsynaptic nicotinic acetylcholinergic receptors

Snake

Chinese cobra (Naja atra)

Purified from venom

1998 (NMPA)

Desirudin (2)

Iprivask

Anticoagulant

Thrombin inhibitor (essentially irreversible)

Leech

European medicinal leech (H. medicinalis)

Recombinant

2003 (FDA)

Enalapril (3)

Vasotec

Antihypertensive, congestive heart failure

Angiotensinconverting enzyme (ACE) inhibitor

Snake

Jararaca pit viper (B. jararaca)

Synthetic

1985 (FDA)

TABLE 8.1

Approved Human Biopharmaceuticals Based on Zootoxins in Venomsdcont’d

Generic name

Trade name

Therapeutic use

Mechanism of action

Species of origin

Production

Approval date (Agency)

Eptifibatide (2)

Integrilin

Anticoagulant

Stops binding of several proteins (fibrinogen, von Willebrand factor) to GPIIb/IIIa on platelets

Snake

Pygmy rattlesnake (Sistrurus miliarius)

Synthetic

1998 (FDA)

Exenatide (2)

Byetta

Insulin secretagogue for diabetes mellitus type 2

Glucagon-like peptide-1 (GLP-1) receptor agonist

Lizard

Gila monster (Heloderma suspectum)

Synthetic

2005 (FDA) 2009 (EMA)

Bydureon

Insulin secretagogue for diabetes mellitus type 2

Glucagon-like peptide-1 (GLP-1) receptor agonist (extended release)

Lizard

Gila monster (H. suspectum)

Synthetic

2011 (EMA) 2012 (FDA)

Leeches (4)

None

Anticoagulant and removal of blood and fluid from engorged tissues during surgery

Many toxin components inhibit the coagulation cascade and platelet aggregation

Leech

European medicinal leech (H. medicinalis) or other species

Whole animal medical device

2004 (FDA)

Lixisenatide (2)

Adlyxin (US) and Lyxumia (Europe)

Insulin secretagogue for diabetes mellitus type 2

Glucagon-like peptide-1 (GLP-1) receptor agonist (extended release)

Lizard

Gila monster (H. suspectum)

Synthetic

2013 (EMA) 2016 (FDA)

Tirofiban (3)

Aggrastat

Anticoagulant

Stops fibrinogen binding to GPIIb/IIIa on platelets

Snake

Saw-scaled viper (Echis carinatus)

Synthetic

1998 (FDA)

Ziconotide (2)

Prialt

Analgesic for severe chronic pain

Antagonist of voltagegated calcium channel (Cav2.2)

Snail

Magical cone marine snail (Conus magus)

Synthetic

2004 (FDA)

Numerical designations in parentheses denote the molecule type: 1 ¼ enzyme, 2 ¼ peptide, 3 ¼ small molecule, 4 ¼ whole venom (toxin mixture). EMA, European Medicines Agency; FDA, U.S. Food and Drug Administration; MHLW, Ministry of Health, Labour and Welfare (Japan); NMPA, National Medical Products Administration (China). Adapted from Bordon KCF, Cologna CT, Fornari-Baldo EC, et al.: From animal poisons and venoms to medicines: achievements, challenges and perspectives in drug discovery, Front Pharmacol 11, 1132, 2020.

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Many systems might be used to categorize zootoxins. Possible taxonomic schemes for classifying zootoxins include grouping by the: • source (animal species [or genus or family] that produces the toxin), • toxin structure, • toxin function, • toxin mechanism of action, and • target tissue(s) for the toxin. For toxicologic pathology, all these classification schemes are reasonable scaffolds for learning the major principles related to animal zootoxicology and zootoxinology. Accordingly, primary concepts for these classification options are reviewed briefly below.

3.1. Zootoxin Classification by Source Taxonomic grouping of zootoxins based on source is a vast endeavor. Such schemes taken to extreme would include thousands of species scattered across all eukaryotic phylada task clearly beyond the scope of this chapter or any single book. Accordingly, this chapter presents a few general themes using major animal groups. Selected poisonous and venomous animals and their principal zootoxins are presented in Table 8.2. Animals that produce medically significant zootoxins are found in both invertebrate and vertebrate phyla (Junghanss and Bodio, 2006). The important invertebrates found in the phylum Cnidaria (e.g., corals and jellyfish) or phylum Arthropoda in either the subclass Arachnida (e.g., scorpions and spiders) or order Hymenoptera (e.g., ants, bees, and wasps). The most relevant vertebrates (phylum Chordata) are members of the class Actinopterygii (rayfinned fishes) and the class Reptilia from the snake families Elapidae (e.g., cobras, coral snakes, and sea snakes) and Viperidae (terrestrial vipers). Exposure to marine zootoxins (e.g., corals and jellyfish) is obviously concentrated along coastal areas, encounters with arthropods (mainly Hymenoptera) are more frequent in densely populated suburban and urban regions, and snakebites are concentrated in rural (usually agricultural and wilderness) settings. Snakebite is the most important medical envenomation globally (Junghanss and Bodio, 2006). Accordingly, the World Health

Organization (WHO) includes antivenom for snakebite in its 21st “Model List of Essential Medicines” for adults (WHO, 2019a) and 7th “Model List of Essential Medicines for Children” (WHO, 2019b). Zootoxin compositions of poisons and venoms vary across geographic domains (GwaltneyBrant et al., 2018). Such differences exist among closely related species and within a particular species (Bernardi et al., 2017; Modahl et al., 2020; Sunagar et al., 2014b). Interestingly, venom disparity among individuals in a species may produce venoms with distinct biochemical profiles that nonetheless synergistically interact to produce the same toxic effect (Grandal et al., 2021). This divergence appears to reflect dietary differences in many poisonous species as the zootoxins are acquired by bioconcentration following ingestion of prey. Zootoxin accumulation by ingestion has been described in marine invertebrates (e.g., saxitoxin in paralytic shellfish poisoning), certain ray-finned fishes (e.g., ciguatoxin in ciguatera), poison dart frogs (batrachotoxin), and a few passerine birds (e.g., homobatrachotoxin in the hooded pitohui) (Nelsen et al., 2014). Alternatively, many marine invertebrates and some fish harbor symbiotic bacteria, and toxin production by these microbes is responsible for the fatal effects (commonly neurotoxicity leading to respiratory failure secondary to muscle paralysis) (Chau et al., 2011). This cohabitation is characteristic of toxin accumulation in pufferfish and the blue-ringed octopus (Hapalochlaena maculosa), both of which harbor lethal quantities of bacteria-derived tetrodotoxin although in different tissues: ovaries, liver, and intestines for pufferfish, and salivary gland for the octopus (Nelsen et al., 2014). The octopus saliva does include genuine zootoxins in addition to bacteria-derived tetrodotoxin and so does qualify as venom. Zootoxins may be concentrated in specific individuals within a species. For example, venoms of female black widow spiders (Latrodectus mactans) and male Sydney funnel web spiders (Atrax robustus) are significantly more toxic than those produced by members of the opposite sex. These sex-related differences are attributed to evolutionary pressures related to sex-specific roles. Female black widow spiders aggressively defend their egg sacs and employ their potent venom in fulfilling this task. Male

II. SELECTED TOXICANT CLASSES

TABLE 8.2

Selected Poisonous and Venomous Animals and Their Principal Zootoxins Key Zootoxin Type(s) Mechanisms

Taxonomy

• Crinotoxins

• Bind to DNA causing apoptosis

Clinical Presentation

Pathology

• Myalgia • Pruritus (itching) • Swelling (skin)

• Edema (skin) • Vesiculation (“blisters”)

P: Porifera

Sponges

P: Cnidaria

• Pore formation C: Anthozoa (corals** • Cytolysins • Necrosis (skin) (cytolysins and hemolysins) • Pain (skin) and sea anemones) • Hemolysins • Neurotoxins • Ion (Naþ and Kþ) • Vesiculation (skin) • Enzymes channel inhibition • Lipid and protein hydrolysis C: Cubozoa (box jellyfish**)

• Cytolysins • Pore formation (cytoly• Hypertension • Hemolysins sins and hemolysins) • Pain (severe) • Neurotoxins • Ion (Naþ) channel inhibition • Respiratory distress and a1-adrenergic receptor • Urticaria (red, itchy agonists welts)

C: Hydrozoa (hydras • Cytolysins and Portuguese man o’ war) C: Scyphozoa (true jellyfish)

P: Echinodermata C: Echinoidea (sea urchins)

• Pore formation

• Pain • Urticaria

• Cardiotoxins • Pore formation (hemoly• Cardiac depression sins and myotoxins) • Hemolysins leading • Myotoxins • Ion (Naþ and Kþ) chanto failure • Neurotoxins nel inhibition • Pain • Enzymes • Lipid and protein hydrolysis • Rash (“bug bite”) • Urticaria • Biogenic amines • Glycosides • Hemolysins • Enzymes

• Altered neurotransmission • Mast cell degranulation • Protein hydrolysis

• Cardiac and respiratory depression and failure • Myalgia (severe local) • Pain

• Hemorrhage (petechial) of skin • Necrosis (skin) • Vesiculation (skin)

• Hyperkalemia • Hemorrhage (minimal) of skin

• Linear skin lesions featuring hemorrhage and vesiculation • Necrosis and ulceration (skin) • Increased serum activities for ALP, ALT, AST, and CK • Serpiginous hemorrhage (skin)

• Rash (via retained spines) • Inflammation (granulomas) of skin and joints

(Continued)

TABLE 8.2 Selected Poisonous and Venomous Animals and Their Principal Zootoxinsdcont’d Key Zootoxin Type(s) Mechanisms

Taxonomy P: Annelida

P: Mollusca

P: Arthropoda

Clinical Presentation

C: Polychaeta (bristle • Neurotoxins • Enhanced AChE activity • Pain worms) • Enzymes • Chitin and protein hydrolysis • Urticaria

C: Clitellata O: Hirudinida (leeches)

• Anticoagulants • Enzymes

• Factor Xa and thrombin inhibition • Fibrinolysis

• Abrasion • Delayed coagulation (skin)

C: Gastropoda F: Conidae (cone snails)

• Neurotoxins • Ion (Naþ, Kþ, Ca2þ) channel • Puncture wounds inhibition (skin) • Paresthesia (burning) • Nicotinic ACh • Respiratory failure receptor blockade

Pathology • Rash (via pieces of venom-filled bristles) • Necrosis () • Increased APTT and PT • Hemorrhage () • Cyanosis (skin, localized near wound)

C: Cephalo• Neurotoxin • Ion (Naþ) channel inhibition • Facial numbness poda (i.e., • Paralysis (leading to O: Octopoda (bluetetrodotoxin cardiac arrest and ringed octopus) from respiratory arrest) symbiotic bacteria)

• None •  Puncture wounds (skin)

O: Hymenop• Alkaloids • Cytotoxicity • Erythema (redness) tera • Formic • Mast cell degranulation • Pustules F: Formicidae (ants) acid (w70%) • Lipid and protein hydrolysis • Histamine • Enzymes

• Edema and hemorrhage • Epidermal necrosis leading to local vesiculation (early) with neutrophil infiltration (late)

O: Hymenoptera F: Apidae (bees)

• Enzymes • Lipid hydrolysis (PLA2) • Ion (Kþ) channel blocker • Neurotoxins • Pore formation (apamin) • Surfactant (mellitin)

C: Chilopoda F: Scolopendridae (centipedes)

• Neurotoxins • Ion (Naþ) channel inhibition • Erythema (redness) • Mast cell degranulation • Edema • Histamine • Lipid and protein hydrolysis • Pain • Enzymes

• Erythema (redness) • Edema • Pain • Anaphylaxis (rare)

• Edema

• Edema and hemorrhage •  Necrosis (cutaneous) and ulceration

TABLE 8.2 Selected Poisonous and Venomous Animals and Their Principal Zootoxinsdcont’d Key Zootoxin Type(s) Mechanisms

Clinical Presentation

Pathology

SC: Arachnida O: Ixodida (ticks)

• Cystatins • Cysteine protease inhibition • Enzymes • Ion (Ca2þ) channel inhibition (PLA2) • Lipid hydrolysis • Neurotoxins

• Fever • Myalgia • Rash • Weakness to paralysis

• Rash

SC: Arachnida O: Scorpiones (scorpions)

• Biogenic • Delayed Na þ channel amines inactivation and increased • Enzymes spontaneous firing rate (PLA2) • Lipid hydrolysis • Neurotoxins • Mast cell degranulation

• Hypertension • Muscle twitching • Nausea • Respiratory distress • Sweating • Tachycardia

• Edema and hemorrhage • Necrosis (cutaneous) leading to ulceration

Taxonomy P: Arthropoda dcont’d

SC: Arachnida • Biogenic • Ion (Naþ, Kþ, Ca2þ) channel • Confusion O: Araneae (spiders) amines inhibition (funnel web spiders) • Enzymes • Lipid hydrolysis • Cramping (widow (PLA2, SMase • Neurotransmitter spiders) receptor antagonists • Edema  erythema D) • Muscle fasciculation • Neurotoxins • Nausea • Pain ( severe) • Sweating • Tachycardia

• Edema and hemorrhage • Inflammation (neutrophilic) • Necrosis (skin and adnexa) leading to deep ulceration and eschar formation (primarily brown recluse spiders)

P: CHORDATA

SC: Chondrichthyes (cartilaginous fishes)

O: Myliobatiformes (rays)

SC: Actinopterygii F: Scorpaenidae C: Teleostei (ray- (lionfish, finned fishes) scorpionfish, stonefish)

• Enzymes

• Altered energy and second messenger metabolism • Digestion of extracellular matrix, membranes, nucleic acids, and proteins

• Cardiotoxins • Vasodilation • Neurotoxins • Altered neurotransmission • Enzymes • Protein hydrolysis • Peptides

• Cardiac arrhythmias • Lacerations (large) with local bleeding, edema, and vasoconstriction • Pain (local)

• Lacerations (large) with acute edema and hemorrhage • Necrosis

• Cardiac and respiratory depression • Pain (severe) • Paresthesia and paralysis • Swelling (lymphedema)

• Edema (skin  lung) • Puncture wounds • Ischemia and cyanosis early with vesiculation and necrosis later at the wound site (Continued)

TABLE 8.2

Selected Poisonous and Venomous Animals and Their Principal Zootoxinsdcont’d Key Zootoxin Type(s) Mechanisms

Taxonomy SC: Actinopterygii F: Tetraodontidae (pufferfishes) C: Teleostei (ray-finned fishes) dcont’d

O: Siluriformes (catfishes)

O: Anura F: Dendroba tidae (poison dart frogs)

• None

• Bioactive peptides • Enzymes

• Edema  cyanosis •  Necrosis (localized)

• Cytolysis (blood cells and skin/subcutis) • Pro-inflammatory signaling

F: Helodermatidae (beaded lizards, Gila monster)

• Edema with erythema • Pain • Puncture wounds

• Ion (Naþ) channel inhibition • Arthralgia • Myalgia • Paresthesia • Paralysis (respiratory) • Vomiting

• Neurotoxins • Irreversible activation of Na þ channels

O: Anura • Neurotoxins • Partial agonists at F: Bufonidae (toads) serotonin receptors

C: Reptilia

Pathology

• Neurotoxin • Ion (Naþ) channel inhibition • Paresthesia (i.e., • Paralysis (bulbar tetrodotoxin and respiratory) from symbiotic bacteria)

O: Perciformes • Ciguatoxin F: Scombridae (from (bonitos, mackerels, bioconcentunas) tration of F: Sphyrae neurotoxins nidae (barracudas) made by dinoflagellates) C: Amphibia

Clinical Presentation

• Hemotoxins • Altered coagulation • Enzymes (with hemorrhage) • Digestion of extracellular matrix, membranes, nucleic acids, and proteins

• None

• Convulsions • Muscle contractions • Paralysis (respiratory failure)

• None

• Hallucinations • Nausea (centrally driven) • Convulsions

• None

• Nausea  vomiting • Pain (intense) • Swelling (localized)

• Edema (localized to bite site) • Hemorrhage (systemic)

TABLE 8.2

Selected Poisonous and Venomous Animals and Their Principal Zootoxinsdcont’d Key Zootoxin Type(s) Mechanisms

Taxonomy C: Reptilia dcont’d

Clinical Presentation

Pathology

F: Elapidae (cobras**, • Hemotoxins • Altered coagulation (but little • Confusion coral snakes, kraits, • Myotoxins hemorrhage) • Cramping sea snakes) • Neurotoxins • Muscle degeneration • Nausea by lipolysis (PLA2), surfactant • Paralysis (leading to respiratory failure) activity (cardiotoxins) • Inhibition of postsynaptic ACh receptor

• Edema (localized to bite site) •  Hepatic necrosis •  Kidney tubular necrosis (from myoglobinuria) • Rhabdomyolysis (skeletal muscle necrosis)

• Hemotoxins • Altered coagulation (with F: Viperidae • Hemorrhage extensive hemorrhage and SF: Crotalinae** (pit • Myotoxins • Pain • Neurotoxins often hemolysis) vipers) • Swelling • Muscle degeneration SF: Viperinae** (Old World vipers) by lipolysis (PLA2), myofiber Naþ channel activation (crotamine)

• Edema (regionally extensive) • Hemorrhage (extensive) • Necrosis (extensive)

C: Aves

O: Passeriformes (some perching birds)

• Neurotoxin

C: Mammalia

O: Monotremata (platypus) O: Primates (slow loris)

• Irreversible activation of Na þ channels

• Cutaneous tingling • Numbness

• None

• Neurotoxins • Increased neurotransmitter release

• Pain (intense and lasting) • Swelling

• Not reported

• Cytotoxins

• Anaphylaxis (protein resembles cat allergen) • Pain • Swelling

• Necrosis

• Not reported

(1) Taxonomy: C ¼ class, F ¼ family, O ¼ order, SC ¼ superclass, SF ¼ superfamily; (2) Zootoxin types: PLA2 ¼ phospholipase A2, SMase D ¼ sphingomyelinase D; (3) Mechanisms: ACh ¼ acetylcholine, AChE ¼ acetylcholinesterase, Ca2þ ¼ calcium ions, Kþ ¼ potassium ions, Naþ ¼ sodium ions. (4) Pathology: ALP ¼ alkaline phosphatase activity, ALT ¼ alanine aminotransferase activity, APTT ¼ activated partial thromboplastin time, AST ¼ aspartate aminotransferase activity, CK ¼ creatine kinase activity, PT ¼ prothrombin time. **Asterisks denote common, medically significant exposures to zootoxins.

568

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funnel web spiders are more toxic because they migrate for extended distances during mating season to find females, which wait in deep burrows (Herzig et al., 2020). Similarly, cantharidin is made only by male blister beetles, although it is transferred to females during copulation (Nikbakhtzadeh et al., 2007). Venom composition in some snake species evolves substantially during the course of life as indicated by substantial differences in the venom components between juveniles and adults (Jackson et al., 2016; Seneci et al., 2021). Some animals co-opt venom produced by other species to their own uses. For example, the European hedgehog (Erinaceus europaeus) enhances the defensive efficiency of its cutaneous spine palisade by smearing toad venom on the spine tips (Brodie, 1977). Similarly, boxer crabs (Lybia spp.) apply sea anemones to their claws and use the cnidocyte complement on the anemone tentacles for mutual defense (Schnytzer et al., 2017). Indigenous peoples in the tropical rainforests of South America prepare their arrows by smearing them with cutaneous secretions of poison dart frogs. Once dried, the toxins retain their potency for approximately a year. Each golden poison dart frog (Phyllobates terribilis), likely the most toxic frog species and among the most toxic animals in existence, possesses enough toxin to kill 10–12 people. Animals may be both poisonous and venomous. The tiger keelback snake (Rhabdophis tigrinus) is venomous. When biting, the snake releases venom into the rear of the mouth. The venom is introduced into the wound by flowing along the grooved surface of the snake’s solidcored opistoglyphous (rear-positioned) fangs. However, the tiger keelback snake is poisonous as well, releasing cardiotoxic steroids from nuchal glands embedded in the skin of the dorsal neck. When threatened, rupture of the glands expels the poison onto the skin surface and into the mouth or onto the face of the predator (Hutchinson et al., 2007). The secretions in the nuchal glands are not produced by the snake but instead are toxins bioconcentrated from their toad prey. Interestingly, tiger keelback snakes that have not consumed a diet of toxic toads are not poisonous, and the toad toxin-free snakes are able to sense whether or not their most effective defensive stance will be flight or presentation of their nuchal region with its zootoxic payload (Mori and Burghardt, 2017).

3.2. Zootoxin Classification by Molecular Structure Zootoxins come in many forms, and most poisons and venoms are a composite of toxins with several or all of these major structures (Fox, 2015). In species with multiple venom glands, the complement of toxins produced by the primary gland and the accessory gland are distinct. For example, in the king cobra (Ophiophagus hannah), 67% of transcripts for the primary gland encode neurotoxic proteins (specifically three-finger toxins [3FTx]), while the main transcripts (43%) for the accessory gland encode lectins (Vonk et al., 2013) that act to inhibit platelet function (Lu et al., 2005). A detailed discussion of zootoxin chemistry is beyond the scope of this chapter, but key structural classes are briefly reviewed here. Many zootoxins are proteins or peptides. Most species use about 50–200 proteinaceous zootoxins (Ainsworth et al., 2018), but the number may be far higher: up to a 1000 peptides in some spiders and 8000 peptides in certain cone snails (Yang et al., 2019). During evolution, zootoxins of this kind often arise by hijacking existing genes (via gene duplication or alternative splicing of gene transcripts) to serve new purposes (Sunagar et al., 2014a). Many proteins have cysteine-rich sequences to facilitate formation of multiple stabilizing disulfide bonds to maintain the correct tertiary folding and sustain bioactivity (Chen et al., 2018). For example, 3FTx are a superfamily of neurotoxins in snake venoms with a primary sequence length of 60–74 amino acids (range, 58–81) and a tertiary structure constructed of three b-strand loops originating from a hydrophobic core bracketed by 4 conserved disulfide bonds (Figure 8.7). Peptides are generally short (mainly 10–30 amino acids [aa] but ranging up to 60 aa) and may comprise up to 95% of dry venom by weight (Karalliedde, 1995). Functions of protein and peptide zootoxins are quite diverse, but key roles include enzymatic degradation of cells and tissues (often freeing bioactive chemical mediators in the process), acting as ligands to alter ion channel or receptor activity, and serving as surfactants to disrupt cell membranes. Application of antimicrobial peptides by some female arthropods that spray venom onto broods protects them against bacterial (broad spectrum) and some fungal infections (Hakim et al., 2015). Poisons and venoms contain many other zootoxins in addition to proteins and peptides. For

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FIGURE 8.7 Crystal structure of a “short” three-finger toxin (3FTx) showing the three b-sheet-containing loops (wide green “fingers” labeled I, II, and III) and the four conserved disulfide bonds in the central core (yellow bridges) that stabilize the molecular conformation. The zootoxin is erabutoxin A, a venom protein of the blackbanded sea krait (Laticauda semifasciata). Image reproduced from Nastopoulos et al. (1998), under a Creative Commons license (CC BY-SA 4.0).

instance, neurotransmitters including biogenic amines and free amino acids alter vascular tone (inducing vasoconstriction), incite mast cell degranulation (histamine), and sensitize cells to other zootoxins (Rash and Hodgson, 2002). Additional constituents include numerous alkaloids, lipids, nucleosides, and polysaccharides (Peterson, 2004; Rodriguez et al., 2017; Watkins, 2013). Alkaloids are organic small molecules with numerous pharmacological activities including but not limited to antibacterial, cytotoxic, immune-modulating, neuroactive, psychoactive, and vasoactive effects. Many lipid variants may be found in venoms including free cholesterol, free fatty acids, phospholipids, and triglycerides (Marie and Ibrahim, 1976). Processed postinjection, these lipid molecules can serve as a substrate pool by which PLA2 might

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rapidly produce pro-inflammatory mediators. Nucleosides (purines and pyrimidines) act as hypotensive mediators for venomous lizards and many snakes (elapid and viperid but not crotalid species), and may be more abundant than proteins in some snake venoms (Aird, 2005, 2008). Polysaccharides in ant venoms have been demonstrated to activate the classic complement pathway in humans (Schultz et al., 1979). Finally, some venom components have no known toxic functions as yet. In some cases, these constituents are still instrumental in maintaining potency. For example, many venoms contain inorganic ions such as metals, which are postulated to act as catalysts to enhance the activities of venom enzymes (Watkins, 2013). Interestingly, a few venom components do not have known functions relevant to initiating or sustaining toxicity. Insulin-like growth factor has been detected in snake venom (Vonk et al., 2013), and nerve growth factor (NGF) has been isolated in honeybee and scorpion (Lipps, 2000) as well as cobra venoms (Li et al., 1999; Lipps, 2000; Vonk et al., 2013). Like their mammalian counterparts, these factors promote cell survival but do not appear to have toxic effects in vitro or in vivo. The evolutionary impetus behind the presence of these growth factors in venom thus is not known.

3.3. Zootoxin Classification by Function Categorization of zootoxins based on function usually results in additional classification based on such major features as the type of toxic effect, the affected cell population, and/or the physiological process that is disturbed by the toxin. The main zootoxin kinds defined by these properties may be classified broadly as coagulotoxins, necrotoxins, and neurotoxins (Bickler, 2020) (Figure 8.8). Some of these toxins have molecular structures that resemble those of endogenous bioactive molecules in vertebrates, and as such represent a rich source for drug discovery (Bordon et al., 2020; Chen et al., 2018; Saez et al., 2010; Xu and Lai, 2015; Yan and Wang, 2015). Zootoxins with the same function may have different amino acid sequences and tertiary structuresdand structurally divergent molecules may elicit similar functional outcomes (Chen et al., 2018; Menez, 1998). Some effects are only produced by venoms since their action depends on penetration of the toxins into deep tissues.

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FIGURE 8.8 Diagram showing the synergistic relationship between enzymatic and nonenzymatic zootoxins in driving local and systemic tissue damage following envenomation. Key aspects include enzyme-mediated coagulotoxicity, leading to hemorrhage and edema; necrotoxicity (or cytotoxicity), which manifests as cell and tissue destruction and further venom dispersion; and neurotoxicity that results in paralysis. Exacerbation of these primary zootoxic effects is supplied by inflammation (which intensifies cell and tissue death) and altered vascular function (which enhances hemorrhage and edema before progressing to shock and cardiovascular collapse). Cell and tissue destruction in turn enhances inflammation, thus establishing a positive feedback loop that maximizes local tissue damage and may lead to gangrene and loss of the affected limb.

Coagulotoxins Coagulotoxins are venom factors that disrupt the coagulation cascade (Debono et al., 2019b; Youngman et al., 2019). The coagulotoxic impact of venom depends on its zootoxin composition. Effects of coagulatoxins often occur with other circulatory anomalies like altered vascular tone (e.g., vasoconstriction or vasodilation), cardiac arrhythmias, and/or hemolysis. For this reason, coagulatoxins sometimes are viewed as a subset of hemotoxins (where the definition of the latter term is expanded to encompass the lysis of erythrocytes [red blood cells], disruption of various clotting factors, and induction of various vascular wall disturbances).

In terms of coagulation cascade disruption, coagulotoxins follow one of three fundamental patterns. First, many zootoxins exert an anticoagulant effect, leading to hemorrhage as a main outcome following envenomation. Principal anticoagulant mechanisms of zootoxins are to (1) limit the initial activation or (2) promote premature inactivation of clotting factors that convert fibrinogen to fibrin or to (3) accelerate removal of fibrinogen and fibrin at the envenomation site (Grashof et al., 2020; Kini, 2006; Kvist et al., 2013). The evolutionary pressure whereby zootoxin actions are focused on modulating this protein stems from fibrin’s central role in forming the protein mesh that stabilizes newly formed platelet

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aggregates. Second, other venoms generate a procoagulant effect, leading to enhanced formation of thrombi within local and often distant blood vessels. For example, crotalid and viperid snake venoms have multiple enzymes that activate clotting factors in the common pathway of the coagulation cascade (Lo¨vgren, 2013; Tans and Rosing, 2001); the common pathway controls fibrin formation. Finally, some venoms impart a pseudo-procoagulant action instead of genuine procoagulant activity (Seneci et al., 2021). This alternative outcome results from conversion of fibrinogen to aberrant fibrin strands, which fail to stabilize the platelet plug effectively (Debono et al., 2019a; Sousa et al., 2018). The poor clot quality leads to sustained fibrinogen cleavage (in a futile attempt to produce enough normal fibrin to anchor the aggregated platelets) and ultimately fibrinogen depletion. The net result of a pseudo-procoagulant microenvironment is an anticoagulant state. Necrotoxins Necrotoxins are cytotoxic molecules (i.e., cytotoxins) leading to cell degeneration and death, ultimately resulting in local to locally extensive tissue necrosis. Hemotoxins are a subset of the broader class of necrotoxins when the definition of the former term is limited to an ability to destroy red blood cells (i.e., hemolysis). Many venoms contain cytotoxic components, but some species are particularly recognized for possessing necrotoxic poisons or venoms. Major species in this regard include blister beetles and brown recluse spiders, which produce epidermal vesicles and locally extensive cutaneous necrosis, respectively, and vipers, which induce necrosis in skin and subcutaneous tissues (especially red blood cells and skeletal muscle). Cell and tissue destruction is mediated by two primary zootoxic activities. The first is the enzymatic degradation of cells and extracellular matrix. Many venom enzymes directly disrupt lipids and proteins of cell and organelle membranes. However, some enzymes spare cells but instead serve as “spreading factors” by dissolving the extracellular matrix, thereby facilitating further dispersion of cytolytic zootoxins within envenomed tissue. The second major avenue for cell and tissue destruction by zootoxins involves nonenzymatic interference with structural and functional integrity of cells. For example, some zootoxins such as mellitin (a major biogenic amine comprising

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approximately 50% of dry bee venom by weight [Figure 8.9]) (Lahiani et al., 2017) and numerous linear peptides in ant, scorpion, and spider venoms (Touchard et al., 2016) act as surfactants that potentiate PLA2-mediated membrane damage by disrupting phospholipid cohesion in plasma and organelle membranes (Watkins, 2013). The affinity of PLA2 for membrane targets may be explained in part by its ability to bind intracellular receptor proteins, such as subunits of mitochon drial cytochrome C oxidase (Sribar et al., 2019). Formic acid, the main zootoxin (approximately 60%–70% v/v) found in fire ant venom (Figure 8.10), is toxic to cells chiefly by producing a locally hyperacidic microenvironment. If taken into cells, formic acid also may induce cytotoxicity by inhibiting cytochrome oxidase, a major enzyme in the electron transport chain of mitochondria that is needed for cellular energy (adenosine triphosphate [ATP]) production (Liesivuori and Savolainen, 1991). Relative to other venomous animals, ant venoms are especially rich in cytolytic zootoxins (Touchard et al., 2016). Neurotoxins Neurotoxins are prominent components of many animal poisons and venoms, and many zootoxin mixtures contain multiple neurotoxic constituents (Aird, 2002; Escoubas et al., 2000; Liu et al., 2012; Touchard et al., 2016; Zambelli et al., 2017). Two primary explanations have been proposed for the rise of such potent zootoxin mixtures during convergent evolution. The first is the need to rapidly immobilize prey, thereby sparing the animal from injury during the struggle to subdue the meal. Fast-acting neurotoxins are essential for this purpose since predators often prey on animals that are bigger and more physically robust (e.g., centipedes eat insects but also amphibians, reptiles, birds, and rodents). The second reason is to deter predators, which is accomplished when neurotoxins excite nociceptors (pain-sensing neurons) and elicit an acute and severe pain sensation. Contact with pain-inducing neurotoxins may occur by venom injection into deep tissues or when zootoxinladen fluids directly contact well-innervated surfaces (e.g., conjunctival mucosa hit by spitting cobra venom or oral mucosa smeared with cutaneous secretions of poison dart frogs and toads). Neurotoxins in venoms typically act to enhance or inhibit proteins associated with synaptic terminals, particularly those at

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FIGURE 8.9 Chemical structure of mellitin, a membrane-disrupting cytolytic peptide constituting the main component of honeybee venom. Image reproduced from Park et al. (2015), under a Creative Commons license (CC BY-SA 4.0).

neuromuscular junctions. Two main categories have been defined. a-Neurotoxins (e.g., a-bungarotoxin, cobrotoxin, erabutoxin A, and pinnatoxins) act on the postsynaptic membrane while b-neurotoxins (e.g., b-bungarotoxin, crotoxin,

FIGURE 8.10 Chemical structure of formic acid, a principal component of fire ant venom. Image reproduced from https://commons.wikimedia.org/wiki/File:Formicacid.png.

notexin, and taipoxin) act on the presynaptic axon terminal. The action of these zootoxins often occurs as a purely biochemical effect with no appreciable structural lesions in the affected presynaptic and postsynaptic portions of the synapse. Instead, exposure is indicated by the clinical presentation with transient to progressive neurological signs (i.e., effects visible to observers) and symptoms (i.e., effects reported by the affected individual). These effects vary with the zootoxin and may include sensory disturbances (e.g., dizziness, headache, numbness, paresthesia [burning], and/or tingling); muscle dysfunction (e.g., asthenia [weakness] or paralysis); or central nervous system (CNS) difficulties (e.g., convulsions or hallucinations). Extremely potent alkaloids (e.g., saxitoxin and

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tetrodotoxin) and peptides (e.g., conotoxins) may be lethal due to paralysis of skeletal muscles essential for respiration. Invertebrate neurotoxins are particularly important zootoxins. Cone snails are predatory marine gastropods that produce myriad conotoxins, which cause paralysis when injected into prey (worms, mollusks, or fish depending on the snail species). Over 80,000 conotoxins have been identified in over 1000 cone snail species (Yang et al., 2019). These short peptides (10–30 aa, ranging up to 60 aa) bind with high affinity and specificity to ion channels (primarily the sodium [Naþ], potassium [Kþ], and calcium [Ca2þ]) or neurotransmitter (typically acetylcholine [Ach]) receptors at synaptic terminals where their synergistic activities decelerate various molecular functions essential for neurotransmission.

3.4. Zootoxin Classification by Mechanism of Action The vast majority of zootoxins act by one of a few fundamental mechanisms (Karalliedde, 1995; Lahiani et al., 2017; Sitprija and Suteparak,

2008) (Figure 8.11 and Table 8.3). Some disrupt molecular cascades, thus altering the normal equilibrium needed to initiate, sustain, and end various physiological events in a timely manner (e.g., coagulotoxins and their impact on the clotting cascade). Other toxins disrupt cell membrane integrity, leading to release of intracellular components and cytolysis (e.g., necrotoxins including surfactant peptides and many enzymes). Some toxins interfere with ionic gradients normally maintained by the cell membrane or impact receptor-mediated signaling pathways needed to sustain critical processes like circulation and respiration (e.g., neurotoxins). Many zootoxins exert their destructive effects through normal physiological processes taken to the extreme. These suprapharmacological consequences may be classified broadly as either pro-inflammatory or vasoactive mechanisms. Some venom constituents are not toxins but instead serve roles that facilitate the actions of true zootoxins. For example, many metal ions are found in venoms as catalysts for enhancing venom enzyme activities (Watkins, 2013). Similarly, pharmacologically active molecules like

FIGURE 8.11 Schematic showing shapes, sizes, and mechanisms of common zootoxin types present in poisons and venoms. The bulk of these agents are peptides and proteins with catalytic activity or peptides and small molecules (i.e., “chemical toxins”) that serve as ligands for surface receptors on host cells. Abbreviations: PSP ¼ paralytic shellfish poisoning; rmu ¼ relative mass unit. Image adapted from Clark GC, Casewell NR, Elliott CT, et al.: Friends or foes? Emerging impacts of biological toxins, Trends Biochem Sci 44, 365-379, 2019, by permission of Elsevier. II. SELECTED TOXICANT CLASSES

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TABLE 8.3 Selected Zootoxin Mechanisms of Actions Effect

Mechanism

Representative Zootoxins

Enzyme-based structural damage (“necrotoxin activity”)

Altered cell adhesion

Gelsolinase

Cytoskeletal disruption

Metalloproteinases (MP)

Extracellular matrix digestion

Collagenase, hyaluronidase

Generation of reactive oxygen and nitrogen species

L-amino acid oxidases (LAAO), sphingomyelinase D (SMase D)

Lipid membrane digestion

Phospholipase A2 (PLA2), SMase D

Lipid membrane disruption (surfactants)

Mellitin

Pore formation in membranes

Pandinin 2

Regulated cell death (e.g., apoptosis) induction

LAAO, MP, PLA2

Hemostasis abnormalities (“coagulotoxin activity”)

Enzyme-based coagulation factor modification Anticoagulants Activation of protein C

Serine proteases (SP)

Conversion of plasminogen to plasmin

Desmokinase (an SP)

Fibrinogen degradation

Elastase, a-fibrinogenase, plasmin, some MP, some SP

Fibrinogen depletion (thrombin-like activity)

SP

Inhibition of factor IX function

Draculin, LAAO

Inhibition of factor X activation

PLA2

Inhibition of thrombin activation

Draculin, PLA2

Procoagulants Activation of factor X

MP, SP

Activation of thrombin

Ecarin, oscutarin

Inhibition of protein C

SMase D

Nonenzymatic coagulation factor modification Anticoagulants Factor IXe and factor Xebinding proteins

C-type lectin-related proteins (CLRP)

Inhibition of factor X activation

Hemextin A (a 3-finger toxin [3FTx])

Inhibition of thrombin activation

Bothrojaracin

Procoagulants Activation of factor X

SP

Activation of thrombin

SP

Platelet aggregation Anticoagulants (prevent aggregation)

C-LRP, peptides (disintegrins)

Procoagulants (promote aggregation)

C-LRP, g-glutamyl transpeptidase (GGT), other SP (Continued)

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TABLE 8.3 Selected Zootoxin Mechanisms of Actionsdcont’d Effect

Mechanism

Representative Zootoxins

Inflammation (“pro-inflammatory activity”)

Complement activation

Polysaccharides, SMase D

Generation of chemical mediators

PLA2, SMase D

Leukocyte recruitment

SMase D (via complement activation)

Mast cell degranulation (i.e., histamine release)

Mellitin, PLA2

Type I hypersensitivity (IgE-mediated anaphylaxis)

Hyaluronidase, PLA2

Altered neurotransmitter synthesis and packaging

b-Bungarotoxin, notexin

Neurotransmission disruption (“neurotoxin activity”)

Altered neurotransmitter release (presynaptic) Inhibition of Naþ channels

Ciguatoxin, d-conotoxin, mconotoxin, saxitoxin, tetrodotoxin

Inhibition of Kþ channels

Apamin, b-bungarotoxin, kconotoxins, dendrotoxin

Inhibition of Ca2þ channels

u-Agatoxins, u-conotoxins

Membrane disruption (presynaptic terminal)

Latrotoxins

Modified presynaptic receptor activity

Candoxin, muscarinic toxins

Altered neurotransmitter clearance (intrasynaptic)

Acetylcholinesterase (AChE, in venom), fasciculins

Altered receptor activation (postsynaptic)

Vascular disturbances (“vasoactive activity”)

Competitive antagonists to block receptors

a-Neurotoxin (e.g., a-bungarotoxin)

Noncompetitive antagonists to stabilize receptors

Crotoxin, histrionicotoxins

Altered signal propagation

Cardiotoxins, inhibitor cysteine knot (ICK) peptides

Exogenous signaling molecules (in venoms)

Biogenic amines, excitatory amino acids

Altered blood vessel diameter and tone Vasoconstriction (hypertension)

Catecholamines, PLA2 (by thromboxane A2 release), sarafotoxin

Vasodilation (hypotension)

Bradykinin-potentiating peptides (i.e., angiotensin-converting enzyme [ACE] inhibitors), calciseptine (a Ca2þ channel blocker 3FTx), Dendroaspis natriuretic peptide, SP

Hemoconcentration

Vascular endothelial growth factor (VEGF)

Increased blood vessel permeability

Adenosine, histamine, PLA2 (by releasing chemical mediators)

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alkaloids, amines, glucosides, glycosides, growth factors, quinones, and steroids are thought to function as ligands controlling many cellular processes, especially the balance among growth, proliferation, and programmed cell death (Peterson, 2004; Rodriguez et al., 2017; Vonk et al., 2013; Watkins, 2013). For some venoms, the pathogenesis appears to be driven in part by altered signaling resulting from protein dimers pairing an endogenous prey molecule and a zootoxin (Bickler, 2020). Taxonomic classification of zootoxins based on the mechanism of action yields numerous possible groupings. Such categorizations are artificial in some sense as many zootoxins exert their effects by disrupting multiple cell processes (Aird, 2002; Ferraz et al., 2019). Given the thousands of zootoxins, the current discussion will scratch the surface of the means by which single toxins or toxin mixtures may injure cells and organisms. Therefore, this section will concentrate on major themes that have been identified during toxinology research during the past few decades. Cell and Tissue Destruction Poisons and especially venoms contain numerous enzymes and some caustic chemicals. A consequential outcome of exposure to these zootoxins is the destruction of structural components in cells or tissues (i.e., necrotoxicity and cytotoxicity). Damage may be directed against a given component (e.g., zootoxic enzymes catabolize particular lipid and protein substrates) or may be nonspecific (e.g., cantharidin and formic acid damage all molecules in the area). For example, envenomation by the terciopelo (Bothrops asper, a crotalid pit viper commonly known as the fer-de-lance) of Central and South America leads to muscle necrosis with substantial inflammation and pain. Myotoxins in this venom increase the intracellular Ca2þ concentrations, thereby initiating programmed cell death cascades. Representative necrotoxic zootoxins in poisons and venoms are briefly reviewed here. PHOSPHOLIPASE A2

PLA2 is a principal necrotoxin in poisonous and venomous animals across invertebrate and vertebrate phyla (Bon et al., 1994; Zambelli et al., 2017). Nearly 500 PLA2 isoforms have been identified (Xiao et al., 2017), and collectively they comprise (as dry venom by weight)

from 32% to 71% of snake venoms (Georgieva et al., 2008; Ziganshin et al., 2015) and 10%– 12% of bee venom (Lee and Bae, 2016). Venom from a single individual may harbor many PLA2 variants (Harris and Scott-Davey, 2013). This enzyme hydrolyzes phospholipids into lysophospholipids and fatty acids (GwaltneyBrant et al., 2018; Pace and Vetter, 2009). Major PLA2 effects are myotoxicity and neurotoxicity (Tonello and Rigoni, 2017). Multiple PLA2 isoforms exist due to convergent evolution. Class I PLA2 variants occur in venoms of elapids (death adders of the genus Acanthophis, cobras, coral snakes) and hydrophids (sea snakes). Class II PLA2 forms are found in venoms for crotalids (pit vipers) and viperids (Old World vipers). Class III PLA2 versions are characteristic of arthropods (e.g., honeybees) and Gila monsters. Regardless of the isoform, PLA2 can drive cell and tissue damage directly. Necrosis of skeletal muscle fibers (rhabdomyolysis), lysis of red blood cells (hemolysis), and damage to blood vessel walls (which leads to hemorrhage) is common at the envenomation site for crotalid and viperid snakes but may occur more widely following bites from certain elapids (e.g., sea snakes) (Xiao et al., 2017). The main effect of PLA2 is lipolysis, leading to membrane destabilization. The lipolytic effect on cell and organelle membranes also generates free radicals, which further damage membrane integrity, and mobilizes intracellular Ca2þ stores (Figure 8.12). Elevated cytosolic Ca2þ levels overwhelm the capacity of mitochondria to regulate this signaling ion, which may drive remaining overly stressed cells toward programmed cell death (Calderon et al., 2014; Santagostino et al., 2021). The degree of rhabdomyolysis often may be estimated by demonstrating elevated serum activities of creatine kinase (CK, a cytosolic and mitochondrial enzyme) and lactate dehydrogenase (LDH, a cytosolic enzyme) released through disintegrating membranes of myofibers and/or higher serum or urine concentrations of myoglobin. CARDIOTOXINS

While numerous 3FTx are neurotoxins, cardiotoxins are the second largest group of 3FTx. These nonenzymatic proteins exert their cytotoxic effects by interacting nonspecifically with membrane phospholipids in cell membranes (Ferraz et al., 2019; Kessler et al., 2017). Hundreds of such necrotoxins have been

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FIGURE 8.12 Diagram showing zootoxic mechanisms of phospholipase A2 (PLA2), a major enzyme in many venoms. Hydrolysis of plasma membranes by PLA2 generates arachidonic acid, a precursor to many eicosanoid mediators with pro-inflammatory and vasoactive effects. Oxidative membrane damage by PLA2 also releases intracellular calcium (Ca2þ) ions, which leads to cell death by disrupting mitochondrial function and invoking regulated cell death (i.e., proapoptotic) programs. Additional abbreviations: 5-HETE ¼ 5-hydroxyeicosatetraenoic acid, 5-HPETE ¼ 5-hydroperoxyeicosatetraenoic acid, LT ¼ leukotriene (e.g., LTA4), NF-kB ¼ nuclear factor kappa B, PG ¼ prostaglandin (e.g., PGE2), PI3 ¼ phosphoinositide 3, TXA ¼ thromboxane.

identified in venoms of cobras. Insertion into plasma membranes brings 3FTx into proximity with phosphatidylserine groups, which allows them to form pores (Konshina et al., 2017). In cardiac myocytes, cardiotoxins interfere with accumulation of Ca2þ in sarcoplasmic reticulum and instead facilitate the rapid release of existing Ca2þ stores (Nayler et al., 1976), which inhibits Ca2þ-activated ATPase and the ability to fuel activity of the contraction apparatus. By transmission electron microscopy, cardiac myofibers have disrupted cell and mitochondrial membranes and visible contraction bands (an indication of hypercontraction consistent with cell necrosis). METALLOPROTEINASES

Venoms are a rich me´lange of enzymatic activity in numerous invertebrate and vertebrate

species. Dozens of enzymes (more than 150) have been identified, and the largest numbers are found in crotalid and viperid snake venoms with potent necrotoxic activity. Metalloproteinases impact the structure and function of cells and tissues both locally and systemically immediately upon introduction into a victim. These zinc-dependent enzymes evolved from ancestral ADAM (“a disintegrin and metalloprotease”) proteins. They range in size from 20 to 110 kDa and are divided into three classes according to their structural configurations. In evolutionary terms, the P-III enzymes with their metalloprotease, disintegrin, and cysteine-rich domains are the oldest. The P-II class (which originated from a P-III ancestor) has only metalloprotease and disintegrin domains, while the P-I class (which arose from a P-II precursor) has a metalloprotease domain only. Crotalid and viperid

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venoms contain enzymes of all three classes while venoms of colubrid (e.g., boomslangs) and elapid snakes have only P-III enzymes (Ferraz et al., 2019). Key effects of metalloproteinases are necrosis of tissues at the envenomation site, including degradation of capillary basement membranes and adhesion proteins attaching endothelial cells to perivascular matrix; the extensive vessel wall disruption leads to hemorrhage (Ferraz et al., 2019). Posttranslational cleavage of some metalloproteinases frees the disintegrin domains to function independently. In general, multiple metalloproteinases and their derivatives are abundant in complex venoms (Correa et al., 2016; Ferraz et al., 2019); for example, disintegrins comprise approximately 17%–18% of venom proteins in crotalid and viperid snakes (Tasoulis and Isbister, 2017). Venom metalloproteinases are able to magnify the tissue destruction by recruiting additional endogenous metalloproteinases from infiltrating neutrophils and resident fibroblasts (Tambourgi et al., 2005). The effects of these enzymes either depend on or are exacerbated by local production of major pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) (Laing et al., 2003). Many enzymes are present in multiple isoforms due to gene duplication during evolution (Ferraz et al., 2019). The multiple enzyme isoforms within envenomation sites likely are synergistic in producing such effects. Many metalloproteinases act by degrading extracellular matrix. For example, collagenases remove collagen fibrils; elastases digest many matrix proteins; and gelsolinases disrupt the actin-binding protein gelsolin, which breaks the cytoskeleton and interferes with cell adhesion. These latter activities coupled with hyaluronidase (a nonmetalloproteinase that dissolves proteoglycans and other matrix glycosaminoglycans) act as “spreading factors” by digesting connective tissue barriers that impede venom dispersion. In addition, these enzymes enhance the effectiveness of other zootoxins by increasing the speed at which they move from the envenomation site to their cellular and molecular targets. SPHINGOMYELINASE D

Besides PLA2, venoms contain several other enzymes that metabolize lipids. The most consequential appears to be sphingomyelinase D (SMase D, also known as phospholipase D), an

important component of brown recluse spider venom. This single enzyme imparts much of the necrotoxic activity to the brown recluse venom through its ability to disrupt lipid membranes and the proteins borne within them (Lajoie et al., 2013; Pace and Vetter, 2009). SMase D has several activities that cooperate synergistically in producing cytolysis. The most important is that the enzyme disrupts lipid asymmetry in membranes, which can drive cell lysis by initiating the classic and alternative complement pathways and the related elements of the innate immune response (Manzoni-deAlmeida et al., 2018; Pace and Vetter, 2009; Tambourgi et al., 2002; van den Berg et al., 2012). Finally, SMase D induces multiple endogenous matrix metalloproteinases that participate in tissue lysis (Correa et al., 2016). Mammalian cells are differentially susceptible to the effects of venom enzymes. In particular, human and pig red blood cells are more sensitive than dogs to hemolysis (Gwaltney-Brant et al., 2018). Circulatory Disturbances Many zootoxins in venoms act on blood vessels. Most outcomes are functional (i.e., vasoactive) effects. The primary functional alterations are to increase blood vessel permeability and to control vessel tone, resulting in vasodilation or less often vasoconstriction. These effects may be local (i.e., at the envenomation site or affecting most of the affected appendage) but not infrequently will affect the entire body. However, some vascular outcomes are truly vasotoxic, causing structural damage to vascular walls. VASCULAR PERMEABILITY ENHANCEMENT

As noted above, enzymatic actions of PLA2 produce numerous vasoactive mediators that increase capillary permeability (Gwaltney-Brant et al., 2018). These signals result from PLA2mediated membrane hydrolysis of various cell types. When mast cell membranes are digested by PLA2 (often aided by mellitin in bee envenomation), the cells die and release their histamine payloads. In other cell types, PLA2 activity acting alone generates arachidonic acid that is then processed into numerous vasoactive leukotrienes and prostaglandins (Figure 8.12). Similarly, adenosine (a common purine component in scorpion, spider, and snake venoms) binds adenosine type 3 (A3) cell surface receptors on viable mast cells, which leads to degranulation

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3. ZOOTOXIN CLASSIFICATION

and release of vasoactive molecules that enhance capillary permeability (Aird, 2002). Venoms also possess biogenic amines (e.g., histamine and serotonin) that can act directly to heighten blood vessel permeability. Some venoms (e.g., the Russell’s viper [Daboia russelii]) have been linked to capillary leak syndrome, a systemic condition in which an increase in vessel permeability leads to massive plasma extravasation (Rucavado et al., 2018). Affected individuals have substantial hypoalbuminemia and exhibit profound hemoconcentration, which compromises perfusion and promotes organ failure. This syndrome has been linked to binding of vascular endothelial growth factor (VEGF) in venom to VEGF type 2 (KDR) receptors on endothelial cells (Yamazaki et al., 2003). This interaction leads to formation of fenestrae in capillary walls (Matsunaga et al., 2009), which potentiates plasma movement into tissues. VASCULAR TONE MODULATION

Many zootoxins rapidly induce hypotension, acting to produce incapacitating shock that immobilizes the prey animal. The mechanisms of action of these zootoxins are mirrored in those of many current antihypertensive treatments (Pe´terfi et al., 2019; Sitprija and Suteparak, 2008; Slagboom et al., 2017). For example, bradykinin-potentiating peptides in snake venoms act as angiotensin-converting enzyme (ACE) inhibitors, thereby facilitating the action of endogenous bradykinin (Pe´terfi et al., 2019). Other venoms have factors that activate endogenous kallikreins, a series of serine proteinases that convert kininogens to vasoactive kinins (Bohrer et al., 2007; Ferraz et al., 2019). Dendroaspis natriuretic peptide (DNP) is a venom component of the eastern green mamba snake (Dendroaspis angusticeps) that exhibits the same strong hypotensive effects of the less stable endogenous mammalian natriuretic peptides (Munawar et al., 2018). Calciseptine and FS-2, two 3FTx in black mamba (Dendroaspis polylepis) venom, serve as L-type Ca2þ channel antagonists (Pe´terfi et al., 2019). Many reptile venoms, including Komodo dragons (Fry et al., 2009b) and snakes (Frangieh et al., 2021), contain cysteine-rich secretory proteins (CRISPs) that inhibit smooth muscle contractions, thus countering vasoconstrictive signals. Adenosine causes vasodilation directly by binding

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adenosine A2 receptors on blood vessel walls and indirectly by binding A3 receptors on mast cells (Aird, 2002). Dipeptidyl peptidase-4 (DPP4)-like serine proteinases degrade vasoconstrictive molecules like neuropeptide Y and substance P (Aird, 2002). Venom of the common vampire bat (Desmodus rotundus) contains a small protein similar to calcitonin gene–related peptide (CGRP) that acts as a potent vasodilator (Kakumanu et al., 2019). This peptide functions selectively to relax arteriolar smooth muscle cells to facilitate consistent blood flow. Finally, lest we forget the obvious, numerous eicosanoid mediators released by PLA2-mediated membrane hydrolysis induce vasodilation (Figure 8.12). Perhaps paradoxically, many zootoxins also induce hypertension. Increased intravascular pressure drives plasma into tissues, which enhances edema and hemorrhage at the envenomation site and ultimately will engender hypovolemia and shock. For example, one eicosanoid released by PLA2-mediated membrane hydrolysis, thromboxane A2, produces vasoconstriction (Figure 8.12). Catecholamines in venom (Owen and Bridges, 1982) or released from endogenous cells by zootoxins in venom (Trejo et al., 2012) increase blood pressure, often producing an organ-specific effect (e.g., decreased renal blood flow and glomerular filtration rate (Thamaree et al., 2000)). Sarafotoxins in venoms of burrowing asps (Atractaspis spp.) are analogs of endothelins, among the most potent of endogenous vasoconstrictors (Mourier et al., 2012). Sarafotoxins have been associated with coronary arterial vasospasm. VASCULAR WALL DAMAGE

Blood vessel walls at envenomation sites are damaged by vasculotoxins (i.e., necrotoxins that disrupt endothelial and/or mural integrity). Destruction will encompass endothelial cells, mural elements, and perivascular tissues. Degradation of blood vessel walls will result in hemorrhage and may delay venom circulation in the blood, although venom constituents are likely to be dispersed in lymphatic vessels at a slower pace. The blood vessel damage is driven by the actions of metalloproteinases more so than PLA2 (Rucavado et al., 2018).

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Hemostasis Abnormalities Hemorrhage is a major pathologic outcome of envenomation because many zootoxins are potent coagulotoxins (Debono et al., 2019b; Slagboom et al., 2017; Youngman et al., 2019). Under normal conditions, coagulation is initiated as one component of an ordered process in which breaks in blood vessel walls are closed first by platelet adhesion and aggregation (termed “primary hemostasis”) and then anchored in place by a protein web (“secondary hemostasis”). This web is generated through the coagulation cascade, an intricately linked series of inactive proteases arranged in parallel “extrinsic” and “intrinsic” pathways that converge on a “common” pathway (Smith, 2009). The extrinsic pathway is initiated by release of tissue factor from injured endothelial and stromal cells, the intrinsic pathway starts when breaks in blood vessel walls provide exposure to collagen in the perivascular connective tissue, and the common pathway is the final effector arm responsible for production of fibrin. Coagulotoxins usually are directed against a specific clotting factor, often a component of the common pathway (i.e., Factor II [prothrombin], Factor V, or Factor X). In contrast, necrotoxic zootoxins cause such substantial cell and tissue damage that they are liable to activate both the extrinsic and intrinsic pathways simultaneously. COAGULOTOXIC ZOOTOXINS

Numerous venom serine proteinases (from the S1 family) and metalloproteinases act by regulating molecular cascades, particularly the balance between hemostasis and thrombosis (Ferraz et al., 2019; Matsui et al., 2000). The serine proteinases range in size from 26 to 67 kDa and are much more common in highly coagulotoxic venoms of crotalid and viperid snakes. Many serine proteinases exhibit thrombin-like capabilities in converting fibrinogen to fibrin, but the fibrin forms are aberrant and thus incapable of producing a stable clot. These venom proteases are not inhibited by anticoagulants like heparin, hirudin (a leech-derived thrombin inhibitor), or endogenous serum protease inhibitors. Some serine proteinases activate other proteins involved in the fibrinolytic system, such as plasminogen (the precursor to

the fibrinolytic enzyme plasmin that aids in thrombus removal) and protein C (an endogenous serine proteinase that catabolizes activated Factors Va and VIIIa and permits sustained thrombin activity to stabilize newly formed thrombi). The thrombolytic protein desmokinase (also called D. rotundus salivary plasminogen activator [DSPA]) found in vampire bat venom is a serine protease that catalyzes the conversion of plasminogen to plasmin and has been tested (as the recombinant protein desmoteplase) as a potential clot-busting treatment for acute ischemic stroke (Elmaraezy et al., 2017; RodeMargono and Nekaris, 2015). Metalloproteinases, such as ADAM17 (Lopes et al., 2020) and other disintegrins (Tasoulis and Isbister, 2017), often exhibit thrombin-like activity and thus promote a procoagulant state. A few metalloproteinases possess affinities for other coagulation proteins including Factor X, protein C, or von Willebrand factor (a soluble glycoprotein that promotes platelet adhesion and sequesters circulating Factor VIII). COAGULOTOXIC MECHANISMS

Procoagulant effects of venoms lead to enhanced formation of thrombi within local and often distant blood vessels. Formation of stable clots is thought to enhance immobilization of prey by inducing acute cerebral ischemia (“stroke”) (Sousa et al., 2020). Serine proteinases in venoms of many snakes (especially crotalid and viperid species) convert the proenzyme Factor X to its active form Xa, which then cleaves prothrombin to thrombin (Tans and Rosing, 2001); some venom metalloproteinases directly convert prothrombin to thrombin (Lo¨vgren, 2013). Thrombin cleaves fibrinogen to make fibrin, which is deposited within platelet aggregates to stabilize the nascent thrombus. Some zootoxins are more potent activators of Factor X and/or thrombin, and therefore drive generation of more stable clots (Sousa et al., 2018). Serum proteinases also boost coagulation by activating other portions of the cascade, such as Factor V (in the common pathway) or Factor VII (in the extrinsic pathway). Some serine proteinases also may induce platelet aggregation, such as the enzyme g-glutamyl transpeptidase (GGT) in many centipede venoms (Liu et al., 2012). This effect is not consistent across species as GGT causes platelet aggregates in

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human blood but elicits hemolysis in mouse and rabbit blood. Anticoagulant effects of venoms produce hemorrhage at the envenomation site, and often in distant tissues into which venom has spread. Anticoagulant toxins often are small proteins (or glycoproteins) and peptides, and they thwart coagulation by several meansdin some cases with a single zootoxin having more than one anticoagulant effect (Grashof et al., 2020; Kini, 2006). For example, draculin is a glycoprotein in vampire bat venom that irreversibly binds Factors IXa and X as well as inhibiting the conversion of prothrombin to thrombin (Basanova et al., 2002). Similarly, the enzymatic action of PLA2 strongly inhibits conversion of Factor X to Xa and prothrombin to thrombin, and its nonenzymatic function is to bind and sequester Factor Xa. Many coagulotoxins act solely as enzymes. Metalloproteinases function to degrade fibrinogen, which prevents formation of fully stabilized fibrin webs over platelet plugs. In like manner, some metalloproteinases (in particular disintegrins) in crotalid and viperid snakes disrupt adhesion between extracellular matrix and platelets, thereby preventing initial formation of platelet aggregates (Munawar et al., 2018; Tasoulis and Isbister, 2017). Serine proteinases have several anticoagulant activities including protein C activation, which shuts down activated clotting factors Va (involved in the common clotting cascade) and VIIIa (a part of the intrinsic clotting cascade), as well as fibrinogenase- and thrombin-like capabilities, both of which lead to fibrinogen depletion. Saliva of medicinal leeches (Hirudo medicinalis) contains elastase and plasmin, which are fibrinolytic enzymes involved in clot destabilization and removal (Kvist et al., 2013). Other nonenzymatic mechanisms of zootoxin-related anticoagulant activity include binding of inactive Factor IX (an intrinsic cascade component) and Factor X as well as inhibition of enzymes involved in conversion of various clotting cascade factors to their activated forms. Interestingly, the anticoagulant activity of venoms depends on the species. For instance, some Australian elapids produce potent anticoagulants for plasma of many species, including humans, while others produce anticoagulant activity only in plasma from prey species (Youngman et al., 2019). Venom of the

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Indochinese spitting cobra (Naja siamensis) has the usual PLA2-mediated anticoagulant activity but lacks some fibrinogenolytic proteins characteristic of venoms from other elapids (Modahl et al., 2020). Pseudo-procoagulant actions of venoms result in generation of aberrant fibrin strands and poor stabilization of platelet aggregates. Further deposition of abnormal fibrin in an attempt to anchor the platelet plug will result in fibrinogen depletion, which produces a net anticoagulant state (Debono et al., 2019a; Sousa et al., 2018). Closely related venomous species differ in their use of a pseudo-procoagulant state (i.e., cleavage of fibrinogen to produce anomalous fibrin) versus true anticoagulant state (i.e., destructive cleavage of fibrinogen) to achieve a net anticoagulant state (Debono et al., 2019b). Venom from a particular species may produce opposite effects depending on demographic attributes of the individual. For instance, the venom of some juvenile rattlesnake species has a strong procoagulant effect, while the venom of adults from the same species has a pseudo-procoagulant activity (Seneci et al., 2021). Inflammation Induction Many zootoxins are effective at initiating and extending inflammation (Ryan et al., 2021). The potency of proinflammatory effects often is linked to the degree of tissue destruction (i.e., necrotoxicity). This relationship stems from both the release of damage-associated molecular patterns (DAMPs) from disintegrating cells and tissues and the generation of proinflammatory chemical mediators from disrupted lipid membranes. For example, myonecrosis caused by terciopelo snake venom leads to leakage of Kþ and ATP from damaged myofibers. Extracellular ATP serves as a DAMP to activate the immune system; the ATP metabolite adenosine causes vasodilation (leading to hypotension) and prevents coagulation (resulting in hemorrhage) (Caccin et al., 2013). The initial inflammatory response to these factors is mounted by the innate immune system, but adaptive immune responses follow if the initial envenomation episode is survived (see Immune System [Vol 5, Chap 6]). Acute stress associated with pain at envenomation sites can modulate the response by initiating the neuroendocrine–immune axis to modulate both

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the pro-inflammatory and antiinflammatory responses (Santhosh et al., 2016). INNATE IMMUNE RESPONSES TO ZOOTOXINS

As one of its principal toxic functions, venom PLA2 hydrolyzes membrane phospholipids into lysophospholipids and fatty acidsdparticularly arachidonic acid (AA), a precursor to numerous pro-inflammatory and vasoactive molecules (Gwaltney-Brant et al., 2018; Pace and Vetter, 2009). Subsequent AA metabolism by 5lipoxygenase and phosphoinositide 3 (PI3) kinase establishes a pro-inflammatory state in the local microenvironment leading to generation of leukocyte chemotactic and activating cytokines (Figure 8.12). Furthermore, venom PLA2 is capable of initiating a self-perpetuating cycle of inflammation by activating endogenous (prey-derived) PLA2 (Bickler, 2020). Bee venom PLA2 alone or together with mellitin causes mast cell cytolysis with release of histamine. In addition, PLA2 but not mellitin is responsible for the IgE-dependent innate immune activation that results in immediate (Type I) hypersensitivity (Lahiani et al., 2017). In fatal bee stings, anaphylaxis rather than envenomation is the cause of death in 95% of cases (Junghanss and Bodio, 2006), and the lethal event is airway obstruction due to PLA2-induced release of histamine along with generation of leukotrienes and prostaglandins (Ennik, 1980). In nonfatal stings, bee venom PLA2 also may participate in priming an adaptive immune response as the enzyme works to generate neoantigens using human skin lipids that are then presented (via a CD1-mediated process) to activate human T lymphocytes (Bourgeois et al., 2015). The companion zootoxic enzyme SMase D also impacts the innate immune response. The primary means, as noted above, is the enzyme’s propensity to launch the classic and alternative complement pathways. The resulting formation of membrane attack complexes (MACs) drives both cutaneous necrosis and hemolysis (Tambourgi et al., 1998). Complement activation by the classic pathway results directly from SMase D-mediated loss of lipid asymmetry in the plasma membrane, thus exposing the inner phosphatidylserine layer (Tambourgi et al., 2002; Tambourgi et al., 2007). In addition, SMase D facilitates complement-based cytolysis by indirectly removing a brake on the alternative

complement pathway; in this case, SMase D activates an endogenous membrane-bound metalloprotease that cleaves cell-surface glycophorins which act as receptors for a factor that limits initiation of the complement cascade (Tambourgi et al., 2000). Activated complement is instrumental in recruiting neutrophils to the envenomation site (Tambourgi et al., 2005). The second SMase D role in modulating the immune response is by promoting the release of fatty acid and cyclic phosphate products from injured cells. These pro-inflammatory mediators augment the innate immune response by activating the respiratory burst of incoming neutrophils, leading to production of reactive oxygen (superoxide anion) and nitrogen (peroxynitrite) species capable of imparting lethal damage to cells (Manzoni-de-Almeida et al., 2018). The action of venom PLA2 on cell membranes generates lysophosphotidylcholine (LPC) as a chemical mediator. This molecule appears to serve as a pathogen-associated molecular pattern (PAMP) that can be sensed by the innate immune system. The response requires participation of the myeloid differentiation primary response 88 (MyD88) protein (Palm et al., 2013), an adaptor molecule that regulates immune cell signaling and production of many pro-inflammatory cytokines (De Nardo et al., 2018). The MyD88 protein and others, including nuclear factor kappa B (NF-kB), mediate the activation of inflammasomes (Man and Kanneganti, 2015). These intracellular multiprotein complexes have a central role in innate immune sensing. Bee venom PLA2 and especially mellitin, activate the nucleotide-binding domain and leucine-rich repeat-containing receptor with a pyrin domain 3 (NLRP3) inflammasome through their abilities to damage cell membranes (Martı´n-Sa´nchez et al., 2017; Palm and Medzhitov, 2013). This activation leads to secretion of IL-1b by primed macrophages (Martı´n-Sa´nchez et al., 2017), which boosts the local inflammatory response, as well as engagement of caspase-1. The rapid cytolysis produced by mellitin limits caspase-1-dependent pyroptosis of injured cells, but caspase-1 activity is needed for neutrophil recruitment to an envenomation site (Ferraz et al., 2019; Palm and Medzhitov, 2013). In snakebites, neutrophils predominate during the first 24 h (approximately 85% of innate immune cells) and attempt

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3. ZOOTOXIN CLASSIFICATION

to limit venom-induced tissue damage through such actions as release of pro-inflammatory mediators, formation of neutrophil extracellular traps (NETosis) and reactive oxygen species, and phagocytosis of debris (Zuliani et al., 2020). The innate reaction is converted to a macrophage-dominated response by 72 h. ADAPTIVE IMMUNE RESPONSES TO ZOOTOXINS

Bee venom, specifically the potent immunogen PLA2, induces a Type 2 (Th2) immune response in mice. Characteristic features of this reaction include generation of IL-4 by T-helper (CD4þ) lymphocytes; IL-5 by type 2 innate lymphoid cells (i.e., ILC2 cells, a lineage that lacks antigen-specific B and T cell receptors); and eosinophilia in response to IL-5 (Palm et al., 2013). The Th2 response depends on the enzymatic activity of PLA2 to release LPC from membranes; arachidonic acid and its downstream eicosanoid mediators do not impact this Th2 response. Production of LPC and the concurrent death of severely injured cells together are associated with generation of IL33. This cytokine supports the Th2 response by binding membrane-bound IL-1 receptor-like type 1 (IL1RL1) receptors (alternative designation: ST2 [serum stimulation-2] receptor) found on the surfaces of Th2 and regulatory T (Treg) lymphocytes as well as ILC2 cells (Griesenauer and Paczesny, 2017). The IL-33-mediated response also requires participation of MyD88 (Palm et al., 2013). Both PLA2 and mellitin in bee venom also elicit production of IgE and IgG1 antibodies (Mu¨ller, 2010; Palm et al., 2013), generation of which are supported by IL-4 secretion. While bee venom allergies are common in humans (affecting approximately 3% of the population (Brown and Tankersley, 2011)), mice do not develop IgE-mediated Type I hypersensitivity (i.e., anaphylaxis) upon a second exposure to venom (Palm et al., 2013). Similarly, the NLRP3 inflammasome has been implicated as a participant in allergic diseases in humans (Xiao et al., 2018) but is reported to have no role in the allergic response to bee venom (Martı´nSa´nchez et al., 2017). Some cases of snake envenomation have been linked to Guillain–Barre´ syndrome (GBS), an immune-mediated polyneuropathy; in one instance, the condition appeared to result from cross-reactivity between

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glycosidic epitopes of proteins in European asp (Vipera aspis) venom and neuronal GM2 ganglioside (Neil et al., 2012). Humoral responses to venom depend on the exposure scenario. Polyvalent antivenoms made by inoculating a single animal with several venoms have higher antibody titers and venom neutralizing capacities than products concocted by mixing antibodies harvested from the plasma of several animals each of which had been immunized with one venom (Dos-Santos et al., 2011). Coinjection of multiple venoms may lead to a reduced response to any single venom, even for potent immunogens like PLA2 and metalloproteinases (Arroyo et al., 2015). Venom zootoxins also are capable of suppressing immune reactions. For example, venom extracts from Chinese scorpions used in traditional antiinflammatory treatments reduce IL-2 release and limit delayed hypersensitivity (Yang et al., 2019). Bee venom PLA2 injected as part of an allergen immunotherapy regimen facilitates differentiation of Treg cells that limit immune responses, limits mast cell degranulation (Mu¨ller, 2010), and moderates the release of IgE-dependent chemical mediators while enhancing IL-10-mediated antiinflammatory effects during Th2 responses (Caramalho et al., 2015; Palm et al., 2013). Neurotransmission Derangement Many poisonous and venomous animals produce one or, more often, many neurotoxins (Ferraz et al., 2019; Karalliedde, 1995). As noted above, these molecules have been selected during evolution to rapidly immobilize prey (to prevent trauma to the neurotoxin-wielding animals) or to inflict substantial pain (to prevent other animals from preying on the wielder). This tendency is exemplified by neurotoxins produced by various orders of arachnids. Those animals with lifestyles centered on predation (to eat or avoid being eaten) are all venomous and have large neurotoxin arsenals (e.g., 99% of w47,000 spider species, 100% of w2300 scorpion species) (Pienaar et al., 2018). In contrast, animals with parasitic or scavenging lifestyles are seldom armed with neurotoxic venoms (e.g., 8% of w900 tick species) (Pienaar et al., 2018). Neurotoxins in animal poisons and venoms are among the most powerful natural toxins.

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The LD50 values (i.e., the lethal dose for 50% of treated animals) for these molecules in mice range from 2 mg/kg for batrachotoxin in the skin of poison dart frogs to 7.9 mg/kg for ciguatoxin in ray-finned fish (Table 8.4). As a point of comparison, the most potent known toxins in nature are two neuroactive clostridial exotoxins: botulinum toxin made by Clostridium botulinum and tetanospasmin (tetanus toxin) produced by Clostridium tetani (see Bacterial Toxins [Vol 3, Chap 9]). Their estimated LD50 values in micedapproximately 0.5 ng/kg for botulinum toxin and 1 ng/kg for tetanospasmin (Gill, 1982)d are at least 2000-fold below the lowest LD50 value for the most potent neurotoxic zootoxin. Neurotoxic zootoxins have divergent chemical structures and act on many processes essential to normal physiological function (Escoubas et al., 2000; Rash and Hodgson, 2002). The common end of these mechanisms is that neurotransmission is disrupted, typically at the neuromuscular junction (NMJ), which is the synapse between the terminal bouton of an axon (i.e., “presynaptic” side) and the associated myofiber (i.e., “postsynaptic” side); similar impacts on neurotransmission also may occur at interneuronal synapses within the CNS or autonomic peripheral nervous system (PNS). Most neurotoxic zootoxins are peptide or small molecule ligands for cell surface receptors or catalytic peptides or proteins (i.e., enzymes) (Figure 8.11). Depending on the species, many amino acid– based neurotoxins share common structural motifs. Most peptides are stabilized by two or more disulfide bonds (Figure 8.7 and Figure 8.13A) so that the conformation is suitable for enzymatic or ligand-binding activity (Cardoso and Lewis, 2019; Grandal et al., 2021; Rash and Hodgson, 2002). Such structures provide high affinity and selectivity. Most peptides in spider venoms possess an inhibitor cysteine knot (ICK) arrangement, a motif containing three disulfide bridges coupled with a cyclic configuration that renders them stable to catabolism by proteases, thereby equipping them with long half-lives (Saez et al., 2010). Neurotoxic peptides in snake venoms primarily occur as “short-chain” 3FTx (57–62 aa with 4 disulfide bonds [Figure 8.7]), “long-chain” 3FTx (66–74 aa with 5 disulfide bonds), and “unconventional” monomers. The divergent structures of these toxins may provide

selectivity for different host cell molecular targets, so complex venoms may elicit neurotoxicity via synergistic activities of 3FTx having different toxic effects. In contrast, small molecule neurotoxins have more variable structures characterized by multiple heterocyclic rings. Some molecules are relatively simple, such as the symmetric low molecular mass (LMM) organic compound nigriventine (in venom of the Brazilian wandering spider [Phoneutria nigriventer]; Figure 8.13B) (Gomes et al., 2011). Agricultural workers frequently encounter these “banana” spiders during harvest season at fruit plantations, and the defensive behavior of these animals when disturbed results in frequent envenomation. Increased c-Fos expression in rat brain following intraparenchymal injection of nigriventine is an “immediate early” response consistent with heightened neuronal activity. A primary clinical manifestation in humans bitten by wandering spiders is convulsions. That said, clinical signs in humans might be induced solely or mainly by many other venom components (Diniz et al., 2018). Batrachotoxin (Figure 8.13C) is a heterocyclic steroidal alkaloid that is the most potent small molecular neurotoxic zootoxin. This pyrrole carboxylic acid has six complete rings including two 2-member rings and one 7-member substituted ring. This molecule interacts with Naþ channels to modify their ion selectivity and voltage sensitivity (see below) (T3DB, 2009). Ciguatoxin is a series of related compounds with heterocyclic rings, many with oxygen atom substitutions, in which the rings are fused (Figure 8.13D); many of the rings have 7 or more atoms. Ciguatoxins alter conditions under which Naþ channels will open. These examples demonstrate that divergent molecular structures nonetheless may yield comparable neurotoxic effects. ALTERED NEUROTRANSMITTER LEVELS

Actions of many neurotoxic zootoxins affect the availability of neurotransmitters within a synapse. The majority of the mechanisms responsible for this effect are focused in the terminal boutons of axons. The NMJ has been used as a model synapse to investigate such mechanisms, and many neurotoxic zootoxins of marine organisms, arachnids, and snakes have been employed in teasing apart the various

II. SELECTED TOXICANT CLASSES

TABLE 8.4 Selected Neurotoxic Zootoxins Demonstrating Diverse Zootoxic Mechanisms for Producing Similar Clinical Effects

Toxin

Toxin structure

Mouse median lethal dose (LD50) Animal

Site

Mechanism of action Clinical presentation þ

Apamin

Peptide

6.0 mg/kg (IV)

Honeybee (Apis mellifera)

Ca -activated K channel

Inhibition lowers • C: swelling threshold for action • N: Pain potential generation at CNS synapses

d-Atracotoxin (robustoxin)

Peptide

160 mg/kg (SC)

Sydney funnel web spider (Atrax robustus)

Nav channel

Slow inactivation yields repetitive firing at NMJ þ autonomic PNS

Batrachotoxin

Alkaloid

2 mg/kg (PO þ SC)

Poison dart frog (Dendrobates spp.)

Nav channel

Persistent • H: Arrhythmias, inactivation leads to ventricular massive ACh release fibrillation at NMJ • N: Paralysis

Brevetoxin

Polycyclic ether

520 mg/kg (PO)

Shellfish (bivalves), by ingesting toxinproducing dinoflagellates

Nav channel

Activation produces • GI: diarrhea, uncontrolled Naþ nausea, vomiting influx • N: Allodynia, numbness, tingling, weakness

Bufotenine

Serotonin analog

290 mg/kg (PO)

Toads (genera Serotonin receptor Anaxyrus, Bufo, and (several subtypes) Incilius)

Partial agonist

• N: Hallucinations, centrally driven nausea, convulsions, autonomic symptoms

a-Bungarotoxin

Peptide

108 mg/kg (SC)

Many-banded krait (Bungarus multicinctus)

Ligand-gated nicotinic ACh receptor at NMJ (postsynaptic)

Irreversible competitive antagonist

• N: Paralysis • R: Distress leading to failure

b-Bungarotoxin

Peptide (PLA2 activity)

108 mg/kg (SC)

Many-banded krait (B. multicinctus)

Membranes in presynaptic terminals

Massive release of ACh leading to exhaustion of ACh reserves

• N: Paralysis • R: Distress leading to failure

2þ

• GI: nausea, vomiting • N: Tingling, facial twitching, confusion, coma

3. ZOOTOXIN CLASSIFICATION

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(Continued)

Selected Neurotoxic Zootoxins Demonstrating Diverse Zootoxic Mechanisms for Producing Similar Clinical Effectsdcont’d

586

TABLE 8.4

Toxin

Toxin structure

Mouse median lethal dose (LD50) Animal

Chlorotoxin

Peptide

250 mg/kg (SC)

Deathstalker Cl channel (Egyptian) scorpion (Leiurus quinquestriatus)

Inhibition

Ciguatoxin

Polycyclic ether

25 mg/kg (IP þ PO)

Ray-finned fish (e.g., Nav channel barracuda, bonito, grouper, red snapper, tuna) e by ingesting fish that ate toxinproducing dinoflagellates

Reduced threshold • GI: diarrfor channel activation hea, nausea, (PNS synapses only) vomiting • N: Allodynia, numbness, tingling

a-Conotoxin

Peptide

12 mg/kg (IP)

Cone snails (Conus spp.)

Nicotinic ACh receptor at NMJ (postsynaptic)

Inhibition

d-Conotoxin

Peptide

5 mg/kg (not stated) Cone snails (Conus spp.)

Nav channel

Delayed inactivation • C: swelling • N: Pain (intense), numbness, tingling, paralysis

k-Conotoxin

Peptide

5 mg/kg (not stated) Cone snails (Conus spp.)

Kv channel

Inhibition

• C: swelling • N: Pain (intense), numbness, tingling, paralysis

m-Conotoxin

Peptide

5 mg/kg (not stated) Cone snails (Conus spp.)

Nav channel

Inhibition

• C: swelling • N: Pain (intense), numbness, tingling, paralysis

u-Conotoxin

Peptide

w60 mg/kg (IP)

Cav channel

Inhibition

• C: swelling • N: Pain (intense), numbness, tingling, paralysis

Site e

• GI: vomiting • H: Arrhythmias, hyper- or hypotension • N: burning, pain • R: Edema

• C: swelling • N: Pain (intense), numbness, tingling, paralysis

8. ANIMAL TOXINS

Cone snails (Conus spp.)

Mechanism of action Clinical presentation

TABLE 8.4

Selected Neurotoxic Zootoxins Demonstrating Diverse Zootoxic Mechanisms for Producing Similar Clinical Effectsdcont’d Mouse median lethal dose (LD50) Animal

Toxin

Toxin structure

Crotoxin (snake PLA2)

Protein (with PLA2 activity)

216 mg/kg (IP)

South American Presynaptic terminal Decreased ACh rattlesnake (Crotalus (target uncertain) release durissus)

• C: Edema, hemorrhage • N: Pain

Dendrotoxin

Peptide

117 mg/kg (IP)

Black mamba Kv channel (Dendroaspis polylepis)

• N: Convulsions, muscle twitching

Domoic acid

Excitatory neurotransmitter

3.6 mg/kg (PO)

Shellfish (bivalves), by ingesting toxinproducing dinoflagellates

Glutamate receptor (brain)

Holocyclotoxin

Peptide (ICK motif) w200 mg/kg (SC)

Ticks (multiple genera) e females

Presynaptic terminal Decreased ACh (target uncertain) release

a-Latrotoxin

Protein

90 mg/kg (PO)

Widow spiders (Latrodectus spp.)

Presynaptic terminal Formation of pores • GI: nausea, leads to Ca2þ influx vomiting and massive release • N: Headache, pain, of ACh (at NMJ) and dizziness, muscle other neurotranscramping (tetany), mitters (at sensory tremors neurons)

Mellitin

Peptide

2.8 mg/kg (IV) 20.8 mg/kg (SC)

Honeybee (Apis mellifera)

Ion exchange pumps Inhibition yields (Naþ/Kþ-ATPase) higher Naþ permeability

Noxiustoxin

Peptide

310 mg/kg (IV)

Mexican scorpion Ca2þ-activated Kþ (Centruroides noxius) channel

Site

Mechanism of action Clinical presentation

Enhanced ACh release

• N: Weakness, paralysis (ascending),  ataxia

3. ZOOTOXIN CLASSIFICATION

Agonist leads to • GI: intestiprolonged activation nal cramping, (excitotoxicity) nausea, vomiting • N: Headache, short-term memory loss, seizures, coma

• C: swelling • N: Pain

(Continued)

587

Inhibition (reversible • H: Arrhythmias, or irreversible for hyper- or various channel hypotension subtypes) leading to • N: burning, muscle increased neurotrans- fibrillation, tingling mitter release • R: Edema, failure

588

TABLE 8.4 Selected Neurotoxic Zootoxins Demonstrating Diverse Zootoxic Mechanisms for Producing Similar Clinical Effectsdcont’d

Toxin

Toxin structure

Mouse median lethal dose (LD50) Animal

Site

Mechanism of action Clinical presentation

PLA2 (snake venom) Protein

750 mg/kg (IV) 6.2 mg/kg (SC)

Multiple species

Presynaptic terminal Damage to synaptic • C: swelling (membranes) vesicle membranes • H: Coaguloleads to immediate pathy, hemorrhage exocytosis (massive • N: Anxiety, headACh release) and no ache, pain, vesicle recycling weakness

Saxitoxin

Alkaloid

10 mg/kg (IP) 263 mg/kg (PO)

Shellfish (bivalves), by ingesting toxinproducing dinoflagellates

Nav channel

Scombrotoxin (histamine)

Biogenic amine

220 mg/kg (PO)

Scombroid fish Histamine receptor (mackerel, tuna) and related species e by bacterial metabolism in spoiling fillets

Conversion of • C: Urticaria histidine to histamine • GI: abdo(agonist) minal pain, diarrhea, vomiting • R: Rhinorrhea, bronchospasm

Tetrodotoxin

Alkaloid

8 mg/kg (IV) 532 mg/kg (PO)

Blue-ringed octopus Nav channel (Hapalochlaena lunulata), pufferfish (several species) e produced by symbiotic bacteria

Inhibition

Reversible inhibition • GI: nausea, vomiting (rapid-onset) • N: Paresthesia, ataxia, weakness, paralysis

(1) “lMouse median Lethal Dose” column: IP ¼ intraperitoneal, IV ¼ intravenous, PO ¼ oral (per os), SC ¼ subcutaneous; (2) “Site” and “Mechanism of Action” columns: ACh ¼ acetylcholine, Ca ¼ calcium, Cl ¼ chloride, CNS ¼ central nervous system, K ¼ potassium, Na ¼ sodium, NMJ ¼ neuromuscular junction, PNS ¼ peripheral nervous system; (3) “Clinical Presentation” column: C ¼ cutaneous, GI ¼ gastrointestinal, H ¼ heart and blood, N ¼ neurological, R ¼ respiratory.

8. ANIMAL TOXINS

• N: Numbness, seizures, weakness, paralysis

3. ZOOTOXIN CLASSIFICATION

589

relationships among the molecular components of this complex system (Karalliedde, 1995; Sitprija, 2006; Watkins, 2013). ALTERED NEUROTRANSMITTER AVAILABILITY A considerable proportion of zootoxin-mediated neurotoxicity is driven by abnormal availability of neurotransmitters within synapses. The biochemical lesions may reflect the presence of neurotransmitters in poisons or venoms or shifts in neurotransmitter metabolism and/or utilization due to zootoxin action. Exogenous signaling molecules Venoms contain many neurotransmitters (Aird, 2002; Rash and Hodgson, 2002). Common molecules (which are identical to neurotransmitters in vertebrate species) are biogenic amines (e.g., histamine, norepinephrine, and serotonin); excitatory amino acids (aspartic, g-aminobutyric [GABA], and glutamic acids); and taurine. In some cases, metabolites of these neurotransmitters are the neurotoxic component. For instance, the psychoactive indolealkylamine 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) that is responsible for the hallucinogenic effects of ingested skin secretions of the Colorado River toad is a metabolite produced by methylation of serotonin (chemical designation: 5-hydroxytryptamine [5-HT]) (Weil and Davis, 1994). In addition to their own intrinsic activities, these neurotransmitters sensitize cells to the effects of other zootoxins. Altered synthesis and packaging At the NMJ, ACh is generated in the cytosol when acetylcoenzyme A (acetyl-CoA) is combined with choline by the action of choline acetyltransferase

=

FIGURE 8.13 Molecular structures of selected neurotoxic zootoxins. (A) Apamin, a globular peptide comprising 2%–3% of honeybee (Apis mellifera) venom that selectively inhibits some subtypes of voltage-gated Kþ channels. (B) Nigriventrine, a recently discovered low molecular mass (LMM) small molecule in the venom of Brazilian wandering spiders (Phoneutria

nigriventer). The molecular target is not yet known. (C) Batrachotoxin, a steroidal alkaloid in skin secretions of poison dart frogs (Dendrobates spp.), acts by opening voltage-gated Naþ (Nav) channels. (D) Ciguatoxin-3, one of several microalgae-derived toxins responsible for ciguatera fish poisoning associated with consumption of several species of large fish, produces sustained activation of Nav channels. Image A reproduced from https://commons.wikimedia.org/wiki/File:Apamin.svg under a Creative Commons license (CC BY-SA 3.0). Images B, C, and D are in the public domain. Image B reproduced from https://commons.wikimedia.org/wiki/File:Nigriventrine.svg. Image C reproduced from https://commons.wikimedia.org/ wiki/File:Batrachotoxin.png. Image D reproduced from https://commons.wikimedia.org/wiki/File:Ciguatoxin3.svg.

II. SELECTED TOXICANT CLASSES

590

8. ANIMAL TOXINS

(ChAT), after which the ACh is packaged in synaptic vesicles. Some neurotoxic zootoxins shift acetylcholine (Ach) production in the axon terminal of the NMJ (Figure 8.14, mechanism 1). For example, a peptide in saliva of the Rocky Mountain wood tick (Dermacentor andersoni) reduces ACh synthesis in axon terminals (Pienaar et al., 2018). The peptides b-bungarotoxin (b-BTX) made by the many-banded krait (Bungarus multicinctus) (Gundersen et al., 1981) and notexin in venom of the eastern tiger snake (Notechis scutatus) (Gundersen and Jenden, 1981) are reported to stimulate synthesis of ACh. Importantly, both b-BTX and notexin also

inhibit release of newly formed ACh. Synaptic vesicles disappear after exposure to b-BTX, and high doses of notexin are associated with lysis of synaptic vesicles (Gundersen and Jenden, 1981). The common theme of these mechanisms is reduced availability of ACh-loaded synaptic vesicles capable of being released at axon terminals. ALTERED NEUROTRANSMITTER RELEASE In a healthy person or animal, arrival of an action potential at the axon terminal leads to plasma membrane depolarization and opening of voltage-gated Ca2þ (Cav) channels on the

FIGURE 8.14 Sites of action for neurotoxic zootoxins in the neuromuscular junction (NMJ). Various toxins can act in the axon terminal (“presynaptic,” mechanisms 1–7); within the synaptic cleft (mechanism 8); or on the myofiber (“postsynaptic,” mechanisms 9 and 10). ACh ¼ acetylcholine. Diagram provided courtesy of Mr. Tim Vojt.

II. SELECTED TOXICANT CLASSES

3. ZOOTOXIN CLASSIFICATION

presynaptic plasma membrane. The Ca2þ influx working via the SNARE (soluble Nethylmaleimide-sensitive factor attachment protein receptor) protein complex drives fusion of ACh-laden synaptic vesicles with the inner leaflet of the presynaptic membrane, resulting in exocytosis (release of ACh) into the synapse (Yoon and Munson, 2018). Many neurotoxic zootoxins modify neurotransmitter release from axon terminals (Figure 8.14, mechanism 2). The affected transmitter is ACh at the NMJ, but other neurotransmitters (e.g., biogenic amines like norepinephrine and serotonin, excitatory molecules like glutamate) may be affected at other synapses. The effect may present as an increased (Karalliedde, 1995) or reduced (Aird, 2002; Pienaar et al., 2018; Tzeng et al., 1995) discharge of neurotransmitters. A number of molecular mechanisms participate in altering the extent of neurotransmitter release. þ Inhibition of Naþ channels Two types of Na channels in cell membranes of electrically excitable cells (e.g., neurons, certain glia, and myocytes) may be targeted by neurotoxins. The two types are classified by the stimuli that trigger their opening. “Ligand-gated” (NaL) channels are controlled by binding a chemical while “voltage-gated” (or “voltage-sensitive,” Nav) channels are controlled by a change in polarity of the plasma membrane associated with transmission of an electrical impulse. The ligand or voltage fluctuation leads to conformational changes in the channels that permit an influx of Naþ, which in the presynaptic terminal results in synaptic vesicle fusion and release of the neurotransmitter payload. Most neurotoxic zootoxins preferentially target voltage-gated (Nav) channels (Cardoso and Lewis, 2019; Hakim et al., 2015) (Figure 8.14, mechanism 3). Various toxins bind different sites on the channel, and the functional effects depend on the binding site (Cestele and Catterall, 2000; Gray et al., 1988; Mouhat et al., 2004; Sitprija and Suteparak, 2008; Watkins, 2013). For example, alkaloids (e.g., saxitoxin and tetrodotoxin) and peptides (e.g., m-conotoxin) that dock at site 1 inactivate the Nav channel. Binding at site 2 (e.g., batrachotoxin [Figure 8.13C]) opens the Nav channel. Attachment at site 3 (e.g., some sea anemone and a-scorpion neurotoxins) slows Nav channel inactivation, leading to sustained depolarization. Interestingly, docking at

591

site 4 can either activate (e.g., b-scorpion toxins) or inactivate (e.g., d-conotoxin) the Nav channel. Binding at site 5 (e.g., ciguatoxin [Figure 8.13D]) maintains channel activation, thus causing persistent depolarization. Acid-sensing ion channels (ASICs) are ligandgated (NaL) channels for which protons (Hþ) serve as the ligand (Gru¨nder and Chen, 2010). These channels are common on nociceptive (pain) neurons. Various neurotoxins of sea anemones, spiders, and snakes can inhibit ASIC activity by locking them in the desensitized state, which blocks the perception of pain (CristoforiArmstrong et al., 2021; Maatuf et al., 2019). Mutations in Naþ ion receptors can reduce or prevent adverse effects produced by venom exposure and Naþ channel activation. For example, bark scorpions (Centruroides spp.) can inflict intensely painful, potentially lethal stings because their neurotoxins activate Nav1.7 channels on nociceptors (pain neurons) in sensitive species to escalate and sustain impulse transmission. In contrast, southern grasshopper mice (Onychomys torridus, often called “scorpion mice”) routinely eat bark scorpions because the Nav1.8 channels on nociceptors actually are inhibited by the scorpion neurotoxins, which thus produce analgesia instead of intense pain (Rowe et al., 2013; Thompson, 2018). Inhibition of Kþ channels Multiple classes of þ K channels exist on nearly all cells, but the ones targeted most often by neurotoxic zootoxins are voltage-gated (Kv) proteins (Figure 8.14, mechanism 4). In excitable cells like neurons, delayed inward flow of Kþ shapes the form of the action potential. Binding of some cone snail, centipede, spider, and snake toxins blocks Kþ channel function (Cardoso and Lewis, 2019; Hakim et al., 2015; Rash and Hodgson, 2002; Watkins, 2013). For example, k-conotoxins from cone snails, apamin (Figure 8.13A) from bees, the noncatalytic B-chain of b-BTX from kraits, and dendrotoxin from mamba snakes inhibit various subtypes of Kv channels. The effect of such neurotoxins is to slow repolarization, thereby enhancing and/or sustaining the release of ACh at synapses (Lahiani et al., 2017). Inhibition of Ca2þ channels Multiple classes of Ca2þ channels exist on various cell types, including both ligand-gated (CaL) and voltagegated (Cav) variants. In a resting state, Ca2þ channels of the presynaptic membrane are

II. SELECTED TOXICANT CLASSES

592

8. ANIMAL TOXINS

closed, but arrival of an action potential results in membrane depolarization and channel activation. The rapid influx of Ca2þ ions incites a series of events that lead to synaptic vesicle movement to the plasma membrane, fusion, and ACh release. A number of channel subtypes (mainly Cav) are found on various populations of excitable cells (neurons, some glia, and myocytes), but the one targeted most often by neurotoxins is the N-type Cav channel (Figure 8.14, mechanism 5). Many cone snail, centipede, spider, and snake toxins interact with various Cav channel subtypes (Bickler, 2020; Cardoso and Lewis, 2019; Hakim et al., 2015; Rash and Hodgson, 2002; Watkins, 2013). Inhibition of Cav channels in axon terminals typically results from direct antagonism of channel function by the toxin. Some neurotoxins with this effect are u-conotoxins made by cone snails, some salivary peptides in ticks (Chand et al., 2016), and u-agatoxins produced by funnel web spiders. Ziconotide (conotoxin uMVIIA) is a U.S. Food and Drug Administration (FDA)-approved nonopioid analgesic for treating chronic neuropathic pain due to its potent inhibitory activity against N-type Cav channels (Rigo et al., 2013). An important ligand-gated portal for Ca2þ entry on nociceptive (mainly types Ad and C) nerve fibers is the transient receptor potential vanilloid 1 (TRPV1) channel. This nonselective cation channel is a homotetramer that forms a central pore which passes Ca2þ preferentially over Naþ and Kþ. The TRPV1 channel is activated by many agonists including capsaicin and many toxins from centipede, scorpion, and spider venoms. Interestingly, activation by these agents and also antagonism by some sea anemone and spider toxins all induce analgesia, possibly by Ca2þ-dependent desensitization of the channel (Maatuf et al., 2019). Modified Presynaptic Receptor Activity Presynaptic nicotinic ACh receptors (nAChRs) (Figure 8.14, mechanism 6) modify the release of ACh at the NMJ and many other neurotransmitters at synapses in the CNS. Binding of ACh to presynaptic nAChRs mobilizes a reserve pool of synaptic vesicles into the pool ready for release, thereby providing a means to stabilize neurotransmitter concentrations within the synapse. This property renders signal

transmission more dependable. The presynaptic nAChRs act as ionotropic (i.e., ion-transporting) autoreceptors at the NMJ where binding of ACh leads to a conformational change in the pentameric protein so that a central channel is opened to permit passage of cations (Naþ and Kþ, and in some cases Ca2þ). As noted above, an influx of Naþ or Ca2þ ions leads to synaptic vesicle fusion and ACh release at the NMJ. Presynaptic nAChRs are rarely impacted by neurotoxic zootoxins. The single example seems to be candoxin, an unconventional 3FTx from the venom of the Malayan krait (Bungarus candidus). Activation of presynaptic nAChRs by this toxin provides positive feedback that allows the nerve terminal to appropriately sustain ACh release in response to action potentials that arrive during periods of high-frequency axon terminal stimulation (Nirthanan et al., 2003). Unlike many neurotoxins in venoms, receptor stimulation by candoxin is reversible. The reason is unknown but may be related to amino acid substitutions that limit its receptor binding affinity. Some neurotoxic zootoxins act via G-proteincoupled receptors (GPCRs). Many appear to be 3FTx with high specificity for their targets (Na¨reoja and Na¨sman, 2012), which often are muscarinic ACh receptors (mAChRs) on presynaptic terminals in the parasympathetic PNS and in various CNS nuclei (Lahiani et al., 2017). Activation of metabotropic (i.e., second messengergenerating) mAChRs produces molecules such as cyclic adenosine monophosphate (cAMP) and inositol triphosphate (IP3) that initiate intracellular signaling cascades controlling many cell processes. Prototypic neurotoxins directed against mAChRs are the muscarinic toxins (MTs), which make up 50% or more of the toxins in venoms of black mamba and green mamba snakes. The mAChRs targeted by MTs control contraction of cardiac muscle and also smooth muscle in the iris, small intestine, and urinary bladder. DISRUPTION OF AXON TERMINAL MEMBRANES

Many neurotoxic zootoxins induce their effects by impacting the integrity of membranes associated with the axon terminal (Ranawaka et al., 2013). Loss of membrane integrity in the synaptic vesicles and/or axon terminal plasma membrane leads to increased neurotransmitter

II. SELECTED TOXICANT CLASSES

3. ZOOTOXIN CLASSIFICATION

release and accelerated commitment, and eventual loss, of neurotransmitter reserves in the presynaptic terminal. The usual targets are the plasma membrane (Figure 8.14, mechanism 7) or synaptic vesicle membranes. Many of these lesions are associated with toxins that possess PLA2 activity, such as b-BTX (Dixon and Harris, 1999); crotoxin (Tzeng et al., 1995), a venom protein of the South American rattlesnake (Crotalus durissus); and taipoxin (Tzeng et al., 1995), a venom protein of the Australian coastal taipan (Oxyuranus scutellatus). A proposed mechanism for PLA2-mediated neurotoxicity is that the enzyme disrupts synaptic vesicle endocytosis (i.e., recycling after ACh release), leading to synaptic vesicle depletion in presynaptic terminals (Montecucco and Rossetto, 2000; Vardjan et al., 2013). In general, these toxins bind irreversibly and the affected axon terminals disintegrate, leading to an extended recovery (Dixon and Harris, 1999). Complete functional restoration depends on regrowth of the axon terminal to rebuild a viable NMJ. Latrotoxins (LTX) are the principal neurotoxins in the venoms of widow spiders. Most LTX are latroinsectotoxins that are active only against insect neural tissues, but a-LTX is aimed against vertebrate neurons (Yan and Wang, 2015). Homotetramers of a-LTX (assembled with the aid of helper proteins termed latrodectins) form large Ca2þ-permeable pores in presynaptic membranes. The incoming flood of Ca2þ induces a massive release of ACh and rapid depletion of synaptic vesicles (GwaltneyBrant et al., 2018); the ACh release is magnified by expulsion of the cytosolic ACh pools, which can pass out through the a-LTX pores. The potency of a-LTX varies among species; the LD50 is 0.0075 mg/kg for guinea pig and 0.9 mg/kg for mice (White et al., 2008). Cats are very sensitive to neurotoxicity, with many animals dying within 5 days from respiratory paralysis. Humans typically recover from intense muscle cramping, tremors, and dyspnea within 72 h, but some sequelae including weakness may persist for weeks or months (Gwaltney-Brant et al., 2018). ALTERED

ACETYLCHOLINESTERASE

ACTIVITY-

Acetylcholinesterase (AChE) is the enzyme within synapses responsible for removing ACh once neurotransmission has been successfully

593

passed from the presynaptic axon terminal to start a postsynaptic action potential. The ACh is cleared from the synapse by AChE and recycled by reuptake into the presynaptic terminal. Neurotoxic zootoxins may alter synaptic AChE activity in two ways (Figure 8.14, mechanism 8). Venoms of elapid snakes (except mambas) but not colubrid, crotalid, or viperid snakes are rich in AChE. In contrast, fasciculins are 3FTx from mamba venom that serve as reversible AChE inhibitors. All these toxins distort the rate of ACh removal, thereby thwarting effective neurotransmission. ABERRANT POSTSYNAPTIC RECEPTOR ACTIVATION

Signal transmission at the NMJ as well as CNS and PNS chemical synapses involves presynaptic exocytosis of neurotransmitters, diffusion across the synapse, and reversible binding to activate receptors on the postsynaptic membrane. At the NMJ, the receptors are ionotropic nAChRs. Receptor activation results in an influx of Naþ into the postsynaptic cell; a dense bed of Naþ channels surrounding the AChRs also supports an inrush of Naþ, which further amplifies the signal. The final outcome is motor endplate depolarization and myofiber contraction. Numerous neurotoxic zootoxins disrupt receptor activation (Figure 8.14, mechanism 9). In general, a-neurotoxins such as a-bungarotoxin in snake venoms (the prototypic a-neurotoxin) and pinnatoxins in contaminated shellfish (Figure 8.15; see Phycotoxins [Vol 3, Chap 5]) bind to one of two sites on nAChRs and keep ACh from docking. a-Neurotoxins function as competitive antagonists of ACh, exhibiting noncovalent and tight (i.e., high affinity) binding. The usual clinical outcome is weakness often leading to death by respiratory muscle paralysis. In contrast, histrionicotoxins (Burgermeister et al., 1977) in poison dart frog secretions function as noncompetitive antagonists at nAChRs to stabilize the receptor, thereby reducing the time the ion channel remains open. Crotoxin (a b-neurotoxin that mainly acts on presynaptic terminals) also is reported to lock nAChRs on the postsynaptic membrane in a desensitized state, and does so without interfering with binding of a-neurotoxins to the receptor (Bon et al., 1979). Some species are resistant to the effects of a-neurotoxins, including certain snakes (e.g.,

II. SELECTED TOXICANT CLASSES

594

8. ANIMAL TOXINS

fibers (Gwaltney-Brant et al., 2018; Krishnan et al., 2009). Some patients bitten by common kraits (Bungarus caeruleus) exhibit nerve conduction defects lasting from 2 weeks to 6 months, sometimes in combination with long-term polyneuropathy (Ranawaka et al., 2013). Interestingly, the NCV in axons is reduced by cardiotoxin (a potent myotoxin) but not cobrotoxin (an a-neurotoxin) in the venom of the Chinese cobra (Naja atra). PLA2 may potentiate the effects of cardiotoxin in this regard (Chang et al., 1972). Protoxin II (ProTx-II), an ICK peptide from venom of the Peruvian green velvet tarantula (Thrixopelma pruriens), inhibits action potential propagation in nociceptors by blocking the Nav1.7 channel (Schmalhofer et al., 2008). Other ICK peptides have a similar effect on impulse conduction. ALTERED MYOFIBER INTEGRITY AND FUNCTION

FIGURE 8.15 Crystal structure showing the interaction between pinnatoxin A (PnTx-A [yellow molecules]), a neurotoxin produced by microalgae (dinoflagellates) and bioconcentrated in shellfish, with sea slug (Aplysia californica) acetylcholine (ACh)binding protein (a pentameric complex with central channel used as a model system for nicotinic ACh receptors on neurons and myofibers). The five protein units are depicted in different colors to showcase binding of PnTx-A between units; binding inhibits channel function. Image reproduced from https://commons. wikimedia.org/wiki/File:4xhe_pinnatoxin_pentamer.png [based on original images in Bourne et al. (2015)] under a Creative Commons license (CC BY-SA 4.0).

Chinese cobra [Naja atra] and javelin sand boa [Eryx jaculus]) and the Egyptian mongoose (Herpestes ichneumon). The nAChRs of these species do not bind a-bungarotoxin because of amino acid substitutions that alter the conformation of the receptor, thus removing the toxin binding site while still permitting normal binding of ACh (Barchan et al., 1992). ALTERED ACTION POTENTIAL PROPAGATION

Some neurotoxic zootoxins have been implicated in disrupting conduction of impulses in axons. For example, weakness in individuals with tick paralysis has been linked to aberrant nerve fiber excitability. Nerve conduction velocity (NCV) and amplitude are reduced due to altered Naþ channel cycling in motor nerve

Some neurotoxic zootoxins act by disturbing electrical conduction in myofibers (Figure 8.14, mechanism 10). In many cases, myofibers represent an additional target site since the toxins often also cause changes in axon terminals, neurons, or glia. Myotoxic effects of neurotoxins are often mediated by altered ion channel activity. For instance, crotamine (a peptide comprising up to 25% of South American rattlesnake venom) is a structurally distinct nonenzymatic defensinlike molecule with neurotoxic and myotoxic activities (Kerkis et al., 2014). To elicit neurotoxicity, this toxin likely blocks some subtypes of Nav and Kv channels. For myotoxicity, the molecule is a cell-penetrating peptide that readily enters cells, where it serves as a necrotoxin by three related effects (Nascimento et al., 2012). First, crotamine liberates intracellular Ca2þ stores, thereby activating cytosolic proteases and launching enzyme cascades leading to programmed cell death. Second, the molecule disrupts lysosomal membranes, releasing more proteases into the cytosol. Finally, crotamine changes mitochondrial membrane integrity, thus quenching the membrane potential needed for energy production. Some scorpion toxins similarly function to increase Naþ influx into muscle cells (Karalliedde, 1995; Lin et al., 1975). Bufadienolides and similar steroidal alkaloids are cytotoxic zootoxins found in the skin secretions of many toads (Tian et al., 2010). Their

II. SELECTED TOXICANT CLASSES

4. CLINICAL PRESENTATIONS AND PATHOLOGIC MANIFESTATIONS

myotoxicity is driven primarily by their cardiac glycoside (i.e., digitalis-like) activity; high doses alter the ion gradients needed to sustain membrane potential in cardiac myocytes and neurons. The molecular target of cardiac glycosides is Naþ/Kþ-ATPase, the enzyme responsible for the exchange of key cations across the myofiber sarcolemma. The electrophysiological microenvironment is maintained by expending ATP to shuttle three Naþ ions outward and two Kþ ions inward against their electrochemical gradients. Digitalis-like glycosides inhibit the Naþ pump, thus raising the intracellular Naþ concentration. This shift in the Naþ gradient activates the Naþ/Ca2þ exchange, a sarcolemmal transmembrane protein that transports three Na þ ions outward for one Ca2þ ion inward. The resulting increase in the intracellular Ca2þ pool is stored in the sarcoplasmic reticulum. Myotoxicity produced by high cardiac glycoside doses releases a sufficient flood of Ca2þ ions to produce myofiber death. The bufadienolides in toad skin secretions are hallucinogens, confirming the neuroactivity of these zootoxins. Interestingly, bufadienolides in certain African plants induce chronic neurotoxicity, manifesting as a paretic/paralytic syndrome in ruminants and horses, known colloquially as “krimpsiekte” (Henn et al., 2019). Affected animals exhibit muscular twitching, staggering, and a characteristic stance (feet together with back arched) clinically, with myocardial degeneration or necrosis upon histopathologic evaluation (van Tonder et al., not stated). To our knowledge, a comparable neurotoxic condition is not induced by exposure to animal-derived bufadienolides, presumably due to the lack of chronic ingestion. Some zootoxins with combined myotoxic and neurotoxic activities act via GPCRs. For example, many frog, Komodo dragon, and snake venoms contain one or more AVIT proteins, named for the four conserved amino acids (A-V-I-T) located at the amino terminus of the molecules (Fry et al., 2009b; Kaser et al., 2003). Binding of some zootoxic AVIT proteins occurs with at least 10-fold higher affinity compared to endogenous mammalian ligands (Kaser et al., 2003). Receptor activation by AVIT zootoxins stimulates intestinal smooth muscle contraction and causes hyperalgesia. This combination of effects evolved as an additional means of immobilizing prey animals.

595

4. CLINICAL PRESENTATIONS AND PATHOLOGIC MANIFESTATIONS OF ZOOTOXIN-MEDIATED DISEASES Clinically significant zootoxins affect many vital organs. Their toxic effects often reflect extensive local damage associated with the actual site at which a poison or venom was applied, but in some cases damage follows widespread dispersion of the zootoxins in the blood. Major targets of zootoxins are blood vessels (including relevant hemodynamic/hemostatic processes), epithelial (cutaneous and mucosal) surfaces, kidney, muscle (cardiac and skeletal), and the nervous system. Many zootoxins attack more than one of these targets at the same time.

4.1. Blood Vessels and Blood Components Many zootoxins are potent hemotoxins, where hemotoxin is defined broadly as an agent that alters blood flow (hemodynamics), destroys red blood cells (hemolysis), disrupts hemostasis (i.e., a coagulotoxin), or injures blood vessel walls (i.e., a vasculotoxin). Many venoms produce several or all these effects due to their complex mixtures of hemotoxins. Hemodynamic alterations often result from changes in vascular wall tone for small to midsized muscular arteries or veins. The usual effect is hypotension due to vasodilation, which may present as shock clinically. The shock is of the “cardiogenic” type (i.e., distributive shock due to insufficient blood circulation) since the main failure is in vascular tone, but the vascular-centered effects may be complicated by superimposed “hemorrhagic” and “inflammatory” types of shock depending on the severity of lesions at the envenomation site. Shock leads to generalized hypoxia, so clinical abnormalities include signs of cardiac collapse (e.g., arrhythmias, delayed capillary reperfusion, increased heart rate, reduced blood pressure); neurological distress (e.g., agitation, confusion); and renal failure (e.g., oliguria). The skin will be cold, clammy, and pale. Metabolic acidosis may accompany shock; gross and histopathologic findings are inconsistent. Zootoxins that induce hemodynamic alterations include small molecules like adenosine (Aird, 2002), peptides such as bradykinin-potentiating peptides

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(Pe´terfi et al., 2019) and dendroaspis natriuretic peptide (Munawar et al., 2018), proteins like the 3FTx calciseptine (Pe´terfi et al., 2019) and the serine protease dipeptidyl peptidase-4 (Aird, 2002), and signaling molecules (e.g., eicosanoids) released by PLA2-mediated membrane hydrolysis. Hemodynamic effects are especially common following exposure to enzyme-rich zootoxin mixtures (e.g., bee, wasp, and snake venoms). Some venom zootoxins instead produce hypertension, which can enhance blood loss into tissues and will increase damage to red blood cells as they squeeze by the jagged edges of any microthrombi within vessels. Examples include venom catecholamines and sarafotoxins (Mourier et al., 2012). Hemolytic crises related to envenomation typically originate from cytolytic zootoxins that attack red blood cell membranes. Among such factors, PLA2 is a major culprit (Arce-Bejarano et al., 2014), so hemolytic episodes are common following envenomation by brown recluse spiders and many snakes. Older red blood cells are more susceptible to PLA2 (Jiang et al., 1989). Pronounced hemolysis often is accompanied by a precipitous decline in the hematocrit, distorted erythrocyte morphology in blood smears (e.g., schistocytes [red blood cell fragments]; Figure 8.16), hemoglobinemia, and hemoglobinuria (Arnold et al., 2017). In most cases, hemolysis does not contribute greatly to morbidity but rather serves as additional evidence with regard to the venom composition. Hemostatic disruption is a common effect of animal-derived coagulotoxins (Debono et al., 2019b; Youngman et al., 2019). Zootoxins may have anticoagulant or procoagulant effects, and venoms often contain constituents that enact both of these processes simultaneously. Most zootoxins that affect the coagulation cascade are enzymes (e.g., PLA2, metalloproteinases, serine proteinases) or small nonenzymatic peptides (e.g., soluble binding proteins that sequester clotting factors). In general, molecular mechanisms involved in these competing outcomes depend on dysregulated activation and deactivation of coagulation factors that catabolize formation or removal of fibrin (Grashof et al., 2020; Kini, 2006; Kvist et al., 2013; Lo¨vgren, 2013; Tans and Rosing, 2001). Some venoms induce a pseudo-procoagulant state in which generation of abnormal fibrin does not

FIGURE 8.16 Hemolytic anemia leading to formation of schistocytes (fragmented erythrocytes [arrows]) in a blood smear of a human patient bitten by a Russell’s viper (Daboia russelii). Image reproduced from Rojnuckarin P, Suteparak S, Sibunruang S: Diagnosis and management of venomous snakebites in Southeast Asia, Asian Biomed 6, 795–805, 2012, by permission of the journal Asian Biomedicine.

stabilize the platelet aggregates, thereby resulting in continued cascade activity, depletion of coagulation factors, and a de facto anticoagulant microenvironment (Seneci et al., 2021). Disseminated intravascular coagulation (DIC) has been reported with bee stings (Cowell et al., 1991) as well as bites of some spiders (Dodd-Butera and Broderick, 2014) and snakes (Rojnuckarin et al., 2012). Patients with coagulopathies generally present clinically with clear evidence of abnormal hemostasis including visible hemorrhage and diminished clotting capabilities. Clinical pathology findings indicative of coagulopathy include increased clotting times (e.g., activated clotting time [ACT], activated partial thromboplastin time [APTT], prothrombin time [PT], 20-minute whole blood clotting test [20WBCT, Figure 8.17]) and the presence of substances indicative of disordered clotting (e.g., extensive fibrin degradation products [FDPs] in plasma) (Clinical Pathology in Nonclinical Toxicity Testing [Vol 1, Chap 10]). Microscopically, thrombi sometimes will be seen in blood vessels, particularly microvessels at the envenomation site as well as organs with dense (e.g., lung) or end-arterial (e.g., glomeruli in kidney) capillary beds. Damage to microvascular walls is a common effect of vasculotoxins. The effects may be purely

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FIGURE 8.17 Results from a 20-min whole blood clotting test (20WBCT) demonstrating the absence of clotting characteristic of a venom-induced coagulopathy (lower tube) in a snakebite victim. The control sample (upper tube) has formed a stable, semisolid clot. Image reproduced from Rojnuckarin P, Suteparak S, Sibunruang S: Diagnosis and management of venomous snakebites in Southeast Asia, Asian Biomed 6, 795–805, 2012, by permission of the journal Asian Biomedicine.

functional, arising as a consequence of increased permeability due to vasoactive amines present in venom and/or discharged from mast cell granules. Classic zootoxins responsible for acute changes in permeability include PLA2 (via membrane hydrolysis to produce leukotriene and prostaglandin mediators) and mellitin in bee venom as well as adenosine, biogenic amines (histamine and serotonin), and cardiotoxins in snake venoms (Miller and Tu, 1989). Minor envenomation yields acute (within minutes) local swelling (a “wheal”) centered on the injection site; the affected tissue is often pale due to abundant extravasated plasma in the absence of hemorrhage (Figure 8.18). Individuals with a preexisting allergy to a venom component (often PLA2) develop more extensive swelling with skin reddening (a “flare”) due to IgE-dependent Type I hypersensitivity (Figure 8.19). The flood of tissue fluid dilutes the venom components, and in the absence of additional insults normal vascular permeability is restored and the extra fluid is removed via the lymphatic vessels. Structural damage to blood vessels is characterized by endothelial cell and/or mural injury leading to altered cell integrity and typically will produce local hemorrhage. The coagulation cascade may be activated if endothelial cell

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FIGURE 8.18 A honeybee envenomation site showing acute formation of a localized, raised focus of dermal edema (“wheal”) around a central wound. The sting occurred 6 min earlier. Image reproduced from https:// commons.wikimedia.org/wiki/File:Bienenstich_25a.jpg under a Creative Commons license (CC BY-SA 3.0).

FIGURE 8.19 A wasp (yellow jacket [Vespula maculifrons]) envenomation site showing extensive regional edema with reddening (“flare”) of one hand and forearm after a single sting 6 h earlier. Image taken by Alan Sirulnikoff (http://www.homeplanet.ca/sirulnikoff/) and reproduced from https://www.sciencephoto.com/media/ 263843/view by license from the Science Photo Library.

injury alters the balance between endothelial cell–derived anticoagulant and procoagulant signaling and/or mural damage exposes tissue factor (also known as coagulation factor III) in the perivascular connective tissue (Yau et al.,

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bilirubin (yellow). If tissue necrosis does not develop, resolution of hemorrhage and removal of extravasated plasma may occur with minimal residual tissue damage as long as blood vessel walls are resealed correctly and perfusion is restored to the region.

4.2. Epithelium (Cutaneous and Mucosal Surfaces)

FIGURE 8.20 Extensive resolving hemorrhage (indicated by yellow-green pigment) due to a bite on the thumb by an unspecified viper that took place 5 days previously. Image reproduced from https://commons. wikimedia.org/wiki/File:The_consequences_of_a_viper_bite5day.jpg under a Creative Commons license (CC BY-SA 3.0).

2015). Injuries to blood vessels are driven more by venom metalloproteinases than PLA2 (Rucavado et al., 2018). Introduction of venoms with larger volumes and/or more necrotoxic zootoxins, as often occurs during bites by crotalid or viperid snakes, may elicit prominent hemorrhage that extends for a considerable distance away from the envenomation site. For example, snakebites on a digit may produce damage that extends partway up or along the entire length of the affected limb (Figure 8.20). Shortly after the bite the affected tissue is red (due to acute hemorrhage) and swollen (due to enhanced vascular permeability). Over time, degradation of the hemorrhage leads to discoloration of the affected tissue ranging from purple to green to yellow. The shift in color reflects conversion of oxyhemoglobin (oxygen-bearing hemoglobin) in acute hemorrhage (red) to deoxyhemoglobin (purple). Subsequently, recycling of red blood cell constituents leads to production of biliverdin (green) and finally unconjugated

Body surfaces, especially the epidermis and dermis of the skin as well as the mucosa and submucosa of the oral cavity (and to a lesser extent the upper alimentary tract), frequently are brought into contact with animal-derived poisons and venoms. Encounters may involve simple contact (e.g., hemolymph from blister beetles or skin secretions from poison dart frogs applied to the skin) or introduction via wounds (e.g., ant bites followed by venom spraying, insect and scorpion stings, spider and snake bites). The extent of epithelial involvement typically is defined by the depth of penetration and dose. The skin receives more exposures due to its greater surface area and relationship to usual sites for bites and stings, but the oral mucosa also is a frequent location for exposure for animals that forage for food. Other mucosal surfaces (e.g., conjunctiva) may be exposed in exceptional cases. Certain zootoxins of arthropods are efficient epithelial cytotoxins. Cantharidin in blister beetle hemolymph is a potent caustic agent. Application of cantharidin to cutaneous or mucosal surfaces, or passage of the unmetabolized blood-borne molecule through nephrons and into the urinary bladder, leads to epithelial necrosis. Fire ant venom induces local necrosis (including hemolysis) and leads to production of painful, sterile pustules (Figure 8.21) using a mixture of formic acid (Figure 8.10) and many cytolytic alkaloids. The alkaloids also induce the local release of histamine due to lysis of mast cells (Lind, 1982). The brown recluse spider is renowned for its ability to produce cutaneous necrosis at the site of envenomation. Clinically, the initial lesion in loxoscelism may be a localized rash, sometimes with visible fang marks within the central portion of the affected region (Figure 8.22A). Over several days, the affected tissue becomes necrotic and

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FIGURE 8.21 Sterile pustules on the epidermis of the lower leg resulting from the swarming attack of a fire ant (Solenopsis spp.) colony that happened 3 days earlier. Image taken by Daniel Wojcik and reproduced from https://commons.wikimedia.org/wiki/File:FireAntBite.jpg as content in the public domain, produced by a government employee of the United States.

often sloughs, leaving a deep ulcer in which the dermis or subcutis is exposed; in exceptional cases, skin may be lost for a considerable distance including the entire covering of the affected appendage. The size and depth of necrotic zones is related to the dose of brown recluse venom (McGlasson et al., 2007). Restoration of the necrotic zone usually involves secondary (“second intention”) healing, with formation of an eschar (thick, dark scab [Figure 8.22B]) over a bed of granulation tissue at the wound base with progressive reepithelialization or cicatrix (scar) formation to close the surface defect. Microscopically, early lesions exhibit coagulative necrosis of epithelial cells in the epidermis and adnexae (glands and hair follicles [Figure 8.23]) while the dermis is edematous and has a modest influx of leukocytes (mainly neutrophils). In general, microscopic evaluation of late-stage lesions is not warranted due to grossly evident necrosis. Snake venoms are effective dermotoxins, acting chiefly to damage the connective tissues of the dermis and subcutis through deeper deposition of venoms. Major changes include locally extensive edema, hemorrhage, and necrosis (see Figure 8.24), especially exposure to necrotoxic

FIGURE 8.22 Evolution of cutaneous lesions induced by brown recluse spider (Loxosceles reclusa) venom. A: One day after envenomation, the epidermis surrounding the fang marks (twin dark spots inside the oval) exhibits a rash characterized by coalescing raised, flat, reddened papules. B: Two months after envenomation, a dark eschar covers a deep ulcer lined by necrotic tissue (indicated by the thin yellow-green rim at the bottom of the ulcer). Image A reproduced from Fegley et al. (2016) as hosted at https://www.ijam-web.org/ viewimage.asp?img¼IntJAcadMed_2016_2_2_256_196867_ f1.jpg by permission of Wolters Kluwer. Image B reproduced from https://commons.wikimedia.org/wiki/File:Necrotic_leg_ wound.png under a Creative Commons license (CC BY 2.0).

(cytolytic) venoms of crotalids and viperids. Lesions are driven not only by direct damage induced by the zootoxins but also by concomitant microvascular effects (e.g., ischemia related to vasoconstriction or vasodilation with systemic

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FIGURE 8.24 Extensive hemorrhage (red) and necrosis (brown and black) of the subcutis and skeletal muscles of a 13-year-old boy bitten on the palm by a 1.5meter-long rattlesnake about 24 h earlier. The wound on the palm is a consequence of the bite, while that on the forearm represents an emergency fasciotomy to relieve extreme pressure caused by the severe regional edema and hemorrhage. Image reproduced by courtesy of Mr. Justin Schwartz from http://www.rattlesnakebite.org/ rattlesnakepics-htm/.

4.3. Kidney

FIGURE 8.23 Histopathologic appearance of skin at a brown recluse spider (Loxosceles reclusa) envenomation site. Extensive acute coagulation necrosis with detachment of the epidermis (Panel A) and deep hair follicles (outlined in Panel B by arrows) associated with widespread dermal edema (increased interstitial space) and infiltrating eosinophils (denoted in Panel A by arrows). H&E. Images reproduced from Machado et al. (2009) under a Creative Commons license (CC BY 4.0).

hypotension) and to a lesser degree inflammation (Laing et al., 2003). Skin necrosis may be sufficiently extensive that surgery (serial debridement and reconstructive surgery) is required to repair large defects (in 60% of cases (Liu et al., 2020)). Gangrene of skin and underlying tissue develops in approximately 8% of victims regardless of whether antivenom is administered (Chotenimitkhun and Rojnuckarin, 2008).

Zootoxins in poisons (e.g., cantharidin) and venoms (unknown constituents) can severely impact renal function. Indeed, acute kidney injury (AKI) resulting in renal failure is a leading cause of death for victims who survive the early phases of snake envenomation (Albuquerque et al., 2013). For snakebites, AKI develops in 2%–50% of survivors depending on the species (Chugh, 1989; Rodrigues Sgrignolli et al., 2011). Renal damage is more likely to develop if the venom dose is high (e.g., multiple insect stings (Yanagawa et al., 2007) or a large snake). The clinical signs and pathologic findings are comparable to those induced by many toxic agents that elicit kidney injury (see Kidney [Vol 5, Chap 2]). The initial clinical picture is related to damage at the envenomation site (e.g., cutaneous and muscle hemorrhage and necrosis) and disruption of key physiological processes (e.g., hemodynamic and hemostatic disruption and/or hemolysis). Renal involvement appears later as oliguria along with various clinical chemistry abnormalities including azotemia (i.e., accumulation of nitrogen-containing waste products like creatinine and urea nitrogen), proteinuria, and hematuria (Albuquerque et al.,

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2013; Priyamvada et al., 2016; Rodrigues Sgrignolli et al., 2011). Both the glomerular filtration rate and urinary output are decreased while the renal vascular resistance is increased (Rodrigues Sgrignolli et al., 2011; Sitprija and Suteparak, 2008). The main zootoxin-related microscopic lesions in affected kidneys are acute tubular necrosis and microvascular congestion (Charoenpitakchai et al., 2018; Chugh, 1989; Rodrigues Sgrignolli et al., 2011). Uncommon renal findings include focal or multifocal cortical necrosis, acute interstitial nephritis, and papillary necrosis. Chronic kidney injury may occur in long-term survivors. In such cases, the typical histopathologic findings seen in renal biopsies are glomerular sclerosis, interstitial lymphocytic infiltration, and tubular atrophy (Herath et al., 2012). A multifactorial pathogenesis has been proposed for zootoxin-induced renal injury, but the contributions of the factors differ with the zootoxin or toxic mixture (Tasoulis and Isbister, 2017). Some molecules, including cantharidin and some venom components, appear to function mainly by direct toxic effects on renal tissue since they are excreted unchanged (Karras et al., 1996; Rodrigues Sgrignolli et al., 2011). Frequently, renal damage is complicated by or originates from hemotoxic effects such as systemic vasodilation, leading to hypotension and renal ischemia, and/or activation of the coagulation cascade, resulting in thrombotic microangiopathy and reduced capillary (including glomerular) perfusion (Gutie´rrez et al., 2009; Sitprija, 2006). Increased levels of reactive oxygen species has been proposed as one cytotoxic mechanism responsible for renal injury (Tohamy et al., 2014). Venoms that produce hemolysis and myonecrosis will result in hemoglobinuria and myoglobinuria, and excretion of these pigments can exacerbate damage to renal tubular epithelium (Chugh, 1989).

4.4. Liver Zootoxins in venoms often induce liver damage. Hepatocytes and endothelial cells are the target cell populations, but the clinical and pathological presentations generally are related to damage inflicted on hepatocytes. Occasionally, acute liver damage may be so severe that the organ will fail.

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Species associated with this condition include hymenopterans (Kolecki, 1999; Watemberg et al., 1995), scorpions (el Nasr et al., 1992; Karalliedde, 1995), spiders (Zambrano et al., 2005), and snakes (Al-Quraishy et al., 2014; Tohamy et al., 2014). As with kidney, the likelihood of venom-related liver damage depends on the dose (Kolecki, 1999; Watemberg et al., 1995). Clinical signs and pathologic findings of venominduced liver injury are similar to those produced by many other toxic agents that cause hepatic damage (see Liver and Gall Bladder [Vol 4, Chap 2]). Clinically, the primary diagnostic features of acute liver injury include icterus (“jaundice”) coupled with elevated serum activities for multiple hepatocyte enzymes (e.g., alanine [ALT] and aspartate [AST] aminotransferases, alkaline phosphatase [ALP], and/or GGT) (AlQuraishy et al., 2014; Jarrar, 2011). Serum total bilirubin (i.e., the yellow pigment that produces icterus) will rise due to accumulation of unconjugated bilirubin; hepatic damage rather than hemolysis is confirmed as the likely cause by the heightened serum enzyme activities and demonstration of few or no damaged red blood cells in blood smears. Histopathological changes induced in liver include hepatocyte degeneration (associated with variable fat accumulation and vacuolation) and necrosis, activation (i.e., enlargement and variable proliferation) of sinusoidal macrophages (Kupffer cells), sinusoidal dilation and congestion, and thrombosis (Jarrar, 2011; Tohamy et al., 2014). Parenchymal inflammation varies from modest to substantial. Increased oxidative stress in conjunction with diminished capacity to detoxify reactive oxygen species has been proposed as a cytotoxic mechanism for venom-related hepatotoxicity (Al-Quraishy et al., 2014; Tohamy et al., 2014).

4.5. Lung Zootoxins in venoms are capable of inducing pulmonary injury. The target sites within the lung include airways and blood vessels; the main vulnerable cells are microvascular endothelium and smooth muscle myocytes. Effects of envenomation depend on the species, with elapid venoms impacting epithelial cells primarily while crotalid and viperid venoms also act by disrupting the coagulation cascade (Gnanathasan and Rodrigo, 2014). Pulmonary

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effects also may be induced by acute scorpion envenomation (Bahloul et al., 2013) as well as venoms of jellyfish and many hymenopterans. Four clinical presentations involving envenomation may induce life-threatening disruption of pulmonary function. All involve mechanisms at least partly unrelated to parenchymal injury. The first is anaphylaxis. This Type I hypersensitivity response is driven by IgE antibodies and pro-inflammatory mediators (e.g., histamine). The classic antigen is PLA2 in bee venom, and a single sting is sufficient to invoke a lethal allergic response. Respiratory distress reflects edema (especially of the larynx, but sometimes of pulmonary alveoli) and/or bronchial constriction. The second presentation is venom-induced interference with the coagulation cascade. Pulmonary manifestations of coagulopathies include hemorrhage and microthrombosis. A third presentation is widespread pulmonary edema, which is seen in routine hematoxylin and eosin (H&E)-stained tissue sections as increased interstitial space and/or eosinophilic (protein-rich) material in alveoli. This effect results from direct damage to pulmonary endothelium or as an indirect consequence of increased intravascular pressure due to acute left-sided heart failure (Bahloul et al., 2013; Deshpande and Akella, 2012). Other microscopic evidence of acute venom-induced direct pulmonary injury includes capillary congestion, multifocal hemorrhage, and inflammation (neutrophils) (Azevedo et al., 2020). The final presentation is respiratory failure arising from generalized neuromuscular paralysis. This condition reflects neuromuscular blockade at the NMJ. The lung parenchyma is not involved in this latter situation. Several mechanisms have been proposed for pulmonary edema following venom exposure. This effect usually is attributed to acute cardiac (left ventricular) failure produced by primary myocardial injury and/or massive catecholamine release associated with severe pain (Bahloul et al., 2013). However, two noncardiogenic factors have been suggested to be instrumental in producing the main lung effects of edema and inflammation. First, induction of inflammation has been credited to generation of pro-inflammatory mediators by either venom-activated alveolar macrophages (Reis et al., 2020) or hypertension-damaged

pulmonary endothelial cells (Deshpande and Akella, 2012). Second, hemodynamic alterations leading to microvascular hypertension have been proposed to reflect an “autonomic storm” (Deshpande and Akella, 2012) where the “storm” represents an acute stress-related PNS response arising from massive catecholamine release in combination with a flood of cortisol (Rabinstein and Wijdicks, 2004). Once edema is present in alveoli, envenomation has been demonstrated to slow the rate at which edema fluid is cleared, potentially by inhibiting the activity Naþ/Kþ-ATPase in the alveolar epithelium (Comellas et al., 2003).

4.6. Muscle (Cardiac and Skeletal) Necrotoxic zootoxins, particularly those of snake venoms, may damage striated (cardiac and/or skeletal) muscle (Ferraz et al., 2019). This capability is seen in some elapid species (e.g., cobras, sea snakes) but is the classic effect induced by crotalid and viperid bites. The most potent myotoxins in venoms are PLA2 isoforms (Tonello and Rigoni, 2017; H. Xiao et al., 2017) and small single-chain peptides like cardiotoxins (Mebs and Ownby, 1990). Zootoxins may induce toxicity to cardiac myocytes (e.g., cantharidin (Zhang et al., 2020)), skeletal myocytes (e.g., sea snake venom (Neale et al., 2018)), or both. Some venoms do impact smooth muscle function (e.g., typically by inducing hyperactivity [“cramping”] of intestinal loops and vasoconstriction) but have not been associated with microscopic evidence of structural damage to smooth muscle myocytes (Fry et al., 2009b; Singh and Sanyal, 1965). When present, clinical signs and pathologic findings of venom-induced muscle injury are analogous to those induced by other cytotoxic molecules that injure myocytes (see Muscle and Tendon [Vol 4, Chap 4]). Local myalgia (i.e., pain without being touched) and tenderness (i.e., pain when touched) at the envenomation site is common, and 10%–15% of patients develop generalized myalgia or tenderness (Silva et al., 2016). Myocyte damage may be detected using serum chemistry analysis of blood, where disrupted sarcolemmal membrane integrity will be indicated by increased activity of AST, CK, LDH, and/or elevated concentrations of one or more troponins; determination

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of CK and troponin isoforms may be needed to definitively state that damage is present in cardiac myocytes, skeletal myocytes, or both (see Clinical Pathology in Nonclinical Toxicity Testing [Vol 1, Chap 10]). Macroscopically, necrosis may be seen as dark (often purple– black due to prior hemorrhage [Figure 8.24]) or pale discoloration in affected muscles. Corresponding microscopic attributes include myocyte necrosis with hemorrhage and inflammation (Figure 8.25). The amount and character of the hemorrhage, myofiber necrosis, and inflammation depend on the age of the envenomation site (Charoenpitakchai et al., 2018). Muscle repair often is incomplete as venom enzymes catalyze the breakdown of laminin and type IV collagen in myocyte basement membranes early after envenomation, thus removing the scaffold needed to support myofiber regeneration (Escalante et al., 2021). Again, myotoxicity related to poisons and venoms has a multifactorial pathogenesis. Direct toxicity is a primary driver due to the vast battery of cytotoxic enzymes injected in most venoms. Cytolytic enzymes (e.g., PLA2) and peptides (e.g., cardiotoxins) may degrade

FIGURE 8.25 Histopathologic demonstration of acute edema, hemorrhage (H), and inflammation (I, consisting of numerous fibrin strands and neutrophils) in the interstitium accompanied by myofiber necrosis (N). Gastrocnemius muscle of a mouse 6 h after injection of 50 mg of terciopelo (Bothrops asper) snake venom. H&E. Image taken by Alexandra Rucavado and reproduced from Zuliani JP, Soares AM, Gutierrez JM: Polymorphonuclear neutrophil leukocytes in snakebite envenoming, Toxicon 187, 188–197, 2020 by permission of Elsevier.

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cell membranes. Low-molecular-weight peptides may activate Naþ channels in the sarcolemma, leading to a fatal disturbance in ionic gradients needed to sustain myocyte viability. Moreover, the outsized venom volumes delivered by large snakes flood the envenomation site with necrotoxins, thus initiating locally extensive necrosis. The degree of tissue death may be severe, especially if muscle mass at the bite site is minimal to start (Gwaltney-Brant et al., 2018). Concurrent necrosis and rupture of blood vessels, formation of intravascular thrombi, and/or vessel occlusion from pressure exerted by tissue edema and hemorrhage compound the deep tissue ischemia, thus enhancing the direct necrotoxic effects of cytolytic molecules.

4.7. Neuromuscular Junction and Other Peripheral Synapses Myriad animals produce potent neurotoxins, which are among the most powerful natural toxins in existence. Species armed with neurotoxins occur in all animal phyla, ranging from relatively simple invertebrates like cnidarians (e.g., jellyfish and sea anemones) and molluscs (e.g., cone snails) to more complex invertebrates like hymenopterans (e.g., ants, bees, and wasps) and arachnids (e.g., scorpions and spiders) to many chordates (e.g., frogs and snakes). As noted above, neurotoxic zootoxins may assault any part of the nervous system via one or more of many molecular handholds, but in general primary neural target sites are localized to PNS structures such as the NMJ, nociceptors, or synapses in autonomic ganglia. The clinical procedures used to evaluate neurotoxic agents are useful for examining the effects of neurotoxic zootoxins, but in general the pathology methods employed to examine CNS and PNS tissue (see Nervous System [Vol 4, Chap 8]) are unable to assess PNS synapses with sufficient sensitivity. Clinical presentations depend on the site of neurotoxin activity. In general, involvement of the NMJ produces weakness (sometimes with ataxia) that may progress to local or systemic paralysis (including death by respiratory failure if the thoracic skeletal muscles [e.g., diaphragm and intercostals] are affected); weakness and paralysis with

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respiratory complications is the single most important clinical manifestation of zootoxininduced neurotoxicity (Ranawaka et al., 2013). An important consideration clinically is that paralysis is a delayed response arising from irreversible NMJ damage and thus is not amenable to antivenom treatment. Activation of nociceptors leads to neurogenic pain that ranges in degree from mild to incapacitating. Altered neurotransmission at autonomic synapses may elicit parasympathetic signs such as bradycardia (slow heart rate), bronchial constriction, hypersalivation, and miosis (pupillary constriction). In general, neurotoxins acting at synapses do not produce lesions that will be detected with conventional anatomic pathology techniques such as light microscopic evaluation, even if special neurohistological procedures are employed. Transmission electron microscopy (TEM; see Special Techniques in Toxicologic Pathology [Vol 1, Chap 11]) can demonstrate changes in synaptic vesicle numbers as well as presynaptic and postsynaptic terminal integrity, but this technique is seldom used for diagnostic purposes. In general, no reliable clinical pathology markers are available for lesions in neural tissue. Intriguingly, however, routine measurement of serum glucose concentration may be informative for certain cases of zootoxin-induced neurotoxicity. For example, severe scorpion envenomation has been associated with hyperglycemia (i.e., increased serum glucose levels) in humans arising from an “autonomic storm.” This stressrelated response results from the massive simultaneous release of catecholamines and cortisol from the adrenal gland. In such cases, hyperglycemia has been linked to such severe sequelae as hemodynamic instability, pulmonary edema, and multisystem organ failure; serious neurological events include altered cognition and paralysis (often leading to respiratory failure) (Bahloul et al., 2018). Collectively, these signs foretell extended need for intensive medical care and an increased risk of death.

4.8. Systemic (Multi-Organ) Failure Simultaneous collapse of several organs is a rare complication of envenomation. The presence of cutaneous necrosis (or hemorrhage) produced by venoms of some wasp species

may occur together with multi-organ failure (Yanagawa et al., 2007). A similar effect has been described following honeybee (Prasad et al., 2020) and scorpion (Khan and Ullah, 2017) stings as well as snakebites (Abohassan et al., 2012). Organ failure typically strikes the kidneys and liver, and to a lesser degree the heart. This outcome seems to be a direct zootoxic effect rather than an allergic response as the clinical severity depends on the dose (number of envenomation sites or volume of venom).

5. DIAGNOSIS AND TREATMENT OF ZOOTOXIN-MEDIATED DISEASES In a practical sense, diagnostic techniques and treatments for zootoxins are grounded in supportive care to treat key signs and symptoms. This pragmatic emphasis is necessary since (1) the animal responsible for the poisoning or envenomation often cannot be identified definitively and (2) specific antivenoms are unavailable for many species. Detailed consideration of diagnostic criteria and treatment approaches to zootoxicoses are beyond the scope of this chapter. Interested readers may explore these topics elsewhere as they apply to medical (Gopalakrishnakone et al., 2015; Gopalakrishnakone et al., 2018; Klaassen, 2013; White et al., 2018) and veterinary medical (Gupta, 2018) zootoxic diseases. The current chapter introduces these subjects and highlights contributions of pathologists to diagnosis and treatment of zootoxic conditions. The section focuses on venoms since they are more likely to produce systemic effects, but the same concepts may be applied to zootoxic poisonings.

5.1. Diagnosis As noted above, the clinical presentation of local or systemic zootoxin exposures involves a range of signs and symptoms, which differ depending on the affected body part(s) and the phase (i.e., acute vs. subacute or chronic) of the disease. The features described previously will not be reiterated here.

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History and Physical Examination The mainstays of initial diagnosis for zootoxic poisoning or envenomation are a thorough patient history and comprehensive physical examination (Knudsen et al., 2021; Valenza et al., 2021). The history should include details regarding the incident such as the location (e.g., lush rural agricultural plantation vs. densely populated urban setting), time (e.g., ambient light levels, temperature), and any known poisonous or venomous species in the area. Ideally, the nature of the offending animal will be determined definitively by visual inspection of either the carcass or a photograph, but a well-crafted narrative description is sufficient if the details permit accurate identification. The length of time since the zootoxic exposure, any signs and symptoms that have developed, and the kinds and vigor of initial first aid should be communicated. The physical examination should begin with a general triage of the patient’s medical needs. Critical concerns like acute hemorrhage, shock, or convulsions may require immediate treatment to stabilize the patient before an in-depth examination can determine appropriate next steps for the therapeutic plan. When feasible, the affected site (e.g., poisoned skin region or envenomation site) should be assessed to determine the likely cause and predict possible sequelae. For example, the presence of fang marks in the center of a reddened skin surface punctuated by raised papules (Figure 8.22A) indicates that the injected venom has widely affected tissues in the region and suggests that substantial inflammation and/or necrosis may occur as the condition progresses. The function of key tissues and organs should be evaluated using routine procedures. Simple and rapid tests appropriate to field and hospital settings include collection of routine vital signs (e.g., pulse [at the wrist and/or ankle, depending on the envenomation site], heart and respiratory rates, blood pressure); auscultation to assess cardiac and pulmonary function; and a neurological examination to confirm mental status and probe neuromuscular and autonomic PNS function. Clinical signs of severe envenomation commonly include erythema (reddening), heat, swelling, and vesiculation (“blistering”) of the skin and hemorrhage of the oral mucosa (Rojnuckarin et al.,

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2012). Some patients develop compartment syndrome in which severe edema produces a marked rise in fascial pressure leading to circulatory impairment and ischemia. This syndrome presents with severe pain when the affected limb is moved or lightly touched. Field Tests In rural areas, the 20WBCT (Figure 8.17) is a simple, rapid, and effective bioassay for detecting clinically significant envenomation (Rojnuckarin et al., 2012; Theakston and Laing, 2014). A blood sample (1.5–2 mL) is aliquoted into a clean glass test tube, which is placed upright in a standard rack and left undisturbed for 20 min. The tube is tilted gently at the end of this incubation period. Normal coagulation is indicated by the presence of a solid clot with a top surface perpendicular to the sides of the test tube. Abnormal clotting (i.e., a venominduced coagulopathy) is confirmed if the blood shifts to flow along the side of the tube. The main disadvantages of this assay are that the sensitivity is low in the clinical setting (Isbister et al., 2013) and many snakes do not induce coagulopathies (e.g., elapids). Assessment of specific zootoxins has been proposed as a means for confirming systemic envenomation (Maduwage et al., 2014). For example, PLA2 is a component of numerous snake venoms, including those noted mainly for necrotoxic effects (e.g., crotalids and viperids) and those with neurotoxic properties (e.g., elapids). While this enzyme is not the principal zootoxin responsible for tissue damage in snake venoms, its presence in the circulation is evidence of a significant systemic envenomation. Serum PLA2 activity is a sensitive diagnostic test due to the very low levels of endogenous PLA2 activity (Maduwage et al., 2014). In addition, use of such tests during the course of treatment would provide a means of monitoring the success of any antivenom doses. Molecular Procedures Fluids (e.g., blood, effusions in aspirates from cutaneous vesicles or fang wounds, urine) or homogenized tissues (e.g., excised envenomation sites) may be used as samples for molecular assays to detect the presence of zootoxins. Such laboratory tests are critical both to confirm

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envenomation as well as guide further treatment. Molecular signatures indicating that the venom components originate from a given species offer the opportunity to provide targeted relief by administration of the correct antivenom (Theakston and Laing, 2014). This ability is essential if the identity of the venomous animal was not confirmed, multiple venomous species exist in the area where the patient was bitten, and available antivenom options are monovalent (i.e., targeted specifically against the zootoxin complement of a single venomous species). Modern molecular methods include enzyme immunoassays (EIAs) and enzyme-linked immunosorbent assay (ELISA). These techniques are designed to detect the presence of specific venom components (e.g., PLA2). Such procedures are relatively inexpensive, sensitive (e.g., limits of detection, 0.2–1 ng/mL (Maduwage et al., 2020; Theakston and Laing, 2014)), and specific. The primary disadvantage is the relatively slow readout (typically 2 or more hours). In general, validated molecular diagnostic kits for venom components are seldom available from commercial sources. Nonspecific ELISA kits to measure circulating cytokine levels (e.g., IL-1, IL-6, interferon gamma [INFg], TNF-a) may be informative in cases of systemic envenomation to demonstrate a generalized proinflammatory state (Sitprija and Suteparak, 2008). Pathology Procedures Conventional laboratory testing is an essential element of diagnostic testing for many poisonings and most envenomations (Knudsen et al., 2021). These tests are nonspecific but nonetheless extremely informative. CLINICAL PATHOLOGY

Conventional clinical pathology analysis is important (Clinical Pathology in Nonclinical Toxicity Testing [Vol 1, Chap 10]). The usual battery of initial tests for envenomation includes hematologic parameters (e.g., complete blood count [CBC], blood smear cytology) and analytes related to coagulation (e.g., ACT, APTT, PT, FDP); electrolyte balance (e.g., Naþ, Kþ, and chloride [Cl–] ion concentrations); and renal function tests (e.g., blood urea nitrogen [BUN], creatinine) (Hifumi et al., 2015; Valenza et al., 2021). Measurement of other analytes (e.g., ALT and

AST activities for liver injury, CK activity and troponin levels for muscle necrosis) may be assessed if clinical evidence of icterus or extensive muscle damage (e.g., large bite wounds or discolored urine) indicates these organs might be impacted. The characteristic clinical pathology profile in many systemic envenomation events by scorpions, spiders, and snakes is increased coagulation times with decreased platelet numbers (i.e., thrombocytopenia) (Rojnuckarin et al., 2012). This effect on coagulation varies with the venomous species; in snakes, the change is prominent for crotalid and viperid snakes and seldom present for elapid snakes. Decreased hematocrit and hemoglobin concentrations, intravascular hemolysis (with schistocytes on blood smears [Figure 8.16]), and DIC (indicated by reduced fibrinogen levels and/or FDP) may be present as well. Neutrophilia with a left shift (indicating release of immature myeloid cells from the bone marrow due to an overwhelming acute pro-inflammatory stimulus) has been reported as an effect of many snake venoms (Lomonte et al., 1993; Sano-Martins et al., 1995; Zuliani et al., 2020) while leukopenia and thrombocytopenia may occur due to rapid removal of these cells into the envenomation site (Tavares et al., 2004). Common nonspecific clinical chemistry findings indicative of severe envenomation include increased serum BUN and creatinine concentrations (indicating reduced renal function) and occasionally elevated serum activities of ALT and AST (if hepatocytes are damaged) or CK and troponins (if cardiac or skeletal muscle fibers are injured). Hemoglobinuria and/or myoglobinuria may be evident after multiple hours to a couple of days, with the late onset reflecting both the delay related to initial venom circulation and the reduced urinary output associated with acute kidney injury. Hyperkalemia (i.e., increased serum Kþ levels) with hyponatremia (i.e., decreased serum Naþ levels) have been described following some cnidarian or toad envenomations due to inhibited Naþ/Kþ ATPase activity in cell membranes. HISTOPATHOLOGY

Microscopic evaluation of excisional biopsy material typically is not needed to diagnose zootoxic poisonings and envenomations. If performed, features seen at the envenomation site

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in routine H&E-stained sections usually will include a combination of edema, hemorrhage, and necrosis. Microvascular thrombosis and neutrophilic inflammation may be observed depending on the degree of coagulopathy and extent of the tissue damage, respectively. Special histological stains are not necessary although in situ molecular procedures such as immunohistochemistry (IHC) to localize zootoxin-specific antigens may be employed to demonstrate the presence of toxins in tissue. Microscopic evaluation to demonstrate zootoxins is prone to falsenegative conclusions due to the small amounts of toxin in small tissue samples.

5.2. Treatment Therapy for zootoxic poisonings or envenomation in the clinical setting (whether medical or veterinary) is focused on supportive therapies rather than curative or prophylactic measures. In general, in most cases supportive therapy is emphasized due to uncertainties in identifying the venomous animal and the limited availability and high cost of curative treatments like antivenom (Junghanss and Bodio, 2006). Species-specific antivenom is the most effective treatment (Utkin, 2015). First Aid for Acute Exposures Most severe cases of envenomation occur in remote settings (e.g., agricultural fields or wilderness), so the goal of first aid is to retard venom absorption and circulation. Historically, many techniques have been proposed for this purpose (Avau et al., 2016). Deactivation has been attempted with cryotherapy, electroshocks, and traditional oral or topical medicines concocted of herbs, oils, and even raw or processed venoms. Eradication has been tried by incision, irrigation, and/or suction (by mouth or device) of the wound. If attempted, short parallel incisions should be used rather than a cruciate pair of cuts crossing the fang marks since this approach will incur less tissue damage. Suction may remove up to 50% of the venom from incised wounds but only if it started within 2 min of venom injection and continued for a minimum of 30–60 min (Karalliedde, 1995). These limitations have caused these first aid approaches to be shelved.

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Current practice is to reduce dispersion of the venom (Avau et al., 2016; Rojnuckarin et al., 2012). The time-tried practice of applying a tourniquet to completely stop blood flow has fallen from favor since amateurs often keep the ligature applied for so long that tissue starts to die from ischemia and not venom toxicity. Instead, recommended practice now is pressure immobilization of the limb by application of a bandage or sleeve with rigid splints that prevent joint movement. The pressure bandage blocks lymphatic drainage but allows sufficient blood flow to keep tissue alive. The immobilization limits movement of venom-laden blood and lymph into the rest of the body. The victim is then transported to a medical center with the envenomation site positioned in a dependent position (i.e., held below the level of the heart) so that entry of envenomed blood into the systemic circulation will be less efficient. Supportive (Nonspecific) Care for Acute Exposures Supportive care is applied sequentially as needed to minimize clinical disease and avoid serious complications. On initial hospitalization, many patients have few or no clinical signs and symptoms and exhibit little or no divergence from physiological norms using standard laboratory tests (Rojnuckarin et al., 2012). A fraction of patients exhibit life-threatening consequences upon arrival, so initial treatments are designed to restore normal circulatory and respiratory functions; common options are fluid therapy to restore blood volume, medications to raise vascular tone, and if necessary positive ventilation for 2–3 days to counter respiratory paralysis. Most patients are admitted for observation, with the length of the stay depending on the expected clinical signs. Neurotoxic venoms typically induce significant neurological signs within the first 12–24 h, while effects induced by coagulotoxic and necrotoxic venoms may occur for as long as 3 days due to the longer circulating half-lives of these latter two venom types (Rojnuckarin et al., 2012). Renal failure may be countered by “forced diuresis” using hydration and drugs to restore urine flow. In most cases, the fluid therapy contains electrolytes to sustain/restore ion gradients. Various drugs are used to treat major signs and symptoms of poisonings and envenomation

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(Rojnuckarin et al., 2012). Analgesics may be administered, in which case acetaminophen is preferred because it has less impact on coagulation relative to aspirin and other common nonsteroidal antiinflammatory drugs (NSAIDs, e.g., ibuprofen and naproxen). Additional agents may be given to maintain ACh concentrations in synapses (e.g., ACh esterase inhibitors) and correct hemodynamic alterations (Karalliedde, 1995). Antibiotics and steroids typically are not required. Routine wound care for deep envenomation sites (e.g., crotalid and viperid bites), such as thorough irrigation and administration of tetanus toxoid, may be warranted as supportive care rather than to directly counter the toxic effects of venom components. Tissue debridement is fairly common for envenomation sites where tissue necrosis and sloughing is extensive. This procedure is commonly done to prepare the wound site for healing and accelerate tissue restoration (Barss, 1984). In exceptional cases, heavily envenomed tissue is removed to limit the cumulative systemic zootoxin dose and subsequent damage imparted by the host inflammatory response (Fujioka et al., 2009).

example, neurological recovery may be delayed for weeks if fast-acting neurotoxins (e.g., krait venom) have destroyed presynaptic axon terminals because the entire terminal will need to be replaced and the NMJ will need to be reestablished correctly before full function is regained. In general, antivenom offers little protection against local necrosis due to the rapid action of proteolytic necrotoxins. The timing of antivenom infusions depends on the envenomation scenario. For snakes with potent neurotoxin arsenals (i.e., elapids), administration is begun immediately if venom is known to destroy axon terminals and induce chronic neurological deficits (e.g., kraits) but may be begun at the first appearance of clinical signs for species where structural damage is minimal or absent (e.g., cobras). For snakes with coagulotoxic venoms (i.e., crotalids and viperids), antivenom often is given once laboratory tests indicate that a substantial coagulopathy is developing. Antivenom treatment may be repeated for venoms that impact the coagulation cascade but are ineffectual for venoms that attack the nervous system. ADVERSE REACTIONS TO ANTIVENOM

Curative (Specific) Therapies and Their Complications Antivenom is the major specific treatment for envenomation (Rojnuckarin et al., 2012). These products are available for many snakes and some arachnid species (e.g., bark scorpions and brown recluse, funnel web, and widow spiders). Antivenom consists of antibodies, directed against various zootoxins, which have been generated by hyperimmunization of large animals with small doses of venom. Adverse reactions to antivenom infusion are a common complication of this treatment in human patients. ANTIVENOM THERAPY

Antivenom is administered by intravenous infusion over 0.5–2 h. In general, antivenom is effective at limiting systemic effects but not local effects at the envenomation site. Antivenom does not immediately correct all clinical signs and symptoms. The first reversal typically is observed in 6–12 h as the coagulopathy begins to recede (Rojnuckarin et al., 2012). Recovery may be prolonged for some target tissues. For

Antivenom is a protein therapy comprised of concentrated antibodies harvested from a hyperimmunized animal. Accordingly, many patients may react to administration of antivenom (Isbister et al., 2008; Rojnuckarin et al., 2012; Thiansookon and Rojnuckarin, 2008). Both acute and delayed reactions may be observed (de Silva et al., 2016). Acute (anaphylactic or pyrogenic), usually mild reactions occur within hours of antivenom infusion and are characterized by fever, chills, headache, nausea, rash or urticaria (i.e., raised, red, itchy rash), and sometimes bronchospasm. This acute response develops within hours of the antivenom infusion in 3%–80% of patients depending on the antivenom product. The proposed pathogeneses for acute antivenom reactions include complement activation mediated through the Fc domain of the foreign antibodies (Malasit et al., 1986) or formation of immunoglobulin complexes (Stone et al., 2013). Infrequently, the response also may include a more severe, IgE-dependent, Type I hypersensitivity reaction. The likelihood of an acute antivenom reaction depends on the product purity

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(processing) and protein content. Acute reactions have been treated empirically in two ways (de Silva et al., 2016). First, premedication protocols (e.g., antihistamines, epinephrine, and/or steroids) have been devised to attenuate the acute innate immune and smooth muscle responses. Second, onset of an acute reaction is managed by halting the antivenom infusion, managing airway patency, administering fluids, and injecting epinephrine. Completion of the antivenom infusion is performed at a slower rate with close monitoring in case additional epinephrine treatments are needed. Chronic (serum sickness) reactions characterized by urticaria with fever, arthralgia, myalgia, and/or thrombocytopenia develop in approximately 35% of patients between 1 and 3 weeks after antivenom infusion (Corrigan et al., 1978; de Silva et al., 2016). This response is attributed to circulating IgG-based immune complexes resulting in sustained complement activation. Current treatment is to administer a 1-week course of steroids with antihistamines and NSAIDs if warranted to control symptoms. Care for Chronic Complications Patients who survive zootoxic poisoning and envenomation may experience long-term sequelae that significantly impact their quality of life (Tasoulis and Isbister, 2017; Waiddyanatha et al., 2019). In general, these chronic effects are treated symptomatically. The most common long-term effect in tropical countries (especially in Africa and Asia) is disability related to effects at the envenomation site. Local pain and swelling may persist. For example, necrosis and tissue loss may arise from direct toxicity of venom or from secondary, untreated or undertreated bacterial infections. Disability may be driven by chronic ulceration, pronounced fibrosis leading to joint contracture, gangrene leading to necrosis of an entire limb, and ultimately amputation to remove the dead tissue. Rarely, the scar tissue at the envenomation site may undergo neoplastic transformation to a locally aggressive and potentially metastatic squamous cell carcinoma (i.e., a Marjolin “ulcer”) (Shah and Crane, 2021; Waiddyanatha et al., 2019). Blindness (venom-induced ophthalmia from spitting cobras) may develop due to chronic conjunctivitis and/or corneal ulcers leading to leukoma (i.e., white opacity of the cornea).

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Chronic effects away from the envenomation site also may develop. Acute tubular necrosis may progress to renal failure. Victims may experience persistent distress due to depression, posttraumatic stress disorder, or somatization disorder (i.e., presence of multiple, recurring symptoms with no discernible organic cause) (Bhaumik et al., 2020; Williams et al., 2011). In general, acute neurotoxic effects (e.g., paralysis) do not result in long-term adverse effects related to the NMJ or autonomic PNS. Prophylactic Measures Prevention or reduction in the severity of unavoidable zootoxicoses may be attempted by prior administration of toxoids. Inactivated but antigenically intact zootoxins are injected to generate an acquired immune response, and periodic boosters often are administered to ensure that immune memory remains robust. Toxoids historically have been manufactured against toxins of common diseases that are either highly communicable, frequently lethal, or both. For this reason, no toxoids against zootoxins are included in lists of common medicines (WHO, 2019a,b) or vaccinations (Medicine, 2011). That said, toxoids do exist and may be given to high-risk individuals (often working dogs). For example, Crotalus Atrox Toxoid is a commercially available veterinary vaccine given to dogs and horses to build immunity against venom of the western diamondback rattlesnake (Crotalus atrox) (Cates et al., 2015). The product also protects against venoms of several related species using a mixture of 15–20 toxic proteins. Annual boosters are recommended to sustain antibody levels. Fewer antibodies develop in response to vaccination compared to natural envenomation (Gilliam et al., 2013). Toxoids are produced against venoms of scorpions and other crotalid and viperid snakes (Aung et al., 1999; Bermu´dez-Me´ndez et al., 2018) but are not widely available.

6. REGULATORY GUIDANCE REGARDING ZOOTOXINS Zootoxins have been used widely as research tools for exploring basic biological processes (Munawar et al., 2018; Rivera-de-Torre et al.,

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2019). For example, a-bungarotoxin from krait venom inhibits activity of nicotinic ACh receptors at the NMJ, b-bungarotoxin from rattlesnake venom inhibits Kþ channels, and a-latrotoxin from black widow spider venom stimulates Ca2þ-dependent exocytosis to release neurotransmitters at PNS synapses (Karalliedde, 1995; Watkins, 2013). Novel molecules with unknown properties can be tested to see if they act via one of these mechanisms by adding a zootoxin with known activity to see if zootoxin interferes with action of the new entity. In general, toxicologic pathologists do not participate in these basic research endeavors. Instead, toxicologic pathologists typically encounter zootoxins in the course of nonclinical development programs for novel chemical or molecular entities with backbones derived from a poison or venom constituent.

6.1. Sources and Major Indications of Medicinal Zootoxins Toxicologic pathologists typically encounter zootoxins in nonclinical development programs for biologic products based on purified, recombinant, or synthetic versions of venom-derived peptides or small molecules (Bordon et al., 2020; Chaisakul et al., 2016; Chen et al., 2018; de Souza et al., 2018; Menez, 1998). These novel molecular and chemical entities may be derived from any animal species but commonly are drawn from poisons or venoms of particular groups with myriad powerful zootoxins including: • cone snails (Utkin, 2015), • leeches (Abdualkader et al., 2013), • honeybees (Lee and Bae, 2016; Lin and Hsieh, 2020; Orsolic, 2012), • centipedes (Fratini et al., 2017; Hakim et al., 2015), • scorpions (Ding et al., 2014; Harrison et al., 2014), • spiders (Saez et al., 2010; Yacoub et al., 2020), • frogs (Arneric et al., 2007; Daly et al., 2000; Mtewa et al., 2018), • lizards (Mtewa et al., 2018; Sanggaard et al., 2015), or

• snakes (Ferraz et al., 2019; Mohamed Abd ElAziz et al., 2019; Xiao et al., 2017; Zambelli et al., 2017). Snakes are particularly useful for such research due to their large venom volumes, which greatly simplifies zootoxin isolation and chemical analysis. A dozen approved medicines are based on zootoxins (Table 8.1), and many more zootoxin mimetics are in development (Bordon et al., 2020). The first zootoxin-like drug was captopril, an ACE inhibitor approved by the FDA in 1981 to treat hypertension. Captopril is a synthetic analog of a peptide in the venom of the jararaca (Bothrops jararaca), a South American pit viper. Drug discovery using zootoxins typically is directed to conditions that fall under the main functional categories for zootoxins described above: blood disorders and cardiovascular diseases for coagulotoxins, cancer therapy and resistant microbial diseases for necrotoxins, and neurological diseases for neurotoxins. A detailed consideration of these indications is beyond the scope of this chapter, but this list provides some relevant citations to guide further reading about the promise of zootoxin-derived molecules as new therapeutic entities that act as: • antimicrobial agents (Clark et al., 2019; Fratini et al., 2017; Samy et al., 2017; Yacoub et al., 2020) that: • exhibit synergistic activity with current antibiotics (Al-Ani et al., 2015; El-Seedi et al., 2020), • penetrate biofilms (das Neves et al., 2019; ElSeedi et al., 2020), and • vanquish antibiotic-resistant pathogens (das Neves et al., 2019; Marques Pereira et al., 2020); • antiinflammatory molecules for autoimmune diseases (Chen et al., 2018; Shen et al., 2017); • antineoplastic therapies (Calderon et al., 2014; Chaisakul et al., 2016; Cohen-Inbar and Zaaroor, 2016; Ding et al., 2014; Mahadevappa et al., 2017; Orsolic, 2012; Upadhyay, 2018); • cardiovascular disease therapies, especially hypertension and congestive heart failure (Frangieh et al., 2021; Mohamed Abd El-Aziz et al., 2019); • coagulation disorder therapies, particularly arterial thrombosis (e.g., cerebral and

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myocardial infarcts) and DIC (Huang et al., 2016; Sanchez et al., 2017); • cosmeceuticals (i.e., cosmetic products with bioactive ingredients purported to have therapeutic properties) (Bordon et al., 2020; Mabrouk et al., 2013; Pandey et al., 2021); and • neurological therapies for conditions including • neurodegenerative diseases (de Oliveira Amaral et al., 2019; de Souza et al., 2018; Gazerani, 2021; Nalivaeva et al., 2012), • neuroinflammatory diseases (Awad et al., 2017; de Souza et al., 2018; Silva et al., 2015), • neuropathic pain (Cura et al., 2002; Maatuf et al., 2019), • neuroprotection following cerebral infarction (Chassagnon et al., 2017), and • polyneuropathies associated with drug therapy (Song et al., 2017; Yoon et al., 2012). Venomous animals may be approved as therapies in their own right. For example, medicinal leeches are approved by the FDA as medical devices for relieving venous congestion (FDA, 2004). Medical maggots, larvae of the common green bottle fly (Lucilla [Phaenicia] sericata [Meigen]), are an FDA-approved medical device to treat chronic cutaneous ulcers (FDA, 2011). While the larvae excrete antibacterial peptides, metalloproteinases, and serine proteinases while working (Poppel et al., 2015), the approval was granted primarily due to their ability to debride necrotic flesh. Next-generation devices have been proposed in which fly larvae are engineered to excrete human growth factors as an additional means of speeding recovery (Linger et al., 2016).

6.2. Practices for Developing ZootoxinBased Medical Products Regulatory guidance differs for developing biomolecule drugs, small molecule drugs, and medical devices. A detailed consideration of all national and international guidance documents is beyond the scope of this chapter, but this section provides an overview of basic principles. More details regarding nonclinical efficacy and safety recommendations may be gained by reviewing Biomedical Materials and Devices (Vol 2, Chap 11), Protein Therapeutics (Vol 2, Chap 6), and Vaccines (Vol 2, Chap 9).

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Certain practices are common to all classes of biomedical products. Nonclinical safety studies typically are performed in compliance with Good Laboratory Practice (GLP) principles (CFR, 2011; FDA, 1987; OECD, 1998) (see Pathology and GLPs, Quality Control and Quality Assurance [Vol 1, Chap 27]). General guidance for nonclinical safety studies relevant to drugs and biologics is provided in the United States by the FDA in its Redbook (FDA, 2000). Prudent sponsors design studies that also follow recommendations of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), a multinational effort to prepare globally accepted guidance for common problems encountered in studies being performed at several study sites in more than one country, to ease acceptance of study data around the globe. The ICH documents usually employed for developing zootoxin-like products apply to agents destined for use in adults; M3(R2) (ICH, 2009a) is well suited to small molecule drugs while S6(R1) (ICH, 2011) is appropriate for biomolecules. Additional ICH guidance is available for special indications, such as S9 for anticancer drugs (ICH, 2009b, 2018) and S11 for pediatric drugs (ICH, 2020). Medical devices usually are developed using guidance given by the International Organization for Standardization (ISO) (ISO, 2006–2017). Externally applied surface devices such as medicinal leeches and maggots still may be investigated by adapting ISO 10993-6:2016 (“Test for Local Effects of Implanted Devices”) and especially Annex A (“Test methods for implantation in subcutaneous tissue”) and Annex E (“Examples of evaluation of local biological effects after implantation”) to evaluate any reactions in the treated tissue; likely systemic exposure to venom constituents may also benefit from studies performed according to ISO 19993-11:2017 (“Tests for systemic toxicity”). In the case of leeches and maggots, their approval as devices followed the FDA’s Section 510(k) “premarket notification” process, which permits marketing of devices without clinical trials if the product performance is “substantially equivalent” to devices already on the market. Innovative zootoxin-based products use technological modifications to maximize delivery and persistence of the therapeutic moiety. For example, nanoparticle-coated zootoxins enter

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TABLE 8.5 Antivenom Products for Selected Species Product mame

Species

Product type

Immunized species Product information

AnaScorp

Scorpion (Centruroides spp.)

F(ab’)2

Horse

Black Widow Spider Antivenin

Black widow spider (Latrodectus mactans)

Polyvalent Ab Horse

Funnel Web Spider Antivenom

Funnel web spider (Atrax Polyvalent Ab Rabbit robustus) e male

https://labeling.seqirus. com/PI/AU/FunnelWeb-SpiderAntivenom/EN/ Funnel-Web-SpiderAntivenom-ProductInformation.pdf

Red Back Spider Antivenom

Red back spider (Latrodectus hasselti) e female

https://labeling.seqirus. com/PI/AU/Red-BackSpider-Antivenom/EN/ Red-Back-SpiderAntivenom-ProductInformation.pdf

ARACHNIDS

Polyvalent Ab Horse

https://www.fda.gov/ media/81093/download https://www.merck. com/product/usa/pi_ circulars/a/antivenin/ antivenin_pi.pdf

SNAKES

AnaVip

• Copperhead F(ab’)2 (Agkistrodon contortrix) • Cottonmouth (Agkistrodon piscivorus) • Rattlesnakes (multiple North American species)

Antivenin (Crotalidae)

Brown Snake Antivenom

Horse

https://www.fda.gov/ media/92139/download

Rattlesnakes (multiple North and South American species) and common lancehead/ferde-lance (Bothrops atrox)

Polyvalent Ab Horse

https://www.bivetmedica.com/sites/ default/files/dam/ internet/ah/vetmedica/ com_EN/product_files/ Antivenin/antiveninlabel.pdf

Eastern brown snake (Pseudonaja textilis)

Polyvalent Ab Horse

https://labeling.seqirus. com/PI/AU/BrownSnake-Antivenom/EN/ Brown-SnakeAntivenom-ProductInformation.pdf (Continued)

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TABLE 8.5

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Antivenom Products for Selected Speciesdcont’d

Product mame

Species

Product type

CroFab

Fab • Cottonmouth/water moccasin (A. piscivorus) • Eastern diamondback rattlesnake (Crotalus adamanteus) • Mojave rattlesnake (Crotalus scutulatus) • Western diamondback rattlesnake (Crotalus atrox)

Immunized species Product information Sheep

https://www.fda.gov/ media/74683/download

North American Eastern coral snake coral snake antivenin (Micrurus fulvius)

Polyvalent Ab Horse

https://labeling.pfizer. com/showlabeling. aspx?format=PDF& id=441

PolySerp-M (MENA ¼ Middle East, north Africa, and Central Asia)

27 snake species of elapids (from 2 families) and viperids (from 8 families)

F(ab’)2

Equine

https:// polyserpantivenoms. com/polyserp-mena/

PolySerp-P (PanAfrica)

F(ab’)2 • Black mamba (Dendroaspis polylepis) • Black-necked spitting cobra (Naja nigricollis) • Puff adder (Bitis arietans) • West African (or ocellated) carpet viper (Echis ocellatus) • Cross-reactivity with 21 other species

Equine

https:// polyserpantivenoms. com/polyserp-panafrica/

Polyvalent Snake Antivenom

• Common death adder Polyvalent Ab Horse (Acanthophis antarcticus) • Inland taipan (Oxyuranus microlepidotus) • King brown snake (Pseudechis australis) • Red-bellied black snake (Pseudechis porphyriacus) • Tiger snake (Notechis scutatus)

https://www.nps.org. au/medicine-finder/ polyvalent-snakeantivenom-concentratefor-infusion

Sea Snake Antivenom

• Beaked sea snake (Enhydrina schistosa) • Cross-reactivity to other sea snakes

https://labeling.seqirus. com/PI/AU/Sea-SnakeAntivenom/EN/SeaSnake-AntivenomProduct-Information.pdf

Polyvalent Ab Horse

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cells more effectively than uncoated molecules (Biswas et al., 2012). Steroid-zootoxin conjugates can deliver peptides to particular tissues by site-specific accumulation based on the steroid structure (Cook Sangar et al., 2020). The scorpion peptide chlorotoxin linked to fluorescent dye is being tested in clinical trials as a “tumor paint” to mark the borders of malignant gliomas (Wulff et al., 2019). Cytotoxic peptides from wasp venom have been joined to poly(L-glutamic acid) polymer (PGA) to selectively drive delivery to tumor cells, where the peptide is freed by the action of proteases overexpressed by the tumor cells (Moreno et al., 2014). These conjugated products are approximately equivalent to immunotoxins, which are antibody–drug conjugates (ADCs) formed by linking a potent toxic payload (e.g., diphtheria toxin) to a recombinant fusion protein that can bind a tumor cell–specific antigen or leukocyte-specific cytokine receptor (see Bacterial Toxins [Vol 3, Chap 9]). Nonclinical safety testing of these complex test articles typically includes evaluation of the complete product as well as the isolated payload (either with or without the attached linker). Pathology endpoints in nonclinical studies for zootoxin-based products are generally similar to those of other biologic and small molecule drugs (see Basic Approaches in Anatomic Toxicologic Pathology [Vol. 1, Chap 9]; Clinical Pathology in Nonclinical Toxicity Testing [Vol. 1, Chap 10]; and Practices to Optimize Generation, Interpretation, and Reporting of Pathology Data from Toxicity Studies [Vol. 1, Chap 28]) with a few exceptions for conjugated products. Toxicokinetic analysis usually must follow distribution of the full product, the recombinant carrier, and the freed payload. Tissue cross-reactivity studies for test article localization in nontarget tissues (see Special Techniques in Toxicologic Pathology [Vol. 1, Chap 11]) may be needed if the carrier is an antibody but are not required for oncology indications. In vitro stability of the conjugated product should be investigated in human and animal plasma (for relevant nonclinical species) as part of the Investigational New Drug (IND)enabling package. The types and designs of nonclinical studies needed to assess the safety of zootoxin-based test articles will need to be defined on a case-by-case basis. Conventional anatomic pathology and clinical pathology data

should be suitable in terms of crafting a registration package for regulatory submission for most injected and oral products derived from or based on zootoxins.

6.3. Practices for Developing Antivenom Products Development of antivenom also requires nonclinical testing suitable to the nature of the product. In the United States, product registration for antivenom is conducted through the FDA’s Center for Biologics Evaluation and Research (CBER). The antibodies or antibody fragments are treated as blood products for this review. Antivenom typically is prepared by plasmapheresis of hyperimmunized horses, sheep, or occasionally donkeys, goats, or rabbits (Theakston et al., 2003; WHO, 2016). For this reason, the preferred designations for these products are now “antivenom” or “antivenom immunoglobulin” rather than “antiserum.” Antibodies are purified to isolate the IgG fraction. This fraction sometimes is processed further to produce antigen-binding fragments that lack the Fc domain that interacts with complement and surface receptors on immune cells. The absence of Fc reduces the potential for initiating undesirable reactions (Ferraz et al., 2019); however, whole immunoglobulin (often aggregated) or Fc-bearing fragments may be found even in highly purified antivenom (Theakston and Smith, 1997). Options to minimize the Fc content involve enzymatic cleavage to produce monovalent Fab molecules (having one constant and one variable domain but no Fc) or divalent F(ab’)2 molecules (possessing two variable domains linked by a hinge region but no constant or Fc domains). The decision to produce intact IgG or fragments depends on the molecular size and toxicokinetics of the principal venom toxin(s) (WHO, 2016). Preferred practice for modern antivenom is to prepare lyophilized formulations to improve the shelf life. Whole venom rather than single zootoxins is preferred as the immunogenic stimulus because venoms are mixtures of many zootoxins. Antivenom primarily is prepared for medically significant scorpion, spider, and snake species (Table 8.5). Antivenom prepared against venom

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from a single species (i.e., a “monospecific” product) in some cases may neutralize venoms from phylogenetically related species. Antivenom that is effective against multiple species in the same geographic region (i.e., a “polyspecific” product) may be prepared by injecting multiple venoms into the same hyperimmunized animal (Sousa et al., 2018). Some antivenoms neutralize the activities of antigens to which they are not targeted; this capability is termed paraspecificity (Ainsworth et al., 2018; Ratanabanangkoon, 2021). Next-generation antivenoms designed using venomic data are predicted to use monoclonal antibodies or small molecules to prevent specific pathologic processes or bind the most damaging zootoxins (e.g., PLA2, serine proteinases, matrix metalloproteinases, 3FTx). Recombinant peptide immunogens targeting conserved toxin epitopes expressed across many species represent one option (Bermu´dezMe´ndez et al., 2018; Ferraz et al., 2019). Another approach will be to reverse-engineer natural toxin-neutralizing factors, such as the opossum (Didelphis virginiana) peptide that is capable of completely neutralizing western diamondback rattlesnake venom (Komives et al., 2017). Expert working groups recommend that antivenom development conform to the production standards of the World Health Organization (WHO) (Theakston et al., 2003; WHO, 2016). In general, preclinical testing focuses on investigating efficacy. The mainstay tests are the median lethal dose (LD50) and median effective dose (ED50) in mice; at present, these two bioassays are the only validated means for confirming antivenom neutralizing capacity. Supplemental testing may be performed for new products or to validate new indications for existing antivenoms by showing that the antibodies can minimalize or eliminate the most clinically significant functional deficits (e.g., coagulation abnormalities, respiratory paralysis) and/or structural damage (e.g., necrosis). Validation studies are required to ensure that animal pathogens (chiefly viruses) will not be transferred to patients. Given the batch-to-batch variation of such products, conventional safety testing generally is not performed. Despite these guidelines, misleading labeling practices often complicate decisionmaking with respect to clinical administration of antivenoms (Simpson and Norris, 2007).

7. SUMMARY Animal-derived toxins, or zootoxins, are complex mixtures of alkaloids, amines, lipids, nucleic acids, peptides, and proteins (especially enzymes) that act locally or systemically to damage cells and tissues. Poisons consist of zootoxic metabolic by-products that accumulate in a nonspecialized tissue and are encountered by direct contact (usually oral). Venoms are zootoxic mixtures generated in a specialized tissue (usually a gland) and injected into another organism using a specialized apparatus (e.g., modified tooth, stinger). Zootoxin exposures in animals and humans usually occur when the poisonous animal is eaten or the venomous animal executes a defensive or predatory attack. The myriad of bioactive constituents in venoms are a fruitful field for drug discovery research, so purified, recombinant, or synthetic zootoxin analogs are ever more likely to enter the medical armamentarium as innovative treatments for underserved medical needs. Toxicologic pathologists will be essential in nonclinical safety testing of these new products. Animals and humans typically exhibit similar clinical signs and pathologic findings when exposed to zootoxins. The noxious effects may be functional abnormalities (e.g., excessive or inhibited coagulation, paralysis) or structural lesions (e.g., hemorrhage, necrosis). Zootoxin exposure frequently induces coagulotoxic, necrotoxic, pro-inflammatory, neurotoxic, and/ or vasoactive effects; many venoms produce multiple additive or synergistic effects. Principal mechanisms of zootoxin action include cell and tissue disruption, altered hemodynamic and hemostatic processes, inflammation, and suppressed neurotransmission. Zootoxicoses are commonly treated by nonspecific supportive care, but antivenom (if available) offers the only zootoxin-specific treatment. Prophylactic injection of a venom-directed toxoid occasionally is used to lessen the threat posed to individuals and communities who must share geographic boundaries with poisonous or venomous animal species. Diagnostic and forensic pathologists are instrumental in performing anatomic pathology and clinical pathology tests required to rationally treat animal and human victims of zootoxin poisoning or envenomation.

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GLOSSARY Antivenom (alternatively Antivenin) an antibody-based therapy for neutralizing one or more toxins in a venom Arachnidism (alternatively Araneism) systematic envenomation due to a spider bite Coagulotoxin a zootoxin that disrupts the coagulation pathway, typically leading to either an anticoagulant or procoagulant effect Convergent Evolutions development of similar traits in different organisms faced with a similar challenge (e.g., venom glands and venoms for food acquisition and defense in spiders and snakes) Crinotoxin a zootoxin produced in a specialized venom cell or venom gland that is released passively into the environment Cytotoxin a toxic molecule that causes biochemical or structural damage to cells, thus leading to degeneration (potentially reversible injury) or death (the outcome of irreversible injury) Envenomation (alternatively Envenoming) the active process of injecting Venom (see definition below) Hemotoxin a zootoxin that destroys blood components, especially erythrocytes (red blood cells) but sometimes expanded to include clotting factors of the coagulation cascade (i.e., a Coagulotoxin [see definition above]) and blood vessel walls (i.e., a Vasculotoxin [see definition below]) Myotoxin a zootoxin with cytolytic activity against striated (cardiac and skeletal) muscle cells Necrotoxin a cytotoxic zootoxin leading to cell degeneration and death, ultimately resulting in local to locally extensive tissue necrosis and structural disorganization/ destruction Neurotoxin a zootoxin, typically a peptide or protein that disrupts nervous system function by blocking action potential propagation or synaptic transmission Paraspecificity the property whereby an antivenom (or other antibody-based product) capable of neutralizing a toxin antigen against which it is directed also can neutralize the effects of other toxins

Poison a mixture of toxic substances (in this case, Zootoxins [see definition below]) produced in a nonspecialized tissue by accumulation of toxic metabolic byproducts and introduced into another organism by direct oral (or occasionally dermal or inhalational) contact Pseudo-procoagulant a setting in which initial procoagulant conditions result in production of abnormal fibrin strands, unstable clots, sustained fibrinogen cleavage, fibrinogen depletion, and ultimately an anticoagulant state Toxicology the study of the effects that toxic substances (including but not limited to toxins) induce in living organisms Toxin a toxic molecule (“toxicant”) of biological origin Toxinology the study of the toxins, poisons, and venoms made by living organisms (animals, plants, and microbes) Toxoid a vaccine prepared by chemical or physical inactivation of a toxin molecule (or a mixture of toxin molecules) to quench its toxic activity while retaining its immunogenicity Toxungen a term proposed for a toxic substance (in this case comprised of one to numerous Zootoxins [see definition below]) introduced onto the external surface of another organism via a noninjurious delivery method (e.g., spitting, spraying) Vasculotoxin a tissue-damaging zootoxin (see definition for Necrotoxin above) capable of disrupting the structural integrity of endothelial cells and/or blood vessel walls Venom a mixture of toxic substances (in this case, Zootoxins [see definition below]) generated in a specialized tissue (often a gland) and introduced into internal tissues of another organism via a specialized, injurious delivery apparatus (e.g., modified tooth, stinger) Venomics study of Venom (see definition above) composition using various “-omics” technologies (typically genomics, transcriptomics, and proteomics) Zootoxicosis a toxicant-induced disease state (or “toxicosis”) caused by exposure to a Zootoxin (see definition below) Zootoxin a toxin produced by an animal

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Acknowledgments The authors thank Ms. Beth Mahler for her assistance in optimizing the hues and resolution of macroscopic and microscopic images, and Mr. Tim Vojt for his superb diagrams and flow charts.

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C H A P T E R

9 Bacterial Toxins Brad Bolon1, Francisco A. Uzal2, Melissa Schutten3 1

GEMpath, Inc., Longmont, CO, United States, 2University of California, Davis, CA, United States, 3Genentech, Inc., South San Francisco, CA, United States

O U T L I N E 1. Introduction

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2. Exotoxins 2.1. Sources of Exposure 2.2. Toxicology

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3. Endotoxins 3.1. Sources of Exposure 3.2. Toxicology

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5. Diagnosis and Treatment of Bacterial ToxineMediated Diseases 5.1. Diagnosis 5.2. Treatment

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6. Regulatory Guidance Regarding Bacterial Toxins 6.1. Food and Beverage Production and Water Treatment 6.2. Manufacturing Biomedical Products 6.3. Safety Assessment of Immunotoxins

4. Clinical Presentations and Pathologic Manifestations of Bacterial Toxin-Mediated Diseases 4.1. Enteric Effects (Intestine) 4.2. Fascial Effects (Connective Tissue) 4.3. Hepatic Effects (Hepatocytes and Hepatic Immune Cells) 4.4. Microvascular Effects (Blood Vessels) 4.5. Myotoxic Effects (Cardiac and Skeletal Muscle) 4.6. Neurotoxic Effects (Brain and Terminal Nerve Synapses) 4.7. Pneumotoxic Effects (Lung)

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1. INTRODUCTION Animals and humans live in delicate balance with myriad microbes in the environment, including enormous numbers that reside permanently within the lumen of the digestive tract (mainly colon) as well as on other body surfaces (e.g., skin, upper and lower respiratory mucosa, urogenital mucosa) (for more information see

4.8. Systemic Effects (Sepsis and Toxic Shock Syndrome) 4.9. Miscellaneous Effects Attributed to Bacterial Toxins

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7. Summary

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Glossary

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Acknowledgments

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Digestive Tract, Vol 4, Chap 1). An individual’s microbiota pattern begins at birth and includes several hundred trillion archaea, bacteria, fungi, protozoa, and viruses; this collection of organisms collectively represents one definition for the term “microbiome.”1 The entire spectrum of microbiota-encoded genes (which is the other definition for “microbiome”) changes throughout each individual’s life

1

A Glossary is included after the Summary section as an easy-to-access means of exploring key and new terms.

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00009-5

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Copyright Ó 2023 Elsevier Inc. All rights reserved.

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with fluctuations in many environmental and physiological factors including age, diet, hygiene, and medication (to name only a few). Interactions between the microbiota and the host in large part determine the health of the host. The remainder of this chapter will focus on the extensive bacterial microflora that constitutes a principal source of microbiomederived toxins. The bacterial flora and host are constantly engaged in a dynamic relationship, leading to the health of both entitiesdhost and microbiomedwhen the interaction is balanced, or disease in one entity if the balance is disturbed. The gut microbiome has been suggested to represent a de facto host organ (Tu et al., 2020). Many xenobiotics may cause gut microbiome toxicity, including antibiotic drugs, artificial sweeteners, heavy metals, and pesticides. Imbalance in the gut microbiome manifests as dysbiosis (dysbacteriosis), leading to suppression of normal microflora and expansion of potential pathogens (Beliza´rio and Faintuch, 2018). The host’s immune system is challenged constantly to discriminate between commensal and symbiotic bacteria (which provide such essential services as aiding digestion and manufacturing vitamins) and pathogenic bacteria (which can cause disease and sometimes death). Dysbiosis results in an altered milieu of microbiota-derived products, the shift in which induces low-intensity but long-lasting activation of tissue-resident macrophages, initiation of inflammation, and ultimately a plethora of degenerative and metabolic diseases including diabetes mellitus, obesity, metabolic syndrome, and neoplasia (intestinal and systemic sites) (Beliza´rio et al., 2018). The detrimental effects of pathogens within the gastrointestinal tract (see Digestive Tract, Vol 4, Chap 1), on other epithelial surfaces, or after traumatic introduction into tissue result from a combination of mechanisms: direct tissue injury due to microbial growth and invasion, a robust antibacterial response by the host immune response, and/or release of bacterial toxins. Recent reports suggest that microbiota-generated toxins (specifically bacterial) may interact with toxic chemicals in the environment to adversely affect human health (Fiorentini et al., 2020; Koontz et al., 2019; Tu et al., 2020).

Toxigenesis is the generic term for the process whereby pathogenic bacteria produce toxic molecules (Todar, 2020b). Bacterial toxins have many means for targeting host cells, including direct damage to cell membranes, inhibited cell metabolism or signaling, and activating an immune response. In many bacterial diseases, the functional and structural consequences of toxin-induced damage to cells and tissues are more incapacitating than the injury wrought by the presence of the pathogens. In this regard, toxemia (i.e., the systemic spread of bacterial toxins) may be as or more debilitating than the systemic dispersion of bacteria; interestingly, “blood poisoning” is a colloquial misnomer applied to blood-borne spread of bacteria (i.e., bacteremia, also called septicemia), not bacterial toxins. The impact of bacterial toxins may be magnified by successful antibiotic therapy as bactericidal agents produce cell lysis and the abrupt release of additional toxin stores. In terms of chemical composition, two broad classes of bacterial toxins are recognized. Exotoxins are metabolic products that can be secreted from viable bacteria or liberated from disintegrating cells, while endotoxins are cell wall structural components that are released by lysis of dead cells. Exotoxins act directly on host cells while endotoxins activate host immune cells to secrete factors that damage or kill nearby host cells indirectly (Figure 9.1). The principal distinguishing characteristics of exotoxins and endotoxins are compared in Table 9.1. The damage caused by exotoxins drives many manifestations of bacterial disease, including pathologic findings in host cells and tissues as well as lethality. This chapter provides a concise overview of the comparative toxicology and pathology of bacterial toxins. The initial sections of the chapter describe the role of bacterial toxins in natural diseases of animals and people. We anticipate that this natural disease setting will be the usual avenue whereby most pathologists and toxicologists encounter bacterial toxins during the course of their lives and careers. Nonetheless, later sections discuss the deliberate adaptation in recent years of selected bacterial toxins for use as therapeutic products or bioterrorism agents. Where warranted, product discovery and development precepts as they apply to bacterial toxin-derived test articles have been incorporated.

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FIGURE 9.1 Mechanisms Used by Bacterial Toxins to Attack Eukaryotic Host Cells. Exotoxins (orange diamonds) are secreted products of many gram-positive and a few gram-negative bacteria that interact with and directly damage host cells. The toxin cell contact may employ several mechanisms, most often a receptor-mediated interaction (shown here). Endotoxins (white hexagons) are lipopolysaccharide (LPS)-rich cell wall components of gramnegative bacteria that are released by death and disintegration of the bacterium. Subsequent binding of the endotoxin to receptors on host effector cells (chiefly immune cells) activates the host cells to produce and secrete factors that damage neighboring host cells that have not experienced direct contact with the endotoxin. II. SELECTED TOXICANT CLASSES

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TABLE 9.1 Comparison of Key Bacterial Toxin Attributes Attribute

Exotoxin

Endotoxin

Gram-negative bacteria (some)

Gram-negative bacteria

SOURCE

Occurrence

Gram-positive bacteria (many) Cellular location

Cytosol (soluble metabolic product)

Outer cell membrane (integral structural element)

Mechanism of release

Secretion by living cells, lysis of dead cells

Lysis of dead cells

Nature

Protein (polypeptide) complexes

Lipopolysaccharide (LPS) eprotein complexes

Size

2e300 kDaa

10e1000 kDaa

Toxic component

Protein

Lipid

Gene location

Plasmid (extrachromosomal)

Chromosome

Heat stability

Labile (60e80
C)ddenatured by boiling (most)

Stable (250
C)dnot denatured by boiling

CHEMICAL CHARACTERISTICS

Stable (250
C)dnot denatured by boiling (some) Removal by filtration

Feasible

Not possible

Toxin specificity

Produced by one (or a few related) bacterial strains

Not specific to any bacterial strain

Potency

Highd1 toxin molecule interacts with many cells

Lowddisease requires large amounts of toxin

Target on host cells

Specific cell membrane receptors

No specific receptors

Immunogenicity

Strong

Weak

Neutralization by antibodies

Feasible

Not possible

Toxicity

Highdfatal in mg quantities

Moderatedfatal in mg quantities

Clinical presentation

Variable; depends on cell target and enzyme action

General symptoms: Fever, diarrhea, vomiting

Detection

Many tests (neutralization, precipitation, etc.)

Limulus lysate assay

Fever induction

No

Yes (via IL-1 and TNF production)

Modification for antitoxin

Feasible (for passive immunity)

Not possible

Modification for toxoid

Feasible (vaccine to yield active immunity)

Not possible

TOXICOLOGY

TREATMENT AND CONTROL

a

Reported sizes vary widely in the literature; these values represent the typical ranges in microbiology reference texts. Abbreviations: IL-1, interleukin-1; kDa, kilodaltons; TNF, tumor necrosis factor.

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2. EXOTOXINS This toxin class consists of several hundred soluble, diffusible bacterial proteins that are released into the extracellular microenvironment to act at a site located at some distance from the bacterial colony (Alouf et al., 2015; Todar, 2020b). Exotoxins are a chemically and functionally diverse class of metabolites, and they disrupt host cell function by one of several mechanisms. Bacterial exotoxins are strongly immunogenic, but their extreme potency often proves fatal to both the affected cells and the host organism before the immune system has time to mount an effective immune response. Indeed, bacterial exotoxins are the most powerful known toxic agents to humans and animals (Todar, 2020b). A particular species may generate a single exotoxin or multiple exotoxins (Barbieri, 2009), but the production of a particular exotoxin is usually limited to one or a few related bacterial species (Todar, 2020b). The ability to produce an exotoxin is a major determinant of bacterial virulence, with more virulent strains capable of toxin production while “nonvirulent” (less virulent) strains are not.

2.1. Sources of Exposure Exotoxins are generated and released by many gram-positive and a few gram-negative bacteria (Todar, 2020b). Humans and animals encounter exotoxins at injurious levels under many situations. Infection An obvious setting for exotoxin exposure is during an infection of the host by toxinproducing bacteria. These infections may present in several ways. First, bacteria might secrete toxins that cause functional changes in host cells without actually invading nearby tissue. For example, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae produce structurally similar heat-labile enterotoxins, and ETEC also generates a heat-stable enterotoxin; these three exotoxins alter intracellular signaling and the electrolyte balance in intestinal epithelial cells, thus leading to massive water efflux into the intestine (Bharati and Ganguly, 2011; Fleckenstein and Kuhlmann, 2019). Similarly, the simultaneous intracellular

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activity of exotoxins from Bordetella bronchiseptica (dermonecrotic toxin, DNT) and Pasteurella multocida (heat-labile toxin) in osteoblasts and osteoclasts induces bone resorption and results in atrophy of the nasal turbinates in young pigs (Magyar and Lax, 2002). Second, bacteria might release exotoxins specifically to bolster bacterial entry and survival within host cells and tissues. Various exotoxins support this purpose, including the adhesins of Helicobacter pylori and Mannheimia haemolytica (Singh et al., 2011), the evasins of E. coli and Streptococcus pyogenes (Smith et al., 2007), and the invasins of many enteric pathogens including Salmonella spp., Shigella spp., and Yersinia spp. (Palumbo and Wang, 2006), to name just a few. Finally, bacteria that have successfully colonized a host tissue might secrete additional rounds of exotoxin metabolites, especially during the phase of exponential bacterial expansion (Todar, 2020b). Exotoxins of this kind include pore-forming toxins and proteases produced during clostridial enteric infections (e.g., Clostridioides difficile, Clostridium perfringens), such as toxins A and B, enterotoxin, and epsilon toxin (Smedley et al., 2004); many proteases and superantigens made by Staphylococcus aureus and Group A Streptococcus (GAS) spp., which produce necrotizing soft tissue infections (“flesh-eating bacterial disease”) and toxic shock syndrome in humans (Shumba et al., 2019); multiple toxins that disrupt host cell signaling that are made by common blood-borne pathogens like Bacillus anthracis, E. coli, Pseudomonas aeruginosa, S. aureus, and S. pyogenes (Ramachandran, 2014); and tetanospasmin, a neurotoxin released by Clostridium tetani carried into poorly oxygenated tissues associated with deep wounds (Hassel, 2013). Tetanospasmin is the second most potent natural toxin (after botulinum toxin) (McClane et al., 2013). Ingestion Exotoxin exposure is common following ingestion of contaminated food or water. Common causative agents of exotoxin-based food-borne illness (“food poisoning”) include botulinum toxin from Clostridium botulinum (Lonati et al., 2020); C. perfringens enterotoxin (CPE), the second or third most prevalent cause of food poisoning in humans in the United States

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depending on the year (McClane et al., 2013); and some two dozen enterotoxins released by S. aureus (Argudin et al., 2010). These toxins are resistant to high heat, low pH, and protease degradation, so they survive bactericidal food processing procedures and passage through the stomach to reach the intestine. The exotoxins must be absorbed to induce toxicity. Botulinum toxin is the cause of botulism in humans as well as domestic and wild animals.

This exotoxin consists of eight antigenically distinct but structurally similar neurotoxins (Figure 9.2), of which types A followed by B and F are the most potent (Nigam and Nigam, 2010). All eight exotoxins are comprised of a heavy chain (100 kDa) that binds at the synaptic terminal and a catalytic light chain (w50 kDa) that acts in the cell cytosol as a zinc-dependent metalloprotease to cleave proteins required for exocytosis (Caya et al.,

FIGURE 9.2 Molecular Structure of Botulinum Toxin (BoNT) Variants A (Panel A) and E (Panel B). Two of eight related Clostridium botulinum neurotoxins that collectively are the most lethal poison known. The BoNTs consist of heavy (H) and light (L) chains linked by a disulfide (S) bond (orange circles) and a “belt” region (blue) that modulates toxin–membrane interactions. The H chain carries a receptor-binding domain (HC–C [green]) to allow toxin uptake by endocytosis, a membrane-translocating domain (HN [yellow]) to enable transfer of the L chain from the acidified endosome into the cytosol, and an additional HC-N (purple) domain whose function is not known but which may serve as a receptor for host cell lipids. Each BoNT variant recognizes a specific host cell receptor (comprised of synaptic proteins and gangliosides). The L chain (L [red]) bears a catalytic metalloprotease domain that degrades proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, which blocks fusion of neurotransmitter-laden synaptic vesicles with the plasma membrane at nerve terminals. The color coding for the crystallographic and schematic depictions is identical. Adapted from Pirazzini M, Azarnia Tehran D, Leka O, et al.: On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments, Biochim Biophys Acta 1858:467–474, 2016. by permission of Elsevier.

2. EXOTOXINS

2004), thereby preventing the release of the neurotransmitter acetylcholine that is needed for neuromuscular transmission. Botulinum toxins exhibit differential toxicity depending on the vertebrate species: types A, B, E, and F are responsible for disease in humans; types A, B, C, D, and E cause disease in domestic and wild animals; and all types are toxic to nonhuman primates (Cope, 2018). Botulinum toxin is the most potent known natural toxin. The main clinical signs of botulism in humans and animals following toxin ingestion are progressive, symmetrical, and flaccid paralysis (e.g., difficulty swallowing, facial flaccidity, ptosis [drooping eyelids], and finally ambulatory and respiratory paralysis) and abdominal distress (e.g., colic, nausea, and/or vomiting). Humans also may show blurred or double vision and speech impairment. Estimated lethal doses are 30 ng following ingestion (about 0.4– 0.5 ng/kg) and 90–150 ng (approximately 1.0– 1.5 ng/kg) after parenteral injection for adult humans, mice, and nonhuman primates (Arnon et al., 2001; Gill, 1982). The staphylococcal enterotoxins (SEs, identified as the “classical” exotoxin group) and enterotoxin-like proteins (SEls, the “new” group) comprise a superfamily of 24 structurally and functionally similar proteins that contribute to the initiation and progression of many human diseases (e.g., necrotizing soft tissue infections, pneumonia, sepsis, toxic shock syndrome) besides food poisoning. These molecules share many attributes including a single chain, nonglycosylated peptide backbone; low molecular weight (19–29 kDa); and globular conformation when folded. The original distinction between the classical SEs (designated SEA to SEE) and new SEls is that the SEs caused emesis in nonhuman primates while the SEls had not been tested; many SEls have since been demonstrated to induce emesis as well. The SEs (especially SEA) are top contributors to food-borne illness, but as better diagnostic tests have emerged the SEls have been confirmed to be powerful players as well (Fisher et al., 2018). Recently, SEB also has been implicated as a contributor to mast cellmediated visceral hypersensitivity (“food allergy”) in ovalbumin (OVA)-induced mouse models of irritable bowel syndrome (AguileraLizarraga et al., 2021). The mix of SEs and

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SEls is regulated by the microenvironmental milieu in which the bacteria reside. Native S. aureus flora on the cutaneous and mucosal surfaces of humans and livestock are considered to be likely reservoirs of food contamination. Relative to nonhuman primates, rodents appear to be less sensitive to the enterotoxigenic effects of SEs and SEls. However, the house musk shrew (Suncus murinus) has been identified as a suitable alternative experimental model to primates based on the shrew’s ability to vomit (Fisher et al., 2018). Studies have shown that SE-induced emesis involves the interaction of SEs with epithelial cells (as portals for crossing the mucosa); the release of 5-hydroxytryptamine (i.e., serotonin, a neurotransmitter) from mast cell granules; and stimulation of the vagus nerve (Fisher et al., 2018). Exotoxins may be delivered deliberately as an instrument of bioterrorism or warfare in contaminated food or water to produce mass poisoning (Patocka and Streda, 2006). However, significant logistical limitations in terms of ensuring rapid and widespread exposure limit the routine use of bacterial toxins as weapons using this route of administration (Caya et al., 2004). Bacterial toxins can undergo bioconcentration in the food chain, thereby causing toxicity in apex predators. A prime example of this phenomenon is accumulation of tetrodotoxin (TTX, Figure 9.3), a neuroparalytic toxin, in

FIGURE 9.3 Molecular Structure of Tetrodotoxin (TTX). This neurotoxic exotoxin can block action potential propagation (“nerve impulses”) by physically obstructing lumens of voltage-gated sodium (Naþ) channels in cell membranes of neuronal processes. This molecule undergoes bioaccumulation in certain viscera of marine and aquatic terrestrial animals.

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some marine fish (see also Phycotoxins, Vol 3, Chap 5 and Animal Toxins, Vol 3, Chap 8). This exotoxin acts to physically occlude voltage-gated sodium (Naþ) channels in cell membranes of neurons and their neurites (i.e., axons and dendrites), thus preventing Naþ ion movements required for propagating action potentials. Consumption of fugu, a Japanese delicacy typically presented in sashimi (raw slices) form, is responsible for several dozen poisonings each year and is an occasional cause of death in humans who dine on improperly prepared puffer fish (Fugu rubripes). Clinical signs of TTX toxicity include abdominal distress (e.g., cramping, nausea, and/or vomiting); headaches; and neurological signs such as incoordination, numbness, weakness, and secondary hyperhidrosis (i.e., diaphoresis [excessive sweating due to sympathetic nerve stimulation]); severe cases are characterized by cardiac arrhythmias, hypotension, and respiratory paralysis leading to death. Toxicity arises from high TTX levels in certain viscera (especially intestines, kidneys, liver, ovaries, and skin) of fish that host any of many bacterial species (Campbell et al., 2009; Lago et al., 2015; Wu et al., 2005). The toxin is thought to reach the target organs by either translocation from the digestive tract (Magarlamov et al., 2017) or through local production by intracellular symbiotic bacteria; the host fish is thought to benefit by using TTX as a means of defense (Lago et al., 2015). Interestingly, TTX also accrues in algae; marine invertebrates (e.g., crustaceans and mollusks); and terrestrial amphibians (especially newts, but also frogs and toads) (Lago et al., 2015; Magarlamov et al., 2017). The oral dose that kills 50% of mice (i.e., lethal dose 50 [LD50]) is reported as 532 mg/kg by ingestion, and male mice appear to be more sensitive (Lago et al., 2015). Toxicity is dose-dependent, and full recovery from nonlethal doses occurs within 24 h. Inhalation Exotoxin exposure may occur through the respiratory tract following inspiration of aerosols. In general, this scenario is unlikely to represent a route for natural exotoxin poisoning. Instead, inhalation of toxigenic bacteria (e.g., B. anthracis spores) and/or purified toxins (e.g., botulinum toxin, staphylococcal enterotoxin B)

is likely to be relevant mainly to aerosol dispersion of bioterror agents (Berger et al., 2016; Roy et al., 2010). Once inhaled, weaponized exotoxins may attack the respiratory epithelium, but more typically they will use the large, highly vascularized surfaces of the nasal passages and lungs as a means for entering the blood. Natural toxins are preferred weapons for some purposes compared to airborne chemicals (i.e., gas warfare agents) since toxins are more potent, have long latency periods, and do not create lasting environmental contamination. Weaponized exotoxins are not contagious, which both shortens the duration of the event and lessens the morbidity and mortality among the populace, and most will break down fairly rapidly under normal environmental conditions, thereby precluding the need for an extended environmental clean-up. B. anthracis is classified by the Centers for Disease Control and Prevention as a Category A bio-terror threat (http://www.selectagents. gov/SelectAgentsandToxinsList.html). The Category A designation applies to agents that can be disseminated readily, will yield high mortality, and have the potential to cause significant public panic and social disruption (FBI, Not stated; Frischknecht, 2003). Anthrax inhalation is an extremely lethal disease that begins with inhalation of spores but is driven by potent exotoxins (Coggeshall et al., 2013); by comparison, classical anthrax in domestic (e.g., cattle, sheep) and wild (e.g., antelope, deer) ruminants follows ingestion or inhalation of bacterial spores (which may remain latent in the environment for decades), although the involvement of exotoxins as a key pathogenic factor is common to all species. Inhaled B. anthracis spores are phagocytized by alveolar macrophages and dendritic cells, after which they are transported to regional lymph nodes. The spores subsequently germinate and begin producing substantial amounts of three exotoxin precursors: edema factor (EF), lethal factor (LF), and protective antigen (PA). The molecules exhibit no stand-alone toxicity, but when correctly paired they form edema toxin (EF þ PA) and lethal toxin (LF þ PA). The PA component binds to cellular receptors, leading to endocytosis of the toxin complexes; the edema toxin and lethal toxin subsequently are released inside endosomes and transferred

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into the cytosol where their catalytic activities disrupt cellular signaling cascades (Liu et al., 2014). Edema toxin and lethal toxin both contribute during the initial stage of infection to downregulating the efficacy of cells involved in innate immunity, especially neutrophils, macrophages, dendritic cells, and mast cells. Continued action of the two toxins late in disease is central to the morbidity and mortality that occur even if viable bacteria are eliminated by antibiotic therapy. The toxins appear to target different cell types, with edema toxin affecting hepatocytes while lethal toxin impacts cardiomyocytes and vascular smooth muscle cells (Liu et al., 2014). As with botulism, the clinical presentation of anthrax in people and animals differs, likely because humans seek medical attention earlier during the course of disease. Signs of inhalation anthrax in humans include nonspecific indications of a systemic infection (e.g., chills, fever, headache, and sweating) as well as specific respiratory effects such as coughing, shortness of breath, and tightness of the chest. In contrast, anthrax in ruminants usually presents as sudden death characterized by hemorrhage from body orifices and incomplete rigor mortis. Botulinum toxin is the only bacterial exotoxin classified as a Category A bioterror threat. Botulinum toxin has been produced by multiple nations beginning during World War II, and it has been deployed in failed bioterror attempts in the last few decades (Berger et al., 2016; Caya et al., 2004); it is easily prepared without the need for specialized equipment. The estimated lethal dose in humans following inhalation is 80–90 ng (Arnon et al., 2001), and the expected onset following a terrorist attack is predicted to be 12–36 h after inhalation. Once inhaled, the toxin crosses the respiratory epithelial surfaces by active transport rather than passive diffusion through the paracellular spaces to reach the blood, after which it disperses quickly and widely to reach its target (synaptic terminals). Inhaled botulinum toxin induces dose-dependent flaccid paralysis and lethality in mice within 3 h (Park and Simpson, 2003). The survival time is inversely proportional to the inhaled dose. Staphylococcal enterotoxin B (SEB) is a Category B bioterrorism agent. The Category B exotoxins are considered to be reasonably easy

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to disseminate, will yield moderate morbidity and low mortality, and thus will have a modest impact on public health and societal activities. This toxin is classed as a biowarfare agent because it is toxic in minute doses, readily disseminated in aerosols, soluble in water, resistant to acidic pH and heat, and can induce a lethal cytokine storm (as in staphylococcal toxic shock syndrome). Lower inhaled doses induce fever within 3–12 h that will persist for several days, while higher doses may incite acute respiratory distress syndrome (ARDS) (Berger et al., 2016). The epsilon toxin (ETX) generated by C. perfringens (types B and D) was once listed as a Category B bioterrorism threat (until 2012). This exotoxin was classified as a potential biowarfare agent because it is the third most potent known natural toxin, after the clostridial products botulinum toxin and tetanospasmin (Berger et al., 2016). In natural disease, ETX is produced in the intestine (especially of lambs and other ruminants fed a predominantly carbohydraterich diet). The predilection for ruminants is fostered by the ability of ETX to bind strongly to vascular endothelium in these species (Xin and Wang, 2019), where it acts locally to increase mucosal permeability before dispersing systemically to elicit endothelial lesions in small blood vessels, chiefly in the brain, heart, and lungs. Widespread endothelial disruption promotes multi-organ edema, again mainly in the brain, heart, and lungs. Direct neurotoxicity to neurons and oligodendrocytes has been demonstrated. The predicted LD50 is 100 mg/kg (Nocera et al., 2016; Uzal et al., 2010). Therapeutic Products Exotoxins are used intentionally for certain medical applications. Such instances occur in pursuing one of four therapeutic objectives: to counter existing toxin exposures (antitoxins), to prevent clinical disease during future toxin exposures (toxoids), to produce toxic effects in diseased tissue, or to utilize toxin actions to modify local physiological responses. Unintentional exposure to exotoxins in therapeutic products is through trace contamination that develops in manufacturing. Antitoxins are designed to counteract circulating toxins, including bacterial exotoxins (and also some animal and plant toxins). Antitoxins

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are available for such extremely potent exotoxins as botulinum toxin, diphtheria toxin (produced by Corynebacterium diphtheriae), and tetanus toxin. Antitoxins to bacterial toxins traditionally were made by inoculating horses with purified exotoxin to induce an acquired immune response, after which blood was collected to harvest serum enriched in toxin-neutralizing antibodies. The toxin-specific antibodies are purified and injected into individuals to provide temporary passive immunity to the exotoxin. Antitoxins can be made from human donors who have survived prior exposure to the toxin. Indeed, in much of the developed world, horse-derived tetanus antitoxin is being replaced by human-derived tetanus immune globulin (HyperTET), which is produced by giving people multiple doses of tetanus toxoid to greatly enhance antibody production. Toxoids are inactivated exotoxins given as prophylactic vaccines generated to prevent clinical disease during future toxin exposures. Exotoxin inactivation is achieved by chemical (formaldehyde) or physical (heat) modification of the peptide. This treatment suppresses the toxic activity but leaves the immunogenic potential intact. Injection of a toxoid does not induce disease but does engender an acquired immune response leading to immunological memory for the toxin antigens; the side effects associated with toxoid administration (e.g., pain and stiffness following clostridial vaccinations) are not evidence of residual toxin activity but of the inflammation associated with the renewed immune response. Important toxoids in modern use exist for botulinum toxin, diphtheria toxin, epsilon toxin, pertussis toxin, and tetanus toxin; in fact, the DTaP childhood vaccination contains diphtheria toxoid, tetanus toxoid, and pertussis toxin in combination, among other ingredients (Medicine Committee to Review Adverse Effects of Vaccines, Institute of Medicine, 2011). Immunity to toxoids wanes over time, so periodic booster vaccinations are needed to maintain anti-exotoxin immunity. Antibody–toxin conjugates (a subcategory of antibody–drug conjugates [ADCs]) have been developed to direct exotoxin-induced cell injury to specific cell populations, commonly tumors (Li et al., 2017). The antibody offers targeted delivery to cells that express a specific antigen of interest, while the potent small molecule payload is the active component. Two

adaptations of this basic approach have been (1) to engineer the binding domain of a highly potent exotoxin to selectively recognize receptors on tumor cells (i.e., a new drug class termed “immunotoxins”) and (2) to fabricate a recombinant fusion protein that links the active region of an exotoxin to the binding domain of a cytokine. Tumor antigens are ideal targets since they usually exhibit limited expression in normal tissues but are highly expressed on tumor cells. Upon antibody binding to a cell surface antigen, the ADC–antigen complex is internalized by endocytosis and trafficked into lysosomes, where the linker is cleaved to release the payload. The payload diffuses into the cytosol and/or nucleus where the toxic molecule acts to damage the cell (Figure 9.4). Bystander toxicity is possible by passage of the toxin to adjacent tumor cells. Off-target toxicity may occur in either an antigen-independent manner, if the ADC enters other cells that lack the target antigen (e.g., when antibody binds to Fc receptors or the intact ADC or toxins are taken up randomly by pinocytosis), or rarely in an antigen-dependent fashion, when an antigen in normal tissue is recognized by the antibody portion of the ADC. Bacterial exotoxins that have been used in antineoplastic immunotoxins include full-length (first generation) and recombinant (next generation) Pseudomonas exotoxin A (PE), diphtheria toxin, and shiga toxin. The first immunotoxins approved by the US Food and Drug Administration (FDA) were recombinant human proteins combining an interleukin (IL) backbonedIL-2 for denileukin diftitox (Ontak) and IL-3 for tagraxofusp-erzs (Elzonris)das the receptorbinding moiety for neoplastic leukocytes with diphtheria toxin as the active element (Allahyari et al., 2017; Shafiee et al., 2019). The two-phase mechanism of action of A-B toxins (i.e., exotoxins pairing “active” [A] and “binding” [B] elements) is of particular interest in cancer therapy research. The general idea is to modify the B component of existing toxins to selectively bind to malignant cells. This approach combines results from cancer immunotherapy with the high toxicity of A-B toxins, giving rise to a new class of chimeric recombinant protein drugs, called immunotoxins (Allahyari et al., 2017; Li et al., 2017). Exotoxins have been harnessed to deliberately modify physiological responses near the site of

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FIGURE 9.4 Antibody–Toxin Conjugates (a subcategory of antibody–drug conjugates [ADCs]) are cytotoxic therapeutics where an antibody (pale blue and green domains) or other protein capable of binding a distinct host cell molecule with high specificity delivers its exotoxin payload (orange stars) to a particular cell population (commonly tumor cells). Target-directed cytotoxicity involves ADC binding to its target antigen on the tumor cell surface, internalization of the ADCs–antigen complex by endocytosis, cleavage of the linker (yellow rectangles) inside lysosomes to release the exotoxin payload, and transfer of the exotoxin into the cell cytosol. Target-enhanced (“bystander”) toxicity to adjacent cells may occur if the exotoxin is passed between cells through gap junctions (long white arrows) or if the exotoxin is cleaved from the ADC outside the targeted cell and instead gains entry into a neighboring cell by diffusing through the membrane (short white arrow) or binding to a toxin-specific surface receptor.

administration. The prototype for this approach is onabotulinumtoxinA (BOTOX), which is used for both cosmetic (e.g., quenching of armpit hyperhidrosis when parading the red carpet,

removal of facial wrinkles) and medical (e.g., treatment of migraine headaches and localized muscle spasticity affecting the head, eyelids, neck, and urinary bladder). Other modifications

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of botulinum toxin A (abobotulinumtoxinA and incobotulinumtoxinA) and one botulinum B variant (rimabotulinumtoxinB) also have been approved for similar uses in humans. Exotoxinbased medical solutions are tremendously diluted (e.g., less than 1 ng per vial for BOTOX, which is an over 200-fold safety margin below the calculated lethal dose in adult humans), so injected doses will be vanishingly small at short distances away from the site of administration. The toxic effects at the synaptic terminals last for 3–4 months on average, and may be modestly more prolonged after repeated injections at the same site. The botulinum toxin variants differ in terms of their diffusion away from the injection site, with onabotulinumtoxinA (BOTOX) reported to remain more highly concentrated at the injection site and thus less likely to produce adverse events, such as ptosis (e.g., drooping eyelid), as an unintended consequence of treating blepharospasm (excessive contraction of eyelid musculature) (Brodsky et al., 2012). Exotoxin contamination may occur regularly albeit at trace levels in therapeutic products (von Wintzingerode, 2017; Williams, 2019). Exotoxin adulteration is more likely in proteinbased medicines (“biologics”), which are produced in living cells grown in vats of nutrient-rich culture media. Removal of impurities is a critical step in manufacturing biologics, and typically employs a number of sizeexclusion steps (chromatography and filtration) to remove unwanted cells and cell products. Heat inactivation of biologics generally is not an option for purification since therapeutic proteins degrade in heat while many exotoxins are heat-stable. Unfortunately, standard filtration membranes have pore sizes (0.02–0.2 mm) capable of removing intact cells but not small cell fragments and exotoxins. Manufacturing processes for biologics thus rely on limiting the bioburden for selected bacterial toxins rather than trying to achieve the unattainable goal of completely eliminating all exotoxins (von Wintzingerode, 2017; Williams, 2019).

2.2. Toxicology The census of bacterial exotoxins is large and expanding. Many systems have been devised to

categorize exotoxins. Some taxonomic schemes used for exotoxins include grouping by the: • bacterium that produces the toxin, • organism(s) that is sensitive to the toxin, • order in which toxins were discovered, • system used for toxin secretion, • toxin stability in hostile environments, • toxin structure, • toxin function, • toxin mechanism of action, and • target tissue(s) for the toxin. The same exotoxin may have multiple names, which have arisen based on their importance in different research fields. For toxicologic pathology, the last three toxin classification schemesdfunction, mechanism of action, and target tissue spectrumdoffer the best scaffold for grasping the major principles related to bacterial toxicology. Exotoxin Classification by Function Taxonomic grouping of exotoxins based on function typically leads to further classification based on such major attributes as the nature of the toxic effect, the target cell population, and/ or the biological process that is disrupted by the toxin. The primary exotoxin categories defined by these active properties are cytolysins and bacterial colonization factors. CYTOLYSINS

Numerous bacterial exotoxins function as cytolysins (an abbreviation for “cytolytic toxins”). These exotoxins are important virulence factors for many gram-positive (e.g., C. perfringens, Listeria monocytogenes, S. aureus, S. pyogenes) and gram-negative (e.g., E. coli, P. aeruginosa, V. cholerae) bacteria. Cytolysin function may be further categorized based on the nature of the toxic effect and the vulnerable cell population. For example, hemolysins act to lyse erythrocytes (red blood cells [RBCs]) (Frey, 2019), while leukocidins kill various white blood cell populations involved in both the innate and acquired immune responses (e.g., dendritic cells, natural killer cells, macrophages, and T-lymphocytes). Many exotoxins that operate as hemolysins at high toxin doses (e.g., for in vitro diagnostic testing of bacterial strain virulence) act at lower (subcytolytic) doses in vivo as leukocidins (Frey, 2019). The

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leukocidin effects tend to be species-specific since exotoxins exhibit binding preferences for the b2 integrins that serve as the membrane receptors on host cells (Singh et al., 2011). The complex biological attributes of cytolysins are indicative of the wide range of potential functions associated with this exotoxin class. For example, a-hemolysin (HlyA) is a prototypic cytolysin made by E. coli strains pathogenic to humans and animals that was first identified and named based on its ability to lyse erythrocytes (RBCs). Membrane receptors for HlyA include glycophorin, a membrane-spanning sialoglycoprotein on RBCs, and integrin aLb2 (also called CD11a/CD18 and lymphocyte function– associated antigen 1 [LFA-1]), a plasma membrane-spanning adhesion protein on leukocytes. The hemolytic activity predominates at high doses of HlyA, while at lower doses HlyA is cytotoxic to other cell types, including leukocytes (mainly neutrophils and macrophages), epithelial cells, and endothelial cells by proteolysis of key intracellular signaling factors, thereby leading to host cell death variously attributed to apoptosis, pyroptosis, and necrosis (Dhakal and Mulvey, 2012; Ristow and Welch, 2016) (see also Morphologic Manifestations of Toxic Cell Injury, Vol 1, Chap 6). Importantly, HlyA is toxic to cells from a range of sensitive species, among them humans, mice, and swine (Dhakal and Mulvey, 2012; Gottschalk and Segura, 2000). In contrast to HylA, listeriolysin O (LLO, a hemolytic cytolysin made by L. monocytogenes) instead exhibits relatively low cytolytic activity at physiologic pH found in the extracellular milieu but rather functions optimally at the low pH found within lysosomes. Accordingly, LLO is instrumental as a virulence factor for allowing this obligate intracellular pathogen to escape from phagosomes into the host cell cytosol without damaging the plasma membrane and killing the host cell (Dramsi and Cossart, 2002). BACTERIAL COLONIZATION FACTORS

Another classification scheme based on exotoxin function sorts toxins according to their physiological role(s) as bacterial colonization factors to promote efficient entry and survival of bacteria into host tissues (de Souza, 2003). The members of this diverse group of toxins

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perform many key tasks in facilitating bacterial invasion and promoting bacterial disease. Adhesins promote bacterial attachment to the target cell population without inducing toxicity per se. Examples include the blood group antigen-binding adhesin (BabA) of H. pylori and transferrin-binding proteins A and B (TbpA and TbpB) for M. haemolytica. A bacterial species may actually produce many different adhesins (Singh et al., 2011). Sustained bacterial contact with the target population of host cells is instrumental in initiating toxin-induced disease. In this regard, adhesins have been shown to be key virulence factors for gastric inflammation and neoplasia associated with chronic H. pylori infection (Wen and Moss, 2009) and for pneumonia and pleuropneumonia (“shipping fever”) incited by M. haemolytica (Singh et al., 2011). Cyclomodulins hijack cell cycle control in host cells, impairing host cell division and altering host cell function (e.g., reducing the immune response and delaying programmed cell death of infected cells). The point of cell cycle arrest depends on the cyclomodulin. For instance, cholera toxin (CTX) of V. cholerae and g-glutamyltranspeptidase (GGT) of H. pylori both arrest progression at the G1 phase, shiga toxin (Stx) of Shigella dysenteriae halts division at S phase, and cycle-inhibiting factors (CIFs) of pathogenic E. coli stop the cycle at G1/S and G2/M transitions (El-Aouar Filho et al., 2017). Regardless of the stage of cell cycle arrest, the result of cyclomodulin activity is that bacteria (as individual organisms and colonies) can survive for longer periods inside or near exotoxin-controlled host cells. Evasins modulate various immunological processes during the course of bacterial infections. Examples include decreasing complement activation (Laabei and Ermert, 2019; Smith et al., 2007), reducing cytokine production (Smith et al., 2007), and formation of neutrophil extracellular traps (NETs) (von Ko¨ckritz-Blickwede et al., 2016). The result of evasin activity is to limit the innate and/or acquired immune responses of the host, thereby promoting bacterial survival. Invasins facilitate bacterial penetration into or between cells to permit microbial invasion of the underlying tissue. These exotoxins typically are secreted by bacteria associated with

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microflora-rich mucosal surfaces such as those lining the gastrointestinal tract as well as the nasal and oral passages, and they bind to receptors expressed on the cell membranes of mucosal epithelium. For example, invasins produced by intestinal Yersinia enterocolitica and Yersinia pseudotuberculosis bind to b1 integrins on the apical membranes of microfold (M) cells, a population of epithelial cells covering the surface of mucosa-associated lymphoid tissue (e.g., Peyer’s patches) that transport luminal antigens to the underlying lymphoid tissue (Kobayashi et al., 2019; Palumbo and Wang, 2006). The Yersinia invasins promote bacterial entry into the Mcells and their transfer into the underlying lymphatic system. Other species of bacterial pathogens including Listeria spp., Salmonella spp., and Shigella spp. also have evolved invasins that utilize M-cells as a portal for entering intestinal tissue. The extensive repertoire of invasins produced by the oronasal pathogen S. pyogenes includes exotoxins that serve as superantigens; as signals to control actin rearrangement in the apical cytoskeleton of host cells, thereby creating a ruffled plasma membrane and/or caveolae that cradle a bacterium as it is engulfed by endocytosis into the epithelial cell; or as proteases to alter normal host cell chemistry to aid in bacterial survival inside the cell (Rohde and Cleary, 2016; Wang and Cleary, 2019). The relative importance of the many invasins for streptococcal entry and disease initiation has yet to be defined. Modulins are exotoxins that control the functions of other cell types. Some modulins act to alter the synthesis of pro-inflammatory cytokines by host cells (Henderson et al., 1996). The most common cytokines measured in this setting are the master cytokines IL-1a, IL-6, and/or tumor necrosis factor-a (TNF-a), and they typically are released by T-lymphocytes or macrophages/monocytes. Depending on the exotoxin, cytokine production may be inhibited (uncommon) or stimulated (frequent). Other modulins regulate the activity of other bacterial proteins. Phenol-soluble modulin (PSM) is a peptide of S. aureus that facilitates the virulence of an S. aureus cytolysin known as alphatoxin (or a-hemolysin [Hla]) (Berube et al., 2014). Loss of PSM attenuates the function of Hla and reduces bacterial survival in colonized tissue.

Segregation of exotoxins by physiological function is somewhat arbitrary since exotoxins often play several roles. For instance, the OmpA outer membrane protein of E. coli has been classed as an adhesin, evasin, and invasin depending on the attribute of greatest interest (Smith et al., 2007). Indeed, some taxonomists consider the term “invasin” to represent a major subset of bacterial colonization factors that encompasses such roles as adhesins and evasins. From a practical perspective, toxicologic pathologists should recognize the major exotoxin roles that contribute to bacterial virulence and not get distracted by the overlapping designations that abound in the literature. Exotoxin Classification by Mechanism of Action Taxonomic grouping of exotoxins based on the mechanism of action yields three major groups. These classes comprise superantigens (SAgs) that bind surface receptors to produce ligandindependent intracellular signaling in host cells (Type I), membrane-damaging toxins (MDTs) that disrupt cell membrane integrity (Type II), and intracellular effector enzymes (A-B toxins) that impede or eradicate critical metabolic processes (Type III). Many bacteria produce an assortment of exotoxins that collectively employ two or all three of these toxic mechanisms. TYPE I EXOTOXINSdSUPERANTIGENS HIJACKING THE IMMUNE RESPONSE

Superantigens (SAgs) are a stable group of membrane-acting bacterial proteins that are the most potent known activators of the immune system (Fraser, 2011). Concentrations of less than 0.1 pg/mL of some SAgs are sufficient to induce fever, shock, and even death. The SAgs bind receptors on the surfaces of host leukocytes to construct immunological synapses in the absence of an antigen. In doing so, SAgs launch nonspecific immune responses rather than properly targeted and limited reactions to recognized bacterial threats (i.e., pathogen-associated molecular patterns [PAMPs]). Amino acid sequences of SAgs differ considerably, but the three-dimensional conformations of SAgs are conserved among bacterial subgroups even though different SAgs exhibit divergent preferences in terms of binding sites and affinities with respect to various host cell

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surface immunoreceptors (Fraser, 2011; Petersson et al., 2004). Dozens of SAgs have been identified, but the prototypes are the multiple variants produced by S. aureus and S. pyogenesdwhich all seem to have evolved from a single primordial ancestor (Proft and Fraser, 2003). A hypothesis to explain the evolution of such a powerful bacterial countermeasure is proposed to be downregulation of the early-stage innate immune response (to permit small numbers of bacteria to successfully colonize a new niche) rather than modulation of later-stage acquired immune responses. Bacterial SAgs commandeer the adaptive (acquired) immune response by circumventing the control mechanisms that regulate the specificity and extent of such reactions during health. Appropriate (i.e., antigen-dependent, specific) antimicrobial immune responses involve bacterial protein uptake and digestion by antigenpresenting cells (APCs) followed by presentation of small peptide sequences on the APC surface together with major histocompatibility complex class II (MHC class II) molecules. The interaction of the combined MHC–peptide complex with Tcell receptors (TCRs) expressed on the surface of helper T-cells stimulates signal transduction that is needed to mount an immune response limited to that specific peptide antigen (Figure 9.5). In contrast, SAgs elicit inappropriate (antigen-independent, nonspecific) activation of adaptive immune cells by binding MHC class II molecules on APCs (without having been phagocytosed and processed first) followed by TCRs on helper T cells (Fraser, 2011). The presence of the SAg bridge in the absence of the peptide antigen activates intracellular signal transduction in T cells (Figure 9.5), ultimately leading to production and release of proinflammatory cytokines. The potency of a SAg (as measured by its ability to stimulate T-cell activity) is the strength of its interaction with the immunological synapse. A key driver is the SAg affinity for the TCR (Arcus et al., 2000). Binding to TCRs usually involves one or several of the complementarity-determining regions (CDRs) of the beta (b) chain variable domain, but a few SAgs exploit portions of the alpha (a) chain variable region instead; the SAgs may be classified into groups based on the particular CDR(s) to which they bind. The most potent

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FIGURE 9.5 Superantigens (SAgs) Nonspecifically Activate the Host Immune System. The normal immunological synapse requires that a major histocompatibility complex (MHC) class II complex (brown heterodimer) on the surface of an antigen-presenting cell displays a peptide antigen (orange circle) to a T cell receptor (TCR [blue heterodimer]) on the surface of a T lymphocyte to yield appropriate (i.e., antigen-dependent, specific) signal transduction that launches a directed immune response. Bacterial SAgs (turquoise moon) facilitate the formation of immunological synapses in the absence of a peptide antigen, thus leading to inappropriate (i.e., antigen-independent, nonspecific) signal transduction that launches an unnecessary immune response.

SAgs have the highest TCR affinities. At least one SAg (staphylococcal enterotoxin B) also binds the costimulatory molecule CD28 (also found on the surface of T cells), which is postulated to provide a more stable immunological synapse and thus an even more prolonged SAg-driven response; knockout mice lacking CD28 are resistant to SAg-driven immune activation (reviewed in Fraser, 2011). Some SAgs also act to cross-link the subunits of MHC class II molecules, which drive APCs to increase production of their own costimulatory molecules and pro-inflammatory cytokines to further bolster the responses of T cells (Proft and Fraser, 2003). In general, SAgs have higher affinities toward human MHC class II molecules than mouse class II molecules. Accordingly, SAgs

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tend to be more potent in activating human T cells rather than mouse T cells (Proft and Fraser, 2003). Individual-specific differences in TCR structure (Proft and Fraser, 2003) or preexisting levels of anti-SAg neutralizing antibodies (Yang et al., 2006) have been proposed as explanations for stronger responses against certain SAgs that develop in people of particular ethnic backgrounds. The immune responses mediated by SAgs are inappropriate since they are not directed against any particular SAg antigen. This lack of specificity undercuts one of the fundamental strengths of the adaptive immune system as a protective response. Moreover, SAg-driven immune responses are excessive. The magnified responses initiated by SAgs are due to the much greater percentage of Tcells (up to 20%) that are triggered by nonspecific binding of SAgs to multiple TCR b chain variants relative to the very few T cells (approximately 0.0001%–0.001%) that are activated in appropriate responses that involve antigen-specific recognition of a particular MHC–peptide complex by a particular TCR single b chain (Proft and Fraser, 2003). The large numbers of activated T cells work in tandem to release a flood of proinflammatory cytokines (i.e., a cytokine ‘storm,’ designated cytokine release syndrome [CRS]), including such master molecules as interferongamma (INF-g), IL-1, IL-2, IL-6, and TNF-a (Proft and Fraser, 2003), among others. These proinflammatory factors not only induce downstream immune effector cells to escalate the immediate immune response but also provide positive feedback to macrophages (IFN-g especially) and lymphocytes to extend and sustain the systemic immune assault. At this stage, macrophages also are stimulated to make and release IL-1, IL-6, and TNF-a. The combined activity of this cytokine stew is to produce fever that may proceed in severe cases to “toxic” shock, multiple organ failure, and death. TYPE II EXOTOXINSdMEMBRANE-DAMAGING TOXINS

Membrane-damaging toxins directly impact membrane integrity by three basic means. The Type II exotoxins may create holes in the plasma membrane by forming channels that connect the cell interior with the extracellular milieu, or they may degrade individual phospholipids or dissolve larger areas of the membrane. The

common denominator of these mechanisms is that they impact plasma membrane permeability, reducing or eliminating the host cell’s ability to maintain the needed internal environmental conditions to support normal cell processes. The first mechanism is to serve as pore-forming toxins (PFTs), creating holes within target cell membranes. The PFTs are the largest class of bacterial toxins (Dal Peraro and van der Goot, 2016). About 30% of bacterial toxins are PFTs, and they are the largest class of virulence factors in antibiotic-resistant bacteria strains (Los et al., 2013). The PFT mechanism is the most common means by which cytolysins damage host cells. These exotoxins are secreted as soluble, inactive monomers that recognize with high specificity a host cell membrane receptor (consisting of lipids, polysaccharides, or proteins variously depending on the exotoxin); known PFT receptors include adhesion molecules (e.g., a disintegrin and metalloproteinase domain-containing protein 10 [ADAM10], glycosylphosphatidylinositol [GPI]-anchored proteins); chemokine receptors (e.g., C–C chemokine receptor type 5 [CCR5 or CD195] on leukocytes); and cholesterol. Captured PFT monomers undergo oligomerization to form multimeric channels that then insert into and finally span the membranes (Figure 9.6). The plasma membrane is the usual target for PFTs, but some do impact membranes of intracellular organelles (e.g., phagosomes, to permit release into the cytosol) instead. Examples of PFTs include exolysin (ExlA) by P. aeruginosa, a-hemolysin (HlyA) from E. coli, listeriolysin O (LLO) by L. monocytogenes, enterotoxin (CPE) and epsilon toxin (ETX) of C. perfringens, and streptolysin O (SLO) from S. pyogenes. Different PFTs form pores with divergent structures and functions. For example, PFTs can be classified as either a-helices or b-barrels based on the secondary structure of the membranespanning channel (Figure 9.7). The diameters of pores range from small (0.5–5 nm) to large (20– 100 nm) depending on the responsible toxin. Different host cell mechanisms are required to fix small versus large PFT-derived pores (Los et al., 2013). The materials that may travel through pores depend on the toxin. Some pores pass only specific ions (e.g., calcium [Ca2þ] or potassium [Kþ]), others permit transit of small substrate molecules like adenosine triphosphate (ATP), and some allow movement of complete

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FIGURE 9.6 Formation of Pores by Membrane-Damaging Exotoxins. Soluble monomers of pore-forming toxin (PFT [purple ovals]) in the extracellular milieu bind to a host cell surface receptor (protein [green] and/or lipid) and undergo oligomerization to generate a circular complex with central lumen. The PFT complex then is inserted into the cell membrane, thereby opening a channel that permits exchange (entrance [white arrow] or exit) of fluids and molecules between the exterior microenvironment and the interior of the cell.

FIGURE 9.7 Structure of a Prototypic Pore-forming Toxin (PFT). Crystallographic depiction of a-hemolysin, a cytolytic exotoxin of Staphylococcus aureus that produces a transmembrane channel characterized by seven identical protein units (a homoheptamer) forming an w1.4-nm-diameter b-barrel pore that readily passes small cations (e.g., Ca2þ, Kþ) and fluid, leading to cell swelling and death. Reproduced under a Creative Commons license (CC BY-SA 3.0) from https://en.wikipedia.org/wiki/Pore-forming_toxin#/media/File:7ahl.png.

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proteins. Most bacterial PFTs are associated with an efflux of Kþ and/or an influx of Ca2þ, which lead to activation of such cellular defensive mechanisms as enhanced extracellular signal– regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) signaling as well as activation of the Nod-like receptor pyrin domaincontaining 3 (NLRP3) inflammasome and cysteine-aspartic protease 1 (caspase-1) (Dal Peraro and van der Goot, 2016; Los et al., 2013). The outcome of such signaling alterations is to stimulate intracellular membrane lipid biogenesis and inhibit protein synthesis to redirect energy consumption until membrane repairs may be completed. In addition, Ca2þ influx facilitates lysosomal exocytosis and the extracellular release of acid sphingomyelinase, a lysosomal enzyme involved in membrane reorganization. If membrane restoration is unsuccessful, the enhanced intracellular ion levels activate proteolysis and caspase-dependent and caspaseindependent programs that ultimately lead to cell death and lysis. Other functions of PFTs also contribute to bacterial colonization and expansion in tissues, primarily by disrupting epithelial and endothelial barriers in many organs (especially the brain, intestine, and lung) as well as evading the host immune response (Los et al., 2013). Barrier dysfunction results from damage to epithelial or endothelial cells either directly by the PFT or indirectly by PFT-induced inflammation (mainly infiltration of neutrophils). Damage and death of barrier epithelial cells lead to edema and hemorrhage, which both provide a nutrient-rich milieu for bacterial growth. Evasion of the host immune response by PFTs may occur by a number of mechanisms. Leukocidins directly kill cells involved in the immune response. Some PFTs (e.g., LLO from L. monocytogenes and early secretory antigenic target [ESAT-6] generated by various Mycobacterium spp.) promote intercellular exchange of bacteria between macrophages and expulsion of phagocytized bacteria into the cytosol. Certain PFTs downregulate the degree of inflammation; S. aureus a-toxin induces mast cell degranulation, which limits inflammation. Finally, some circulating PFTs alter vascular resistance via induction of eiscosanoid-mediated vasoconstriction (via thromboxane A2 or various leukotrienes depending on the exotoxin). This response

leads to both reduced influx of circulating leukocytes and tissue hypoxia, both of which can bolster the invasiveness and survival of bacteria. The second mechanism by which MDTs damage host cell membranes is by enzymatic digestion (hydrolysis) of phospholipids. The enzymatic activity is directed preferentially against the lipids in the outer portion of the bilaminar membrane, such as phosphatidylcholine and sphingomyelin. Degradation of the lipid components leads to weakness and eventual loss of membrane integrity (Figure 9.8). The biological consequences of exotoxin binding depend on the host cell type (Oda et al., 2015). Examples of this cytolysin class include a-toxin (phospholipase C activity) of C. perfringens, b-toxin (sphingomyelinase C) from S. aureus, and multiple phospholipases by Vibrio spp. The third principal mechanism by which MDTs injure host cell membranes is by acting as surfactants. These molecules are amphipathic (also called amphiphilic) lipopeptide detergents, which allow them to attach directly and nonspecifically (i.e., without a receptor) to the plasma membrane and integrate into the lipid-rich (hydrophobic) shells of both

FIGURE 9.8 Lipid Hydrolysis as a Mechanism of Exotoxin-Induced Membrane Damage. Bacterial proteins (gray globules) with lipase activity destroy lipids in host cell membranes by hydrolytic cleavage to separate the charged hydrophilic “heads” (yellow circles) from the hydrophobic fatty acid “tails” (yellow corkscrew lines), thereby opening irregular holes that permit entrance and egress of key molecules needed to sustain cell homeostasis and survival. Adapted from Sonnen AF, Henneke P: Role of pore-forming toxins in neonatal sepsis, Clin Dev Immunol 2013:608456, 2013 under a Creative Commons license (CC BY 3.0).

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prokaryotic cells (i.e., act as an antibiotic) and eukaryotic cells (including selected fungi and some mammalian cell lineages) (Otto, 2014). Entrance of these surfactant molecules into cell membranes alters membrane integrity (Figure 9.9), leading to dissolution and local disintegration of the lipid bilayer leading to formation of irregular holes in the membrane. The presence of such holes may or may not lead to cytolysis. Examples include phenolsoluble modulins like d-toxin of S. aureus as well as kurstakin and surfactin from Bacillus subtilis and Enterobacter cloacae.

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For example, B. anthracis produces a three-part A-B toxin comprised of two “A” subcomponents (edema factor [EF] and lethal factor [LF]), and a single “B” element (protective antigen [PA]), while the six-part “AB5 toxins” of Bordetella pertussis (pertussis toxin [Ptx]), S. dysenteriae (shiga toxin [Stx]), and V. cholerae (cholera toxin [CTX]) have one “A” domain and five “B” subunits. Binding of the “B” domain to the host cell receptor leads to a conformational change that facilitates endocytosis of the toxin–receptor complex (Figure 9.10). The “A” domain of the

TYPE III EXOTOXINSdINTRACELLULAR EFFECTOR ENZYMES

The Type III exotoxins, usually designated A-B toxins, are two-domain polypeptides that alter host cell function by delivering an active enzyme into an intracellular compartment to modify a cellular process necessary for normal cell function or survival. The “A” part of the toxin is the enzymatic (“active”) region, while the “B” part engages (“binds”) a receptor on the host cell. The two domains may consist of multiple units.

FIGURE 9.9 Surfactant Degradation as a Mechanism of Exotoxin-Induced Membrane Damage. Bacterial lipopeptides (gray corkscrews) with an amphipathic tertiary structure (i.e., having both hydrophilic and hydrophobic domains) attach directly and nonspecifically (i.e., without a receptor) to the plasma membrane, where they act as detergents to dissolve the lipid layers and release intact phospholipid molecules having both hydrophilic “heads” (yellow circles) and hydrophobic fatty acid “tails” (yellow corkscrew lines). Adapted from Sonnen AF, Henneke P: Role of pore-forming toxins in neonatal sepsis, Clin Dev Immunol 2013:608456, 2013 under a Creative Commons license (CC BY 3.0).

FIGURE 9.10 Mechanism of Receptor-Mediated Host Cell Damage by A-B Toxins. These paired polypeptides consist of a binding (“B”) domain (orange diamonds) that interacts with a surface receptor to initiate endocytosis and an active (“A”) domain (orange circles) that is released in the endosome and translocated into the cytosol where it acts enzymatically to inhibit (red inverted “T”) host cell processes by modifying substrates necessary for normal metabolism and cell survival (shown here for ribosomal translation of mRNA into protein [jagged green line]). After release of the A domain, the B domain usually is expelled from the cell by exocytosis.

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toxin then is released and translocated into the cytosol, while the “B” part usually is expelled from the cell by exocytosis (Figure 9.10). Once inside the cell, the “A” enzyme inhibits cell function by modifying substrates needed for cell metabolism and survival. A primary mechanism is to introduce posttranslational covalent modifications to proteins (Simon et al., 2014). The primary mechanism by which A-B toxins accomplish this task is to act as poly(adenosinediphosphate)-ribosyltransferases that add ADP-ribose to various proteins. The impact of ADP-ribosylation depends on the molecular site of covalent modification. For example, diphtheria toxin and exotoxin A of P. aeruginosa both phosphorylate eukaryotic elongation factor 2 (eEF2), which inhibits protein synthesis by keeping eEF2 from stabilizing the ribosome and facilitating ribosome conformational changes needed for mRNA translation. In contrast, CTX, Ptx, and heat-labile E. coli enterotoxin ADP-ribosylate certain G proteins, locking them in a conformation that constitutively stimulates host cell adenylate cyclase to produce a persistent rise in levels of the second messenger molecule cAMP (cyclic adenosine monophosphate) and uncoupling of cAMPregulated intracellular processes. Multiple exotoxins utilize ADP-ribosylation to activate the NLRP3 or pyrin inflammasomes (Greaney et al., 2015). Multiple Type III clostridial exotoxins including C. botulinum C2 toxin and C. perfringens iota toxin attach ADP-ribose to actin, which inhibits actin polymerization and destroys cytoskeletal integrity. Any of these disruptions will produce cell stress, and if sustained they may lead to cell death. Exotoxin Classification by Target Organ Spectrum Bacterial exotoxins exert their cytotoxic effects in many organs, and may affect cell types differentially within those organs. This section briefly introduces key target organs and major bacterial exotoxins that affect them. A given bacterial species may produce multiple exotoxins that attack a particular target organ or cell type, and many exotoxins are capable of assaulting a variety of cell types and thus may be included among several of the target organ categories that are given below.

ENTEROTOXINS

Enterotoxins are produced by many bacteria that reside within the gastrointestinal tract, but they also may be found as contaminants in foods or liquids. Major enterotoxin producers include S. aureus and many clostridial species (notably C. difficile and C. perfringens). Staphylococcal enterotoxins (SEs) and enterotoxin-like proteins (SEls) comprise a superfamily of two dozen low-molecular-weight superantigens that characteristically cause emesis in many model species including cats, dogs, ferrets, pigs, primates, and shrews (Fisher et al., 2018). Enterotoxemia is by definition a disease in which bacterial overgrowth in the intestine leads to high levels of intraluminal and circulating bacterial enterotoxins and ultimately to toxicity in distant organs. A classic example of this phenomenon is “overeating disease” of young sheep, goats, and occasionally cattle, produced mainly by excessive consumption of a carbohydrate-rich (mostly grain-based) diet. Permissive conditions in the intestine permit the overgrowth of C. perfringens type D and generation of high levels of epsilon toxin (ETX), a PFT that acts primarily on endothelial cells to alter vascular permeability. Primary tissue findings in ruminants induced by ETX include extensive fluid leakage (i.e., edema) and, in severe cases, hemorrhage. Blood-borne spread of ETX is responsible for edema in multiple organs and, in subacute to chronic cases, focal symmetrical encephalomalacia (i.e., bilateral necrosis of the corpus striatum, cerebellar white matter, and other brain regions). Enterotoxemia due to staphylococcal SE type B is one major cause of system-wide immunological activation (i.e., toxic shock syndrome). HEMOLYSINS

Hemolysins are cytolytic exotoxins that are capable of destroying RBCs (Frey, 2019). The ability to lyse RBCs is a dose-dependent property. Many exotoxins are hemolytic at high toxin doses, such as those encountered during in vitro diagnostic testing of bacterial strain virulence, but do not produce RBC lysis at lower concentrations (Frey, 2019). This dichotomy is true of E. coli a-hemolysin (HlyA), the prototypic toxin that targets RBCs, and is typical of its activity in such sensitive species as humans, mice, and

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pigs (Dhakal and Mulvey, 2012; Gottschalk and Segura, 2000). These exotoxins may act as PFTs (e.g., S. aureus a-hemolysin); hydrolytic enzymes (e.g., S. aureus b-hemolysin, which acts as a sphingomyelinase); or surfactants (e.g., S. aureus d-hemolysin and phenol-soluble modulins [PSMs]) (Trstenjak et al., 2020; Vandenesch et al., 2012). LEUKOCIDINS

Leukocidins are cytolytic exotoxins that destroy white blood cell lineages involved in both the innate and acquired immune responses (Frey, 2019). Innate immune cells like neutrophils and macrophages are common targets, and highly virulent bacteria (e.g., S. aureus) tend to make three to five leukocidins (Alonzo and Torres, 2014). Many also function as hemolysins at high concentrations. Leukocidins bind to species- and cell-type-specific b2 integrins (Singh et al., 2011; Trstenjak et al., 2020) and subsequently act as bicomponent b-barrel PFTs comprised of ligand-binding S components and pore-forming F components (e.g., S. aureus ghemolysin and Panton Valentine leukocidin [PVL]) or amphipathic surfactants (e.g., S. aureus d-hemolysin and PSMs) (Trstenjak et al., 2020; Vandenesch et al., 2012). MYOTOXINS

Myotoxins are bacterial exotoxins that serve as key drivers of “histotoxic” infections. Two classic histotoxic diseases are clostridial-induced rhabdomyositides: blackleg in cattle, and gas gangrene in humans and several animal species (particularly guinea pig and mouse models) (Uzal et al., 2015). In blackleg, blunt trauma without penetration induces necrosis of skeletal muscle; the resulting anaerobic conditions permit germination of C. chauvoei spores that are latent within the tissue (after having arrived in the blood following uptake in the intestine). In contrast, gas gangrene results from contaminated wounds that penetrate the deep subcutis or underlying skeletal muscle and lead to compromised microvascular circulation, reduced oxygen tension, and growth of clostridial spores (chiefly C. perfringens [80% or more of cases in humans] but also C. septicum in other species) in the devitalized or dead tissue, and finally the release of multiple exotoxins with divergent functions. Many

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myotoxins are proteolytic enzymes (e.g., C. perfringens a-toxin [phospholipase C or lecithinase], collagenase, and hyaluronidase). Others are cholesterol-dependent cytolysins (CDCs), which use membrane cholesterol as receptors to assemble transmembrane pores (e.g., C. perfringens perfringolysin [PFO]). Together, these exotoxins dissolve cells and connective tissue to facilitate bacterial spread. Interestingly, a-toxin and PFO prevent infiltration of host leukocytes into the affected tissue, thereby protecting the bacterial horde from immune-mediated lysis. Some bacterial exotoxins have been shown to injure cardiomyocytes. This “cardiotoxin” role has been suggested to be a contributing factor in human patients with septic cardiomyopathy secondary to pneumonia (Anderson et al., 2018; Monticelli et al., 2018) and it has been seen experimentally in mice and immunosuppressed rhesus macaques treated with pneumolysin (PLY, a CDC-type PFT released by the death of Streptococcus pneumoniae) (Alhamdi et al., 2015; Brown et al., 2014) and zebrafish embryos exposed to C. difficile toxin B (Hamm et al., 2006). The toxic properties of PLY depend on the exotoxin level in tissues (Anderson et al., 2018). High PLY concentrations incite irreversible pore formation in many cell typesdblood cells (leukocytes, platelets, RBCs), cardiomyocytes, epithelial cells, and endothelial cellsdthat culminates in programmed cell death by apoptosis and necroptosis (Anderson et al., 2018). Low PLY concentrations produce transient pore formation and influx of Ca2þ that is followed by membrane repair. However, this sequence of sublethal events activates both neutrophils and platelets, thereby producing a pro-inflammatory and prothrombotic state that increases the likelihood of ischemia and indirect damage to stressed cardiomyocytes. The pathogenesis of C. difficile toxin B involves disruption of cytoskeletal and tight junction integrity, which progresses to cell rounding and ultimately cell death (Di Bella et al., 2016). In cardiomyocytes, cell demise is reported to occur by caspase-3-mediated apoptosis (Hamm et al., 2006). NEUROTOXINS

Many bacterial species produce neurotoxic exotoxins, but by far the greatest numbers

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are made by clostridial species (Popoff and Poulain, 2010). The main clostridial neurotoxins include botulinum neurotoxin (BoNT) from C. botulinum (Cope, 2018), tetanospasmin (i.e., tetanus neurotoxin [TeNT]) from C. tetani (Hassel, 2013), and epsilon toxin (ETX) from C. perfringens types B and D (Xin and Wang, 2019). Recent work has shown that BoNT also is produced by a few nonclostridial species of bacteria (Popoff, 2018; Poulain and Popoff, 2019). Other less potent but nonetheless lethal bacterial neurotoxins include saxitoxin (Chun et al., 2018; Orr et al., 2013) and tetrodotoxin (TTX) (Magarlamov et al., 2017; Wu et al., 2005). The clostridial neurotoxins also are the most potent neurotoxic exotoxins, and indeed are the most lethal poisons yet identified. Lethal doses in mice for clostridial neurotoxins range from 0.0003 mg/kg for BoNT and 0.001 mg/kg for TeNT to 100 mg/kg for ETX (Popoff and Poulain, 2010; Uzal et al., 2010). In contrast, mouse lethal doses of other bacterial neurotoxins (e.g., C. difficile toxins A and B, E. coli and S. aureus enterotoxins, and V. cholerae cholera toxin) are higher by at least 200-fold (Popoff and Poulain, 2010). Bacterial neurotoxic exotoxins act by several different mechanisms (Popoff and Poulain, 2010). Staphylococcal enterotoxins serve as superantigens (Type I exotoxins). C. perfringens ETX and S. pneumoniae PLY are pore-forming molecules (Type II exotoxins). Most other bacterial neurotoxins disrupt intracellular processes (Type III exotoxins). For example, BoNT and TeNT are enzymes that degrade proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex; damage to the SNARE complex prevents neurotransmitter secretion by blocking fusion of synaptic vesicles with the plasma membrane (Yoon and Munson, 2018). Toxins A and B from C. difficile are glucosyltransferases that inactivate many Rho GTPases, which are essential players in many physiological processes and signaling pathways (Chen et al., 2015). E. coli enterotoxins and cholera toxin increase the intracellular concentrations of second messengers, thus disrupting signaling cascades. Saxitoxin and TTX are selective blockers of voltage-gated Naþ channels. The final outcome of these neurotoxic exotoxins is that neurotransmitter secretion is altered, with some acting to

inhibit release (BoNT, TeNT, toxin B) while others stimulate release (cholera toxin, ETX, E. coli and S. aureus enterotoxins, toxin A). The neurotransmitter that is affected and the clinical presentation of neurotoxic disease depend on the cell population that has been targeted (Popoff and Poulain, 2010). The unique biochemistry of the bacterial neurotoxins and their receptors helps to explain their distinct effects. Comparison of the clostridial products BoNT and TeNT, the most (BoNT) and second-most (TeNT) potent natural biotoxins, provides a useful example of this principle. Both toxins share a common structure: production as an inactive single-chain precursor protein (w150 kDa), with activation after release (by clostridial or host cell proteases) to paired light (LC, w50 kDa) and heavy (HC, w100 kDa) chains linked by a disulfide bridge (Figure 9.2). The LC harbors the catalytic domain, while the HC ensures endocytosis of the toxin. The eight BoNT variants and TeNT share a conserved amino acid sequence that ranges from 34% to 97%. Each type of BoNT and TeNT recognizes specific receptors (involving both gangliosides and synaptic proteins) on terminal nerve endings, mainly through the HC C-terminal (HCC) domain. The high affinity of the BoNTs and TeNT for axonal presynaptic membranes at the very low tissue concentrations needed for toxicity likely reflects the multiple and synergistic interactions of this dual-receptor apparatus. Interestingly, the identified protein receptors are expressed on multiple cell types including intestinal crypt epithelium, so at high toxin levels these neurotoxins also elicit toxicity (i.e., inhibited vesicle trafficking) in nonneuronal cells. Neurotoxin bound to its receptor is internalized by receptor-mediated endocytosis, by different mechanisms. The BoNTs are taken up directly in recycling synaptic vesicles or clathrin-coated vesicles at the axon terminus, after which acidification leads to translocation of the LC into the cytosol. In contrast, TeNT instead enters nonacidified endocytic vesicles at the axon terminus that carry the toxin by microtubule-dependent retrograde transport to the neuronal cell body in the spinal cord gray matter; subsequent release of TeNT into the extracellular space allows the toxin to migrate into nearby inhibitory interneurons via acidified vesicles,

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finally releasing the LC. These differences explain why BoNT acts in the peripheral nervous system while TeNT acts in the central nervous system.

3. ENDOTOXINS The technical designation for these bacterial toxins is lipopolysaccharide (LPS). These large molecules function to make the bacterial cell wall of gram-negative bacteria (GNB) both less permeable (to large hydrophobic molecules and microbicidal enzymes) and more stable (Todar, 2020a). As noted implicitly in the LPS designation, endotoxin is a complex molecule consisting of a lipid anchor within the outer membrane of the cell wall and long polysaccharide chains that project into the extracellular environment; toxicity results from exposure to the lipid. Toxicity requires substantially larger levels of circulating endotoxin (w2.5 mg/kg) compared to exotoxins, which are lethal at doses in the mg/kg and even ng/kg range. Endotoxin quantities are expressed as endotoxin units (EUs) since the function and structure of endotoxins differ substantially among GNB species based on variations in both the lipid and polysaccharide portions of LPS. Endotoxin contamination formerly was assessed in vivo by parenteral administration to rabbits, but EU calculations now are performed in vitro using the limulus amebocyte lysate (LAL) assay. The main reagent for this assay is a lyophilized homogenate of pericardial immune cells (amebocytes) harvested from the hemolymph of the Atlantic horseshoe crab (Limulus polyphemus). Endotoxin (specifically the lipid component) will, when mixed with the lysate, activate a cascade of amebocyte serine proteases that in turn cause other lysate proteins to gel (coagulate). Other horseshoe crab species (e.g., Tachypleus gigas or Tachypleus tridentatus) are employed similarly in Asia to prepare reagents for the tachypleus amebocyte lysate (TAL) test. These gel-clot techniques are recognized by the US FDA as suitable tests for assessing pyrogen contamination (i.e., endotoxin at pg levels) that is likely to occur during production of biologics, drugs, and medical devices (FDA, 2012b; Hurley, 1995).

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3.1. Sources of Exposure Endotoxins are released from disintegrating gram-negative bacteria but are essentially absent in gram-positive bacteria (Todar, 2020a). Humans and animals are exposed to endotoxins in several settings. Infection Gram-negative bacteria (GNB) cause disease in almost all body systems of humans and animals due to their proclivity to invade the bloodstream (bacteremia). Key GNB pathogens include the Enterobacteriaceae (E. coli, Klebsiella spp., Salmonella spp., Shigella spp., Vibrio spp., Yersinia spp.) as well as nonenteric species like Neisseria meningitides and P. aeruginosa. Mortality from GNB bacteremia is high in both humans and animals. Endotoxemia is a major factor in this mortality even though the circulating endotoxin levels are not well correlated with more severe clinical disease and vary for different bacterial species (Hurley and Opal, 2013). The principal organs targeted by GNB are the intestine and lungs due to local overgrowth of resident flora as well as the nervous system (meninges) and urinary tract during episodes of bacteremia. Intervention to remove the source of infection (e.g., antibiotic therapy, surgical debridement) is essential to treating GNB disease but typically cannot quench any endotoxin already released into the circulation. In fact, successful antibiotic therapy may exacerbate endotoxemia since dying bacteria will release even more cell wall fragments as they disintegrate. The stability of endotoxins with respect to enzymatic and thermal degradation means that endotoxins will remain active for long periods even after the initial infection has been resolved. Ingestion Endotoxin may be present in food and water when comestibles are contaminated by GNB (e.g., E. coli, Salmonella spp., Shigella spp., Vibrio spp.). Common sources of GNB and thus endotoxin ingestion include food supplements based on live bacterial cultures (Wassenaar and Zimmermann, 2018), produce (Uhlig et al., 2017), and fecal-adulterated or improperly purified water (Stojek and Dutkiewicz, 2011; Stojek et al., 2008). Nonetheless, direct ingestion of endotoxin will have a negligible impact (if any)

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on health because the quantity of endotoxin in food is dwarfed by the amount produced in the intestinal tract (Wassenaar and Zimmermann, 2018). Instead, food poisoning linked to GNB contamination is attributed to exotoxins rather than endotoxin (Herna´ndez-Cortez et al., 2017). Inhalation Endotoxin exposure may occur via the respiratory tract. Resident GNB in the nasal and oral cavities produce endotoxin, although their low numbers during health and disease (e.g., periodontitis, sinusitis) ensure that a minimal amount of endotoxin will be inhaled. Endotoxin is found ubiquitously on the surfaces of animals, plants, soils, and structures in environments where GNB are common, including both occupational settings (e.g., agricultural operations with high densities of animal feces or grain dust, textile factories, waste processing plants) and conventional housing (e.g., air-conditioned, well-sealed spaces with absorbent materials [carpets, mattresses, pillows, etc.] and/or pets). Levels of particulate-adsorbed endotoxin in workplaces (thousands of EU/m3) are several hundred-fold higher than those aerosols encountered in domestic settings (90%) of pyrogen testing in qualifying therapeutic product lots is performed using the LAL assay. Disadvantages of this assay are that the composition of the amebocyte lysate varies from lot to lot, the manufacturing process for certain products includes steps that may yield false-negative results (e.g., aluminum hydroxide adjuvants in vaccines interfere with the LAL assay) or false-positive results (e.g., the glucan in cellulose filters used in vaccine clarification [purification] can stimulate the gelling reaction of the amebocyte lysate). A final disadvantage in pyrogen testing of therapeutic products is that neither of the classic pyrogen testsdthe in vitro LAL assay and the in vivo rabbit pyrogen test (RPT)drespond to nonendotoxin pyrogens (e.g., mainly exotoxins and fungal toxins) with the sensitivity shown by human blood leukocytes. Alternative pyrogen tests developed in recent years to address these disadvantages and reduce animal use include (1) the monocyte activation test (MAT), which uses sensitive human blood cells (fresh or frozen) and therefore is capable of assessing all pyrogens, and (2) the recombinant factor C (rFC) assay, which is based on a cloned version of the horseshoe crab clotting factor that reacts with endotoxin. These new assays have yet to achieve widespread use. Endotoxin limits vary depending on the route of administration and the therapeutic product. For example, the endotoxin limits for injectable therapies (bolus dose or cumulative dose in a 1-h period) for human use are 0.2 EU/kg for intrathecal (or equivalent) delivery into the cerebrospinal fluid (CSF) and 5.0 EU/kg for parenteral routes that do not offer direct access to the brain (FDA, 1985). These limits represent the estimated thresholds for inducing fever in humans and rabbits, which are highly sensitive to endotoxins. In contrast, the endotoxin allowances for implanted materials are 2.15 EU/ device if placed in direct contact with the CSF

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and 20 EU/device for other locations (USP-NF, 2016). No endotoxin limits apply to materials slated for oral administration.

3.2. Toxicology Endotoxin is not encoded by genes but rather is assembled enzymatically from lipid and polysaccharide building blocks. The structure of endotoxins from various bacterial species shares a common scheme in which multiple polysaccharide motifs extend outward from a phospholipid moiety that anchors endotoxin in the outer lipid layer of the outer cell wall (Figure 9.11) (Todar, 2020a). Furthermore, endotoxin from all GNB species is sufficiently similar that they execute a comparable set of molecular events that abolishes the normal immune function of affected hosts. Therefore, in practical terms endotoxin may be viewed as a single entity regardless of the GNB that produce it. Structure and Functional Attributes of Endotoxin Endotoxin is known alternatively as lipopolysaccharide (LPS) due to its chemical structure. Most research performed to elucidate the basic composition of endotoxin has used various E. coli strains and Salmonella species. The polysaccharide portion of endotoxin consists of an outer oligosaccharide sequence (O-antigen) and a deeper core (Figure 9.11). The O-antigen consists of repeating units each comprised of three to five monosaccharides, often unique dideoxyhexoses not found in nature except in the cell walls of GNB. Organization of the individual O-antigens varies in terms of length (up to 40 repeat units) and structure (branching or linear); the composition of Oantigens varies considerably among GNB species, strains, and serovars. This endotoxin region is hydrophilic and negatively charged, thereby maintaining moisture to support bacterial processes while repelling potential microbicidal proteins; the region also has the antigenic epitopes against which a weak host immune response may be raised. The core connects the O-antigen to the lipid. Organization of the core is conserved with minor variations for all members of a given bacterial genus, but the cores of different bacterial genera are distinct. The inner core links to the lipid, is more highly

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conserved, and is built of both heptulose monosaccharides (the most common of which is L-glycero-a-D-manno-heptopyranose) and noncarbohydrate components (e.g., amino acids, ethanolamine, and phosphate groups); the outer core links to the O-antigen, is less conserved, and is constructed as a chain of three hexoses (D-glucose, D-galactose, and/or D-mannose). The Kdo (2-keto-3-deoxyoctonoic acid) element of the inner core is chemically unique and always present in LPS, so it can be used as a reliable indicator for the presence of endotoxin. The phosphate groups of the inner core increase the overall negative charge of the cell membrane, which helps to stabilize the membrane and repel positively charged microbicidal molecules. The conformation of the O-antigen is one factor that determines virulence for GNB. Small differences in composition of the O-antigen yield major alterations in virulence. Bacteria with intact O-antigens grow as “smooth” colonies when cultured, while disruption in the O-antigen (especially the terminal monosaccharides that are primary contributors to its recognition by host immune cells) leads to expansion as “rough” colonies. Possible mechanisms whereby O-antigen disruptiondleading to a loss of “smoothness”dmay lessen GNB virulence include decreased adherence to target cells, especially in epithelial tissues like intestine; heightened vulnerability to phagocytosis; and reduced capacity to fend off microbicidal host immune proteins like antibodies and complement. Alterations to the O-antigen do not impact the structure of the core and Lipid A domains, and thus do not alter the inherent toxicity of endotoxin. The lipid portion of endotoxin, termed Lipid A, is the hydrophobic domain that anchors the entire LPS molecule to the outer layer of the bacterial cell wall. Lipid A is a phosphorylated Nacetylglucosamine (NAG) dimer with six or occasionally seven fatty acid chains (Figure 9.12); the fatty acids are long and saturated, and thus insert readily into the lipid layer of the outer cell wall. The structure of Lipid A is highly conserved among GNB, and is virtually identical among the Enterobacteriaceae (e.g., E. coli, Salmonella spp., Shigella spp., Vibrio spp., Yersinia spp.). In disease, however, pathogenic GNB can alter the structure of Lipid A in order to evade the host immune response (Bishop, 2005).

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FIGURE 9.11 Structure of Bacterial Endotoxin. Endotoxins from gram-negative bacteria (GNB) are lipopolysaccharides (LPSs) characterized by a multidomain polysaccharide portion anchored by a lipid moiety embedded in the outer membrane (cell wall). The polysaccharide portion is the superficial hydrophilic, negatively charged, weakly antigenic, chemically variable O-antigen (composed of up to 40 linear or branching repeated units) and a deep GNB genus-specific core; the inner core that links to the Lipid A domain is more conserved, and its unique Kdo (2-keto-3-deoxyoctonoic acid) element is a reliable biomarker for the presence of endotoxin. The hydrophobic Lipid A portion embedded in the outer membrane is comprised of multiple long fatty acid chains suspended from two N-acetylglucosamine (NAG) molecules, is highly conserved among all GNB, and is the LPS domain that is responsible for endotoxin activity. The schematic representation depicted here is for Salmonella enterica serovar Typhimurium, a primary enteric pathogen for humans and animals.

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FIGURE 9.12 Structure of Lipid A, the Toxic Portion of Bacterial Endotoxin. The injurious lipid region of endotoxin consists of a phosphorylated N-acetylglucosamine (NAG) dimer that is attached to six (or rarely seven) long, saturated, linear or branching fatty acid chains. The fatty acid chains extend into the outer phospholipid layer of the outer membrane where they serve to anchor the entire endotoxin molecule to the cell wall. The schematic diagram shown here is for Salmonella enterica serovar Typhimurium, a primary enteric pathogen for humans and animals.

As noted above, Lipid A is the toxic component of endotoxin, and its biological activity is thought to depend on the structure of the combined Lipid A/inner core, which are not present in eukaryotes. Lipid A must be intact to sustain bacterial viability, likely to aid in assembling and stabilizing the outer layer of the cell wall barrier. In this regard, Lipid A is not a virulence factor in the traditional sense because it is only toxic when released from disintegrating GNB. On the other hand, Lipid A may be viewed as a virulence factor since its potent modulation of the host immune response presumably improves the prospect that other still-viable GNB will survive to continue the invasion. Endotoxin-Mediated Cell Signaling Upon reaching the blood, endotoxin is captured by LPS-binding protein (LBP) (Morris et al., 2014; Pa˚lsson-McDermott and O’Neill, 2004; Park and Lee, 2013). An acute-phase protein made in the liver, LBP forms a highaffinity complex with endotoxin in all its forms: free-floating molecules, cell wall fragments, or cell walls of intact bacteria. The LBP–endotoxin complex subsequently docks with CD14, a protein expressed mostly by macrophages, dendritic cells, and monocytes and available in both a membrane-bound form (mCD14) anchored to myelomonocytic cells by a glycosylphosphatidylinositol tail and a soluble form

(sCD14) that circulates in the blood. Both CD14 variants are pattern recognition receptors (PRRs) that provide early warning of bacterial PAMPs indicative of GNB invasion; mCD14 presents endotoxin to LPS receptors on the same myelomonocytic cell while sCD14 delivers endotoxin to LPS receptors that lack mCD14 (e.g., endothelial cells). The ternary LBP–endotoxin–CD14 complex delivers endotoxin to LPS receptors on the surfaces of many cell types. In doing so, CD14 reduces Lipid A aggregates into Lipid A monomers that will fit within the recognition site of the LPS receptors borne by innate immune cells. The LPS receptor complex is comprised of myeloid differentiation factor-2 (MD-2), an extracellular adapter protein, and Toll-like receptor-4 (TLR4), a transmembrane signaling receptor (Morris et al., 2014; Pa˚lsson-McDermott and O’Neill, 2004; Park and Lee, 2013). Endotoxin binds first to MD-2, which is an essential event for endotoxin recognition. The endotoxin–MD2 complex then associates with a TLR4 molecule, which induces TLR4 aggregation on the membrane surface. Aggregation of multiple TLR4 brings their intracellular Toll/IL-1 receptor (TIR) homology domains into proximity. The TIR domains then recruit intracellular adapter molecules like myeloid differentiation factor 88 (MyD88). The clustering of MyD88 death domains (DDs) in turn enlists multiple proteins

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with both DDs and serine/threonine kinase domains (e.g., IL-1 receptor associated kinases [IRAK2 and IRAK4]) to form a large towerlike complex termed a Myddosome. Assembly of the Myddosome launches signal transduction (De Nardo et al., 2018). Early endotoxin-related signaling events are dependent on MyD88 and include activation of tumor necrosis factor receptor–associated factor 6 (TRAF6) followed by transforming growth factor-b–activated kinase 1 (TAK1, a mitogen-activated protein kinase [MAPK]), the latter of which serves as a common activator of three key signaling cascades: the MAPKs jun N-terminal kinase (JNK) and p38, and the transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NFkB). Later endotoxinrelated signaling events, which employ the intracellular adapter molecule TRAM (TIRdomain-containing adapter-inducing interferon-b [TRIF]-related adapter molecule) instead of MyD88, include activation of several interferon regulatory transcription factors (IRFs, especially IRF3) and NFkB. Endotoxin may act via a TLR4–TRIF interaction to exert an antiinflammatory signaling cascade (Morris et al., 2014). The shape of the endotoxin molecule determines its TLR specificity (Netea et al., 2002). Conical Lipid A (e.g., from E. coli and other Enterobacteriaceae) is a specific ligand for TLR4. In contrast, cylindrical Lipid A (e.g., Rhodobacter spp.) prefers TLR2 and may actually act as an antagonist for endotoxin binding at TLR4. This phenomenon runs counter to the usual TLR2 preference for bacterial-derived ligands, which is for lipoproteins in the outer membranes of gram-positive bacteria (Oliveira-Nascimento et al., 2012). Entry of endotoxin into the cell occurs by receptor-mediated endocytosis. Uptake may exploit the LPS receptor (CD14–MD-2–TLR4) for this purpose or employ another receptor (e.g., the low-density lipoprotein [LDL] receptor to remove endotoxin-laden chylomicrons from the blood) (Ghermay et al., 1996). Clearance of circulating endotoxin may reduce mortality from septic shock; in most species, this uptake is mediated chiefly by the liver (sinusoidal macrophages [Kupffer cells] followed by hepatocytes) (Munford, 2005; Nolan, 2010). Inhaled endotoxin is removed by alveolar macrophages

in the lung. However, subsequent translocation of endotoxin into the cytosol may initiate the NLRP3 inflammasome by a noncanonical pathway with caspase-11 acting as a cytosolic LPS receptor and an effector protein that promotes pyroptotic cell death (Deng et al., 2018; Vanaja et al., 2015). Chronic or massive endotoxin exposure that overwhelms clearance and detoxification mechanisms may initiate or exacerbate liver, lung, or renal disease (Nakatani et al., 2001). Pathogenesis of Endotoxin-Induced Immune Dysfunction Endotoxin is a potent modifier of the host immune response (Morris et al., 2014; Pa˚lssonMcDermott and O’Neill, 2004; Park and Lee, 2013; Radon, 2006). The chief culprit is the Lipid A portion, and its primary effect is to induce a systemic inflammatory reaction. The immune-enhancing stimulus is extensive. Even very small amounts of endotoxin lead myelomonocytic cells (including tissue-specific variants like microglial cells in the nervous system) to generate and release numerous factors that recruit and activate leukocytes. Proinflammatory cytokines induce multiple master regulatory molecules like INF-g, IL-1, IL-6, IL-8, and TNF-a as well as chemoattractant chemokines to attract neutrophils (e.g., CXCL1, CXCL12); monocytes (CCL2, CCL5, CCL7, CCL8); and lymphocytes (CXCL9, CXCL10, CXCL16) (Thomson et al., 2020). Endotoxin exposure increases the quantities of several cell adhesion molecules including soluble forms of L-selectin, P-selectin, and vascular cell adhesion molecule (VCAM)-1 (Wilson et al., 2001), all of which impact leukocyte migration during inflammation. Finally, endotoxin also increases the production of alternative pro-inflammatory messengers by upregulating the expression and activity of enzymes involved in the metabolism of arachidonic acid metabolism (e.g., cyclooxygenase-2 [COX-2], 5-lipoxygenase) (Hanna and Hafez, 2018) and nitric oxide (e.g., inducible nitric oxide synthase [iNOS]) (Brown and Tepperman, 1997). The immune response to endotoxin exposure is particularly robust because Lipid A initiates autoamplification of pro-inflammatory signaling in myelomonocytic cells (Cavaillon, 2018). Complement (via a cleavage product of factor C3) may enhance

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this autoamplification in innate immune cells by upregulating the expression of caspase-11 (the cytosolic LPS receptor), which in turn can lead to more lethal outcomes (Napier et al., 2016). Furthermore, preexposure to bacterial exotoxins tends to boost the endotoxin-induced cytokine production, thus launching a potentially lethal ‘cytokine storm’ (cytokine release syndrome) upon subsequent encounters with LPS (Cavaillon, 2018). People with different LPS receptor (specifically TLR4) haplotypes exhibit divergent responses to endotoxin exposure, with some individuals (primarily those of African descent) exhibiting an enhanced pro-inflammatory response and a predisposition to develop septic shock (Ferwerda et al., 2008). Pro-inflammatory mediators may be measured systemically in some but not all septic patients, while the antiinflammatory pathways are activated more frequently (van der Poll et al., 2013). Mechanisms available for reducing inflammation during sepsis include the release of antiinflammatory cytokines (e.g., IL-10) and the availability of soluble cytokine inhibitors that either bind pro-inflammatory mediators (e.g., soluble IL-1 and TNF receptors) or antagonize their receptors (e.g., IL-1 receptor antagonist). Circulating levels of soluble cytokine inhibitors are elevated more substantially, indicating that the system seeks to control excessive function of IL-1 and TNF-a as a primary means of controlling the exuberant endotoxinmediated, systemic inflammation. Endotoxin activates the coagulation cascade in parallel to stimulating production of proinflammatory mediators (Foley and Conway, 2016; van der Poll et al., 2013). Endotoxin stimulates expression of tissue factor on the surfaces of many cells, including monocytes and endothelial cells. Tissue factor is a key initiator of coagulation, so its widespread upregulation readily leads to disseminated intravascular coagulation (DIC). Enhanced coagulation also is driven in part by several actions of the LPS receptors TLR2 and TLR4. First, these receptors regulate expression of tissue factor. Second, both TLR2 and TLR4 also assist in activating platelets. Third, these LPS receptors boost coagulation in response to the presence of extracellular histones. Endotoxin provokes histone release by activated neutrophils (and possibly monocytes) as they form neutrophil extracellular

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traps (NETs) to ensnare blood-borne bacteria. The same NETs enhance thrombosis by providing a scaffold on which clots may form, and the NETs also aid in activating endothelial cells and platelets to sustain the burgeoning thrombus. Activated caspase-11 (the cytosolic LPS receptor) also modulates expression of tissue factor (Yang et al., 2019). Prior exposure to low levels of circulating endotoxin can induce tolerance to subsequent exposure at higher concentrations (Cavaillon, 2018). Healthy people and animals commonly have measureable levels of endotoxin in the intestinal tract due to basal production by resident microflora (90%) is performed by the limulus amebocyte lysate (LAL) assay (FDA, 2012b). Animal welfare concerns and the unresponsiveness of the LAL assay to non-endotoxin pyrogens (e.g., exotoxins and fungal toxins) have driven the development of new pyrogen screens like the human monocyte activation test (MAT) and the recombinant factor C (rFC) assay. The MAT is especially useful since it is more responsive to grampositive bacterial pyrogens than either the LAL assay or the in vivo rabbit pyrogen test (RPT, which was the gold standard until replaced by the LAL assay). The MAT has been accepted as a pyrogen test since 2010 by the European Pharmacopeia and since 2012 by FDA (FDA, 2012b). As noted above, acceptable endotoxin contamination depends on the product type and its intended use. Endotoxin limits for injectable therapies (for a rapid bolus or 1-h infusion) are set at 0.2 EU/kg for intrathecal delivery and 5.0 EU/kg for parenteral administration (FDA, 1985). This specification means that a 1-mL parenteral injection to an adult human (70 kg) could contain up to 350 EU and still be acceptable for use (Wassenaar and Zimmermann, 2018). Endotoxin allowances for implanted materials are 2.15 EU/ device if placed in direct contact with the CSF and 20 EU/device for other locations (USP-NF, 2016). Recent research suggests that “hidden” endotoxin (i.e., molecules bound to protein, masked by other product constituents, or present at levels that cannot be detected by routine analytical methods) may be capable of stimulating the immune system, though the relevance of this hypothetical response for human risk assessment has yet to be established (Williams, 2017).

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Exotoxin levels in biomedical products are not specified in current regulatory guidance, so their relevance must be assessed on a case-by-case basis (von Wintzingerode, 2017). Exotoxins may be treated as potential “critical components” in the safety assessment of biopharmaceutical products. Sterilizing filters (pore size, 0.02–0.2 mm) used to reduce the bioburden of organic material from bacterial (e.g., E. coli) or cell (e.g., Chinese hamster ovary [CHO]) fragments in reactor vat culture medium will remove most bacteria (size range, 0.2–2 mm in diameter or width) but likely will not preclude passage of bacterial exotoxins (size range, 2–3 to 300 kDa) (Alouf et al., 2015). An additional source of contamination in some lots might be contaminating bacteria that are known or presumed to make exotoxins based on their taxonomic pedigree. In either of these situations, exotoxins should be considered as potential contaminants in the bulk raw material; a default assumption is that 10% of exotoxins might be retained by nonspecific binding to the desired protein (von Wintzingerode, 2017). Downstream processing (multiple rounds of ion-exchange chromatographic purification) used to concentrate the desired protein should remove many of these exotoxins since the charges and sizes of these molecules typically will allow the exotoxins to elute in a different fraction from the therapeutic protein. Chemical (e.g., acid– base hydrolysis, oxidation) and physical (heat) methods for pyrogen removal cannot be used for exotoxin inactivation since the desired protein therapeutic would be destroyed by these procedures while some heat-stable exotoxins would likely survive the treatment.

6.3. Safety Assessment of Immunotoxins As noted above, immunotoxins are a variant of antibody–drug conjugates (ADCs) whereby a potent exotoxin (“payload”) is targeted to a specific cell population using a recombinant fusion protein (“ligand”) that binds a tumor cell–specific antigen or leukocyte-specific cytokine receptor (Figure 9.4). Safety assessment of these complex test articles includes an evaluation of the entire molecule as well as the payload either with or without the attached linker.

Regulatory Guidance for Immunotoxin Safety Assessment Historically, little regulatory guidance has been given for designing the safety assessment of immunotoxins (or other ADCs). A brief section on evaluation of conjugated proteins slated for oncology indications is included in the International Council for Harmonisation (ICH) S9 guidance (ICH, 2009). Subsequent clarification in a question-and-answer format updated S9 expectations for characterizing the stability, toxicokinetics, and toxicity of oncology products (ICH, 2018). Briefly, toxicity of the intact test article and isolated payload should be evaluated in both rodents and nonrodents if possible, although testing in one species is feasible if the targeting protein is not bound and pharmacologically active in both species. The isolated toxin typically is assessed using procedures appropriate to a small molecule test article; if the toxin remains attached to the linker for testing, cytotoxicity (and genotoxicity, if any) of the payload is presumed to override any additional cytotoxicity or genotoxicity associated with the linker. The nature of the exotoxin and the results obtained in pilot studies with the payload or payload–linker will dictate the tests required for safety assessment as well as whether the unconjugated toxin will need further testing as a separate arm of the Good Laboratory Practice (GLP)-compliant general toxicity studies. In general, pathology endpoints in nonclinical studies for immunotoxins are comparable to those of other biopharmaceutical test articles (see Basic Approaches in Anatomic Toxicologic Pathology, Vol 1, Chap 9; Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10; and Practices to Optimize Generation, Interpretation, and Reporting of Pathology Data from Toxicity Studies, Vol 1, Chap 28), with a few exceptions. Toxicokinetic analysis must follow distribution of the intact ADC, the free ligand, and the unconjugated payload. Tissue cross-reactivity studies for test article localization in nontarget tissues (see Special Techniques in Toxicologic Pathology, Vol 1, Chap 11) are not required for oncology indications. In vitro stability of the ADC should be investigated in human and animal plasma (for relevant nonclinical species) as part of the Investigational New Drug (IND)enabling package. Because dose-limiting toxicity

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7. SUMMARY

generally requires intermittent administration (e.g., once every 3 or 4 weeks), the ICH S9 clarification (ICH, 2018) recommends that INDenabling nonclinical studies include at least two doses every 3–4 weeks to support initial clinical dosing regimens of once every 3 or 4 weeks to account for the extended half-life of ADCs and the potential cumulative toxicity of the conjugated exotoxin. The kinds and designs of nonclinical studies needed to assess the safety of an ADC test articles will need to be determined on a case-by-case basis. Toxic Effects Associated with Immunotoxin Administration Toxicity induced by immunotoxins reflects a combination of specific and nonspecific effects. Specific (“on-target”) toxicity follows appropriate binding of the ligand to its antigen or receptor (Figure 9.4), while nonspecific (“offtarget”) toxicity results from receptorindependent uptake. Two common manifestations of off-target toxicity in nonclinical studies and clinical trials are immunogenicity and vascular leak syndrome. Hepatic and renal toxicity also have been reported. Immunogenicity is a common effect of immunotoxins in animals because the entire molecule is foreign to the test species. The ligand portion typically will be a humanized (i.e., animal/ human chimera) or fully human molecule, but it may be engineered to have reduced immunogenicity. In contrast, the exotoxin payload typically must remain intact to maintain its activity, which means that it will be relatively more immunogenic. Almost all patients develop ADAs at some point during their treatment with these molecules. Based on a range of clinical trials, the incidence of immunogenicity after a single immunotoxin cycle ranges from 50% to 100% for solid tumors and from 0% to 40% for hematologic tumors. A potential explanation for this response is that patients with hematologic malignancies generally are immunosuppressed and thus develop antidrug antibodies (ADAs) much later, if at all. Formation of ADAs may lead to immune complex formation with deposition in and around blood vessels (often with intramural or perivascular mononuclear leukocyte accumulation), especially slow-flowing microvessels like hepatic sinusoids and renal glomerular capillaries. Immune complexes also may lead to early clearance of some

immunotoxins by leukocytes (by binding of the Fc domain of the test article to Fc receptors). This outcome may limit the clinical efficacy by preventing the toxin payload from reaching the target tumor cells as well as increase the degree of immunosuppression if the toxin levels inside leukocytes reach lethal levels. Vascular leak syndrome (VLS), a frequent dose-limiting toxicity of immunotoxins, is a potentially life-threatening off-target toxicity resulting from toxin-induced endothelial damage. The clinical presentation may include body weight gain, generalized edema (in tissues and/or body cavities), hypoalbuminemia, and orthostatic hypotension. Endothelial injury permits fluid to escape blood vessels. The resulting increase in interstitial tissue pressure leads to vascular compression and regional hypoxia. Efforts to reduce VLS include both engineering to limit the circulating half-life of immunotoxins (to reduce the cumulative exposure) as well as ancillary antiinflammatory therapy (e.g., steroids to reduce the number of toxin receptors on endothelial cells available to bind the test article). In general, edema and hypoalbuminemia have been modest and self-limiting if fluid accumulation does not occur in body cavities. Accordingly, VLS has not prevented clinical development of immunotoxins. Other toxic manifestations of immunotoxins described in clinical trials occur chiefly in the liver and kidney. Hepatotoxicity is attributed to binding of the basic residues on the Fv portion of the ligand to the negatively charged surfaces of hepatocytes although it also may be facilitated by local cytokine production in sinusoidal macrophages (Kupffer cells). Renal toxicity has been ascribed to this organ serving as the likely route of excretion for the unconjugated toxin after catabolism of the test article. Clinical signs related to immune complex deposition in hepatic sinusoids or renal glomeruli typically have not been reported during either nonclinical studies or clinical trials.

7. SUMMARY Bacterial toxins are potent virulence factors that act locally or systemically to injure host cells, either to provide a suitable environment for bacterial invasion and expansion or as a means of modulating the host immune response to promote bacterial survival. The two main classes

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of bacterial toxins are exotoxins (secreted by many gram-positive and a few gram-negative bacteria) and endotoxins (produced only by gram-negative bacteria). Exotoxins are more potent, resulting in death at doses several hundred-fold below a lethal endotoxin dose. Humans and animals encounter bacterial toxins during active infections, by ingestion of contaminated food and water, and by inhalation. Eukaryotes have developed numerous physiological mechanisms to meet these toxic attacks, thereby permitting the host and its microflora to generally coexist in a state of de´tente. Humans also are exposed increasingly to bacterial toxins as incidental contaminants (mainly endotoxin) in biopharmaceuticals as a class and also as the main active component (exotoxins) of an antineoplastic protein–toxin conjugate (called an immunotoxin). Animals and humans exhibit similar clinical signs and pathologic manifestations when exposed to bacterial toxins. The main mechanisms for bacterial toxin–induced cell injury include enzymatic disruption of host cell chemistry, membrane damage to host cells, and nonspecific immune cell activation. Diverse bacterial toxins affect different target cell populations depending on the distribution of toxin receptor complexes comprised of several proteins or a mixture of proteins and lipids. Diagnosis of bacterial infections currently is founded in the spectrum of clinical signs, definitive identification of the organism(s) using bacterial culture and/or molecular assays, or demonstration of toxin-encoding genes or toxin itself by molecular or (for exotoxins) antibody-based flow cytometric analysis. Toxemia (i.e., circulation of high bacterial toxin levels in the blood) is treated using antibiotics and/or surgery to vanquish the source of infection, adjuvant therapies (e.g., antibody treatments to inhibit toxin action and/or ex vivo filters to remove circulating toxin). Prophylactic injection of an antitoxin or a toxoid is an effective means for preventing exotoxin-induced disease.

GLOSSARY Adhesin a bacterial virulence factor that maintains bacterial attachment to a host cell Antitoxin an antibody-based adjuvant therapy that provides temporary passive immunity to one or several circulating bacterial exotoxins

Bacteremia presence of bacteria circulating in the blood Cyclomodulin a protein exotoxin that assumes control of the host cell cycle Cytolysin a protein exotoxin that kills the host cell Dysbiosis (alternate designation: dysbacteriosis) an imbalance in the expected kinds and/or numbers of bacteria on an external or internal surface Endotoxin (alternate designation: lipopolysaccharide) a bacterial toxin composed of lipids and oligosaccharides that is found within the cell walls of gram-negative bacteria and is released by cell lysis Enterotoxemia a condition in which overgrowth of enterotoxinproducing bacteria in the intestine leads to high blood levels of enterotoxins with toxicity in distant organs Enterotoxin a protein exotoxin that targets the epithelial lining of the intestinal tract Evasin a bacterial virulence factor that controls host immune function to aid bacterial survival Exotoxin a bacterial toxin composed of proteins that is secreted by many gram-positive and a few gram-negative bacteria Hemolysin a protein exotoxin with cytolytic activity against erythrocytes (red blood cells) Immunotoxin a special type of antibody–drug conjugate (ADC) in which a bacterial exotoxin is linked to a protein ligand that binds specifically to a particular host cell population (usually a tumor cell) Inflammaging (or inflamm-aging) the concept that chronic inflammation due to persistent stimulation of the innate immune system can accelerate cell senescence and thus initiate or aggravate agerelated diseases Invasin a bacterial virulence factor that enhances bacterial penetration into or between host cells Leukocidin a protein exotoxin with cytolytic activity against leukocytes (white blood cells) Microbiome depending on the context, this term refers to either (1) the full constellation of microorganisms (e.g., bacteria, fungi, protozoa, and viruses) or (2) the complete mass of microbial-derived genetic material in a given body environment Modulin a bacterial virulence factor that acts to control the functions of other cell types Myotoxin a protein exotoxin with cytolytic activity against muscle cells (skeletal and cardiac) Neurotoxin a protein exotoxin that disrupts either nervous system function (by blocking action potential propagation or synaptic transmission) or kills selected neural cells (usually neurons) Sepsis an exaggerated systemic immune response to an extensive local or systemic bacterial infection Septic shock a medical emergency in which unresolved sepsis results in severe hypotension and multi-organ failure Septicemia widespread distribution of bacteria in the blood (i.e., severe bacteremia) Superantigen a bacterial exotoxin that drives exuberant and antigenindependent (nonspecific) stimulation of the immune system (characterized by polyclonal T cell activation and massive cytokine release) Toxemia the spread of bacterial toxins in the blood Toxic shock syndrome a medical emergency in which bacterial toxemia, specifically of superantigen exotoxins, leads to an exaggerated, nonspecific systemic immune response Toxigenesis the process whereby pathogenic bacteria produce toxic molecules (toxins) Toxin a toxic molecule (“toxicant”) of biological origin Toxoid a vaccine prepared by chemical or physical inactivation of a toxin molecule to quench its toxic activity while retaining its immunogenicity

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Acknowledgments Noah’s Arkive images are provided by courtesy of the Charles Louis Davis and Samuel Wesley Thompson DVM Foundation for the Advancement of Veterinary and Comparative Pathology (hosted at https://davisthompsonfoundation.org/ noahs-arkive/) and are reproduced under a Creative Commons BY-NC-SA license. The authors thank Ms. Beth Mahler for aid in optimizing all figures and Mr. Tim Vojt for his superb medical illustration skills in crafting detailed schematic diagrams (Figures 9.1, 9.4, 9.5, 9.6, 9.8, 9.9, 9.10, and 9.11).

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Nolan JP: The role of intestinal endotoxin in liver injury: a long and evolving history, Hepatology 52:1829–1835, 2010. Oda M, Terao Y, Sakurai J, et al.: Membrane-binding mechanism of Clostridium perfringens alpha-toxin, Toxins 7:5268– 5275, 2015. Oliveira-Nascimento L, Massari P, Wetzler LM: The role of TLR2 in infection and immunity, Front Immunol 3:79, 2012. Orr RJ, Stuken A, Murray SA, et al.: Evolution and distribution of saxitoxin biosynthesis in dinoflagellates, Mar Drugs 11:2814–2828, 2013. Otto M: Staphylococcus aureus toxins, Curr Opin Microbiol 17: 32–37, 2014. Pa˚lsson-McDermott EM, O’Neill LA: Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4, Immunology 113:153–162, 2004. Palumbo RN, Wang C: Bacterial invasin: structure, function, and implication for targeted oral gene delivery, Curr Drug Deliv 3:47–53, 2006. Park BS, Lee J-O: Recognition of lipopolysaccharide pattern by TLR4 complexes, Exp Mol Med 45:e66, 2013. Park J-B, Simpson LL: Inhalational poisoning by botulinum toxin and inhalation vaccination with its heavy-chain component, Infect Immun 71:1147–1154, 2003. Patocka J, Streda L: Protein biotoxins of military significance, Acta Med 49:3–11, 2006. Petersson K, Forsberg G, Walse B: Interplay between superantigens and immunoreceptors, Scand J Immunol 59:345– 355, 2004. Pirazzini M, Azarnia Tehran D, Leka O, et al.: On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments, Biochim Biophys Acta 1858:467–474, 2016. Popoff MR: Multifaceted interactions of bacterial toxins with the gastrointestinal mucosa, Future Microbiol 6:763–797, 2011. Popoff MR: Botulinum neurotoxins: still a privilege of clostridia? Cell Host Microbe 23:145–146, 2018. Popoff MR, Poulain B: Bacterial toxins and the nervous system: neurotoxins and multipotential toxins interacting with neuronal cells, Toxins 2:683–737, 2010. Poulain B, Popoff MR: Why are botulinum neurotoxinproducing bacteria so diverse and botulinum neurotoxins so toxic? Toxins 11:34, 2019. Proft T, Fraser JD: Bacterial superantigens, Clin Exp Immunol 133:299–306, 2003. Quinn PJ, Markey BK, Leonard FC, et al.: Veterinary microbiology and microbial disease, ed 2nd, Ames, IA, 2011, WileyBlackwell. Radcliff FJ, Waldvogel-Thurlow S, Clow F, et al.: Impact of superantigen-producing bacteria on T cells from tonsillar hyperplasia, Pathogens 8:90, 2019. Radon K: The two sides of the “endotoxin coin”, Occup Environ Med 63:73–78, 2006. Ramachandran G: Gram-positive and gram-negative bacterial toxins in sepsis: a brief review, Virulence 5:213–218, 2014.

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C H A P T E R

10 Metals Sharon M. Gwaltney-Brant Veterinary Information Network, Mahomet, IL, United States

O U T L I N E 1. Introduction

680

2. Antimony 2.1. Sources and Exposure 2.2. Toxicology 2.3. Manifestations of Toxicosis 2.4. Diagnosis and Treatment

680 680 680 682 683

3. Arsenic 3.1. Inorganic Arsenic 3.2. Organic Arsenic 3.3. Arsine 3.4. Diagnosis and Treatment 3.5. Chemical Warfare Considerations

683 683 686 687 687 688

4. Beryllium 4.1. Sources and Exposure 4.2. Toxicology 4.3. Manifestations of Toxicosis 4.4. Diagnosis and Treatment

688 688 688 688 690

5. Bismuth 5.1. Sources and Exposure 5.2. Toxicology 5.3. Manifestations of Toxicosis 5.4. Diagnosis and Treatment

691 691 691 691 692

6. Cadmium 6.1. Sources and Exposure 6.2. Toxicology 6.3. Manifestations of Toxicosis 6.4. Diagnosis and Treatment

692 692 693 695 699

7. Chromium 7.1. Sources and Exposure 7.2. Toxicology 7.3. Manifestations of Toxicosis 7.4. Diagnosis and Treatment

700 700 701 701 702

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00010-1

8. Lead 8.1. Sources and Exposure 8.2. Toxicology 8.3. Manifestations of Toxicosis in Animals 8.4. Human Exposure and Disease 8.5. Diagnosis and Treatment

702 702 703 705 706 708

9. Mercury 9.1. Sources and Exposure 9.2. Toxicology 9.3. Elemental Mercury 9.4. Inorganic Mercury 9.5. Organic Mercury 9.6. Diagnosis and Treatment

709 709 709 711 711 712 714

10. Plutonium 10.1. Sources and Exposure 10.2. Toxicology 10.3. Manifestations of Toxicosis 10.4. Diagnosis and Treatment

715 715 715 716 716

11. Thallium 11.1. Sources and Exposure 11.2. Toxicology 11.3. Manifestations of Toxicosis 11.4. Diagnosis and Treatment

716 716 717 717 719

12. Uranium 12.1. Sources and Exposure 12.2. Toxicology 12.3. Manifestation of Toxicosis 12.4. Diagnosis and Treatment

719 719 719 720 721

13. Summary and Conclusions

721

References

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Copyright Ó 2023 Elsevier Inc. All rights reserved.

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1. INTRODUCTION Less than half of known metals have posed significant toxic risks to humans and animals (Tokar et al., 2013). The toxicity of some metals, such as lead and arsenic, has been recognized for hundreds of years, while the hazards of others, such as cadmium and beryllium, have only more recently been acknowledged. Contamination of the environment by some metals, such as mercury and lead, is of continuing medical and political concern. The ability of wildlife to accumulate and concentrate toxic metals increases the threat of toxicosis to those further up the food chain. Because metals are so widely utilized in everyday life, they have the potential to pose a continuous risk for human and animal health. Although the clinical presentations of toxicoses from the different metals may be quite varied, most metalsinducedamagethroughsimilarmechanisms, either by binding of the metals to vital enzymes or cellular macromolecules or by substitution of the metals for other elements in biochemical reactions (Table 10.1). Differences in clinical disease induced by metals more often reflect differences in absorption, distribution, or metabolism between the metals rather than significant differences in toxic mechanism. Because of this, discussion of the toxicologic pathology of heavy metals must include pertinent toxicokinetic information, for by knowing the fate of a given metal within the body one can then understand the pathogenesis of the signs and lesions induced by that particular metal. Toxicologic pathology of specific organ systems is covered in Volumes 4 and 5.

2. ANTIMONY 2.1. Sources and Exposure Antimony (SB, atomic number 51) is a metalloid that exists in trivalent and pentavalent forms. Antimony is present in soil and aquatic sediments where it can sorb onto clay minerals, hydroxides, and oxides. In addition to contamination of waterways through anthropogenic action, antimony can

enter waterways due to natural weathering of soil. In most areas of the world, natural exposure to very small amounts of antimony occurs via water, food, and ambient air; in areas where antimony is being mined, processed, or used in manufacturing, exposure levels increase to over 50 times that of natural background antimony. Antimony is used in the metals industry and in the production of fireproofing materials, semiconductors, diodes, ceramics, glassware, and pigments (Sundar and Chakravarty, 2010). Antimony has been used as an insecticide, and the inclusion of antimony in rodenticides served as an emetic in the case of accidental ingestion by pets and humans. Medicinally, antimony is used to treat parasitic diseases such as schistosomiasis and leishmaniasis.

2.2. Toxicology Absorption of antimony varies among the antimonial salts as well as by route of exposure, with absorption of poorly soluble forms (e.g., antimony trioxide) being lower than more soluble forms (e.g., antimony potassium tartrate) (Gebel, 1997). Antimony is poorly and slowly absorbed from the gastrointestinal tract following ingestion. Because it is such a potent gastrointestinal irritant, vomiting of ingested antimony often limits oral exposure. Inhaled antimony is poorly absorbed, with most inhaled particles remaining in the lungs. Absorbed trivalent antimony concentrates in the red blood cells and liver, whereas pentavalent forms are carried primarily by plasma proteins. Antimony distributes widely throughout the body with highest concentrations found in the lungs, gastrointestinal tract, erythrocytes, liver, kidney, bone, spleen, and thyroid. Although some slow in vivo conversion of pentavalent antimony to trivalent antimony appears to occur via nonenzymatic thiol reduction, there is currently no evidence that the scope of this interconversion is of toxicological significance (ATSDR, 2019; Ferreira et al., 2003). Antimony is not appreciably metabolized in the body, with methylation of trivalent antimony (generally considered a detoxification pathway) occurring at low levels

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TABLE 10.1

Toxicity of Metals

TABLE 10.1

Toxicity of Metalsdcont’d

Primary organ(s)/ system(s) affected

Metal

Mechanism

Antimony Enzyme inhibition through binding of sulfhydryl groups

Gastrointestinal tract

Mercury

Arsenic

Inorganic arsenic: Gastrointestinal tract, vascular endothelium (acute); skin, peripheral nerves, liver (chronic) Phenylarsonics: Brain, spinal cord, peripheral nerves Arsine: Red blood cells

Enzyme inhibition through binding to sulfhydryl groups; alteration of membrane transport channels; glutathione depletion

Metal

Mechanism

Impairment of cellular respiration through binding to sulfhydryl groups on cellular enzymes

Beryllium Corrosive tissue injury (acute) Type IV hypersensitivity

Gastrointestinal tract, respiratory tract Respiratory tract

Bismuth

Kidney (acute), gastrointestinal (chronic), central nervous system

Alteration of fluid and electrolyte transport; interference with sulfur-containing enzymes

Cadmium Enzyme inhibition through replacement of zinc in metalloenzyme systems; competitive replacement of calcium in metabolic systems

Gastrointestinal tract, respiratory tract (acute) Kidney, bone, testicle (chronic)

Chromium Oxidative stress due to reduction of Cr(VI) to Cr(III), which generates reactive intermediates

Oral cavity, esophagus, gastrointestinal tract, respiratory tract, skin

Lead

Central nervous system, gastrointestinal tract, kidney, red blood cells

Impairment of cellular metabolic pathways through binding to sulfhydryl groups; glutathione depletion Competition with calcium ions

(Continued)

Plutonium Enzyme inhibition and membrane damage through binding to cellular proteins and phospholipids

Primary organ(s)/ system(s) affected Elemental mercury: Respiratory tract, kidney (acute); gastrointestinal tract, central nervous system (chronic) Inorganic mercury: Gastrointestinal tract (acute), kidney (subacute), central nervous system (chronic) Organic mercury: Central nervous system Kidney, bone marrow

Thallium

Gastrointestinal tract, Replacement of kidney, skin potassium in potassiumdependent processes, binding to sulfhydryl groups on enzymes and structural proteins

Uranium

Enzyme inhibition and membrane damage through binding to cellular proteins and phospholipids

Kidney

Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table I, p 702, with permission.

(e.g., 1–2% in rodents) primarily in the liver (Wu et al., 2018). Antimony undergoes some degree of enterohepatic recirculation. Antimony is rapidly excreted through the urine and feces, and no appreciable storage occurs, although inhalation exposure may result in concentration of antimony within the interstitium of the lungs.

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Pentavalent antimony is rapidly excreted with 50% eliminated in urine in the first 6 h following intramuscular or intravenous injection, whereas trivalent antimony takes longer, with 25% being excreted within 24 h of injection (ATSDR, 2019). The half-life of antimony is triphasic, characterized by a rapid phase of elimination with a 2 h half-life followed by a slow phase with a halflife of 72 h and a terminal phase of >30 days. Like other heavy metals, the mechanism of toxicity of antimony is due to the binding of sulfhydryl groups with subsequent enzyme inhibition (Gebel, 1997). Enzymes involved in cellular respiration and carbohydrate or protein metabolism, such as phosphofructokinase, are particularly susceptible to inactivation by antimony (Poon and Chu, 1998). High affinity of antimony for thiol groups can result in depletion of antioxidants such as glutathione, predisposing to oxidative cellular injury. Inhaled antimony has been associated with pulmonary tumors in laboratory rodents, but the mechanism of antimony-induced genotoxicity has not been elucidated (Boreiko and Rossman, 2020; De Boeck et al., 2003). Antimony does not appear to directly interact with DNA, and investigations into indirect pathways such as generation of reactive oxygen species, alteration of DNA repair mechanisms, interactions with cellular proteins, and alteration of gene expression are areas of current research.

2.3. Manifestations of Toxicosis Human exposures to toxic levels of antimony are most commonly associated with occupational exposure during mining, processing, or manufacturing operations, with nonoccupational exposure occurring at a much lower level (Boreiko and Rossman, 2020). Occupational human exposures to antimony occur primarily via inhalation and exposure to antimony levels exceeding 0.5 mg/m3 may result in rhinitis and bronchitis, and high levels of inhalation exposure may cause acute pulmonary edema (ATSDR, 2019). Chronic changes from inhaled antimony in mammals include pneumoconiosis, pulmonary interstitial and peribronchiolar fibrosis, emphysema, and myocardial necrosis

with fibrosis and mineralization. In contrast to antimony-induced pulmonary fibrosis in rodents, which is often progressive and results in significant pulmonary compromise, human pneumoconiosis from antimony tends to result in benign, nonprogressive fibrotic lesions. “Antimony spots,” a rash of pustules, eczema, alopecia, and epidermal eruptions of the trunk and limbs, have been described in workers exposed to airborne antimony; the rash was found to be associated with high ambient temperatures, as transferring affected workers to cooler environments resulted in rapid recovery (ATSDR, 2019). Similar dermatologic lesions have been reported in animal studies. Chronic inhalation of antimony trioxide increased the incidence of cataract and corneal irregularities in rats (ATSDR, 2019). Similar ocular lesions have not been reported in humans, although eye irritation and corneal injury has occurred with occupational exposure to airborne antimony. Female laboratory rats exposed to chronic levels of inhaled antimony have reduced fertility, reduced litter sizes, uterine metaplasia, and ovarian abnormalities. An epidemiological study of reproductive effects of inhaled antimony in humans found menstrual cycle abnormalities, spontaneous abortions, and other reproductive abnormalities; however, the lack of a welldefined control group makes interpretation of the study difficult (ATSDR, 2019; Belyaeva, 1967). Oral exposure or repeated inhalational exposure to antimony results in gastrointestinal effects such as vomiting, abdominal pain, diarrhea, and gastrointestinal ulceration (Sundar and Chakravarty, 2010). Antimony is highly irritating to the gastrointestinal tract, and because it is actively secreted into the gastric lumen following systemic absorption, it can cause profuse vomiting even when administered parenterally. In addition to severe vomiting, corrosive injury to the alimentary tract may occur following antimony ingestion, resulting in ulcerative stomatitis, esophagitis, hemorrhagic gastroenteritis, and colitis. Severe acute oral exposures may lead to hypovolemia, cardiac insufficiency, shock, and multi-organ failure. Alterations in cardiac function may

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3. ARSENIC

lead to arrhythmias, such as torsade des pointes and ventricular fibrillation, that can contribute to cardiovascular compromise in acute antimony toxicosis. Lesions of acute antimony toxicosis in animal models include multifocal ulceration of gastrointestinal mucosa with submucosal edema and hemorrhage (ATSDR, 2019; Sundar and Chakravarty, 2010). There may be extensive centrilobular hepatocellular necrosis in severe cases. Myocardial degeneration, renal glomerular congestion, proximal renal tubular degeneration, and necrosis may also be present. Degeneration of hair and supporting cells within the organ of Corti have been reported in rats and guinea pigs administered antimonial antibilharzial agents. Altered evoked otoacoustic emissions suggesting similar cochlear injury have been reported in humans receiving meglumine antimoniate therapy for leishmaniasis (de Oliveira Bezzera et al., 2017). Chronic oral exposure of rats to 0.35 mg/kg/ day of antimony resulted in alterations in blood glucose and cholesterol levels, and pancreatitis is known to be an adverse effect of the use of therapeutic levels of pentavalent antimonials in humans (Sundar and Chakravarty, 2010). In Fischer and Wistar rats, chronic inhalation of antimony trioxide has resulted in an increase in lung tumors in some studies, but other studies failed to establish a link between antimony exposure and lung tumors (ATSDR, 2019). Antimony trioxide is currently classified as a possible human carcinogen by the International Agency for Research on Cancer (IARC), while the US Department of Health and Human Services has categorized antimony trioxide as reasonably anticipated to be a human carcinogen (IARC, 2015; NTP, 2018). Conversely, antimony trisulfide is currently listed as unclassifiable as to its carcinogenicity (IARC, 2015).

2.4. Diagnosis and Treatment Urine testing is the most accurate and reliable test for measuring antimony levels in humans, with elevations of urine antimony concentrations above established background ranges being indicative of recent exposure, although not necessarily diagnostic for toxicosis (Sundar and Chakravarty, 2010). Blood antimony concentrations may be elevated during exposure to antimony;

however, determination of antimony toxicosis must evaluate the clinical signs seen in light of the blood and/or urine antimony concentrations. Analysis of hair samples for antimony content is not considered a reliable method to determine antimony exposure or toxicosis (ATSDR, 2019; Sundar and Chakravarty, 2010). There is no specific treatment for antimony toxicosis beyond removal of the antimony source and symptomatic care (e.g., antiemetics, antiulcer medication, etc.). As most significant exposures to antimony are due to occupational exposures, adherence to governmental standards on air quality is required to prevent exposure to potentially toxic levels of antimony. Prevention of toxicosis from the therapeutic use of antimonial compounds entails strict adherence to established dosing guidelines, regular monitoring of patients, and, when possible, substitution of other drugs for antiparasitic therapy.

3. ARSENIC Arsenic (As, atomic number 33) is a metalloid with a complex chemical structure, being present in elemental, gaseous (arsine), trivalent (þ3, arsenite), and pentavalent (þ5, arsenate) inorganic forms, as well as trivalent and pentavalent organic forms. Inorganic arsenicals of interest due to their potential to cause toxicosis in humans and animals include the trivalent arsenic trioxide, sodium arsenite, and arsenic trichloride, and the pentavalent arsenic pentoxide, arsenic acid, and arsenates (lead arsenate, calcium arsenate, etc.). Organic arsenic compounds of toxicologic interest include phenylarsonics such as arsanilic acid. Organic arsenicals may also be trivalent and pentavalent and some organic arsenicals are also methylated. Trivalent arsenic compounds are more soluble, and therefore more toxic, than pentavalent arsenic compounds.

3.1. Inorganic Arsenic Sources and Exposure Sources of exposure to inorganic arsenicals include air and water contamination from glass and chemical manufacturers and from copper, zinc, and lead smelters (where arsenic is

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a by-product); occupational exposure to arsenical compounds occurs in these industries as well. Naturally occurring arsenic in bedrock that contaminates groundwater is a concern for human health in many parts of the world particularly in areas of southwestern United States, parts of Chile and Argentina, much of Bangladesh and western India, and many regions in southeast Asia (Cambodia, Vietnam, China, Thailand, and Taiwan) (Nurchi et al., 2020; Prakash et al., 2016). In areas with low levels of arsenic in drinking water, the most important source of arsenic exposures is via the diet, particularly seafood (Luvonga et al., 2020). Seafood can accumulate arsenic from contaminated water or due to bioaccumulation up the food chain. Much of the arsenic in seafood is stored as arsenobetaine, an organoarsenical that is considered much less toxic than inorganic arsenic. Grains, such as rice, planted in arsenic rich soil or watered with arseniccontaminated water can contain relatively high levels of arsenic. Arsenic compounds used as herbicides, pesticides, and fungicides are other sources of environmental arsenic contamination, although these uses have greatly declined in the Western world in recent years as environmental concerns have risen in many countries. Historically, arsenic was used commonly medicinally, and arsenic-based pharmaceuticals were a significant source of arsenic exposures; currently, the pharmacologic use of arsenicals in the United States is now almost exclusively limited to veterinary medicine. Up until the 1980s arsenic was used in the preservation of natural history animal specimens (skins, hair, fur, and feathers), and these tissues are a potential source of chronic arsenic exposure to those who regularly handle them (Strekopytov et al., 2017). More nefariously, arsenic has a long history of being used as a malicious poison against both animals and humans. Toxicology Inhaled arsenic, usually in the form of trivalent arsenic oxide, is deposited in the respiratory tract and absorbed from the lung in a manner that is dependent upon particulate size (ATSDR, 2007). Ingested arsenic is well absorbed from the gastrointestinal tract. Absorption of arsenic through the skin is minimal; however, loss of dermal integrity may result in enhanced transdermal arsenic

absorption. Once absorbed, 95% of arsenic is bound to red blood cells, where it is widely distributed throughout the body. In acute exposures, highest tissue levels are present within liver, kidney, heart, and lungs, with lesser amounts accumulating in brain and muscle (Luvonga et al., 2020). Arsenic has a predilection for sulfhydryl-rich keratin, and in chronic exposures arsenic tends to concentrate in the skin, nails, sweat glands, and hair. Arsenic can also deposit in bones and teeth in chronic cases. Inorganic arsenic does not readily cross the blood– brain barrier. Passage of arsenic through the placenta can result in arsenic levels in fetal cord blood that approximate maternal blood levels. Elimination of absorbed inorganic arsenic requires in vivo transformation of inorganic arsenic to methylated organic forms. Pentavalent inorganic arsenicals must be reduced to the more soluble (and more toxic) arsenic trioxide prior to transformation to dimethylarsenate, which is rapidly excreted via the kidneys. Exposures to levels of inorganic arsenic that exceed the rate of biotransformation can result in toxicosis. The mechanism of arsenic toxicity is related to its effects on enzyme systems within the cell, primarily by binding to sulfhydryl groups on enzymes and other cellular proteins (ATSDR, 2007). Accumulation of arsenic in mitochondria results in the inhibition of pyruvate oxidases and phosphatases, interference with NADlinked substrates within mitochondria, inhibition of succinate dehydrogenase activity, and uncoupling of oxidative phosphorylation by replacing phosphates in high-energy phosphorylated substrates (arsenolysis). The consequences of these effects are functional and morphological cellular abnormalities secondary to impairment of cellular respiration and depletion of energy stores. Generation of reactive oxygen species and reactive nitrogen species can result in damage to cellular organelles, cytoskeleton, and DNA (Sattar et al., 2016). Tissues most severely affected by arsenic are those rich in oxidative enzymes, i.e., the alimentary tract, liver, kidney, lung, endothelium, and epidermis. Arsenic can also induce thiamine deficiency by limiting thiamine bioavailability, and it can elevate plasma lactic acid levels. Arsenicinduced genotoxicity is secondary to inhibition of DNA repair pathways and induction of oxidative DNA injury.

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3. ARSENIC

Manifestations of Toxicosis Peracute respiratory failure and asystole may occur within hours following ingestion of massive amounts of arsenic (ATSDR, 2007). Some victims may display profound depression prior to death; alternatively, no antemortem signs may be noted. In cases causing death within 24 h of exposure, lesions may be few and are generally limited to mild splanchnic congestion and edema. A latent period of up to 48 h may precede the development of signs in acute arsenic toxicosis. The initial signs are acute onset of profuse vomiting, diarrhea, colic, and salivation. Extensive inflammation and necrosis of gastrointestinal mucosa leads to severe hemorrhagic gastroenteritis. Increased vascular permeability due to endothelial damage results in fluid loss to the interstitium, which, coupled with intestinal blood loss, contributes to intravascular volume depletion, dehydration, hypotension, and hypovolemic shock. Damage to pulmonary capillary endothelium may initiate fulminant pulmonary edema. Hepatic and renal failure may result from direct effects of arsenic on these organs or may be due to multi-organ failure secondary to acute cardiovascular collapse. Cardiac arrhythmias, skeletal muscle fasciculations, and weakness may occur, especially in victims surviving longer than 48 h. Terminally, there may be delirium, dementia, or seizures. Inhalational exposure may result in cough, dyspnea, chest pain, and pulmonary edema with acute respiratory distress syndrome (ARDS) and respiratory failure. Cutaneous erythema (“flushing”) may occur in acute arsenic exposures. Subacute arsenic toxicosis may result in milder manifestations of the above signs as well as lethargy, anorexia, fever, hepatomegaly, polyuria progressing to anuria, weakness, paresis, tremor, hypothermia, stupor, cardiovascular failure, and death. Those surviving may later develop polyneuropathy, bone marrow depression, encephalopathy, cardiac arrhythmias, and visual disturbances. Chronic arsenic toxicosis is well documented in humans, though it is less commonly recognized in domestic animals (ATSDR, 2007). The effects of chronic arsenic exposure include dermal, gastrointestinal, bone marrow, and

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hepatic manifestations. Mee’s lines (semilunar white bands on nails) may develop along with hyperkeratosis of skin on the extremities, acrocyanosis, cutaneous vesiculation edema, and ulceration. In extreme cases, gangrene of the feet may occur. Brick-red discoloration of mucous membranes, ulcerative stomatitis, anorexia, chronic diarrhea, and cachexia may develop. Reversible bone marrow suppression may lead to leukopenia and anemia. Generalized hepatomegaly associated with elevations in liver enzymes may progress to cirrhosis, icterus, and ascites. Peripheral neuropathy with neural pain and weakness and dementia due to encephalopathy have been described in humans. Periarticular fibrosis resulting in stiff gait and asymmetrical joint enlargements has been reported in cattle chronically exposed to arsenic. Epidemiologic evidence suggests that arsenic may play a role in cardiovascular disease in humans, and it has been associated with exacerbation of inflammation and atherosclerotic plaques in murine models. Abortions may occur in pregnant humans and animals. Many of the lesions of arsenic toxicosis are secondary to severe vascular injury leading to congestion, edema, and hemorrhage (ATSDR, 2007). Gastrointestinal lesions of arsenic poisoning include generalized reddening of gastrointestinal mucosa, mucosal hemorrhage and necrosis, and gastric ulceration. Extensive thickening of stomach wall due to edema fluid may occur. Gastrointestinal contents are fluid, have a foul odor, and frequently contain blood and tags of shredded intestinal mucosa. In humans, pseudomembranous enteritis has been described. Histopathologic lesions in the gastrointestinal tract include severe congestion, denudation of mucosal epithelium, submucosal hemorrhage and edema, and thrombosis of submucosal vessels with multifocal infarction. The liver is soft and yellow. Histopathologically, there is generalized hepatocellular swelling, severe fatty change, glycogen depletion, and focal necrosis; ultrastructurally, severe swelling of mitochondria is present. In rats, enlargement of the common bile duct up to 10 times normal diameter has been described as a specific lesion of arsenic toxicosis. Fatty change in heart and kidney and cerebral edema with petechiation

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may occur in mammals surviving several days. Dermal lesions in humans range from erythema, necrosis, and ulceration to hyperkeratosis with hyperpigmentation. Histologically, dermal blood vessels may show evidence of endothelial swelling, necrosis, and thrombosis. Degeneration of myelin and axons occurs in those cases where peripheral neuropathy is evident. Degeneration of the ganglion cell layers of the retina has been reported in humans from the use of pentavalent arsenicals in the treatment of syphilis. Arsenic can cause fetal deaths and resorptions in humans and animals. Experimentally, exposure to arsenic in utero has resulted in genitourinary deformities in animals, but no teratogenic effects have been attributed to arsenic exposure in humans. Inorganic arsenic is classified as a known human carcinogen with increased incidence of basal cell tumors and squamous cell carcinomas in chronically exposed humans (ATSDR, 2007). Other neoplasms associated with arsenic exposure in humans include epidermoid bronchogenic carcinoma, hepatic hemangiosarcoma, lymphomas and leukemias, and cancers of the nasopharynx, kidney, colon, and urinary bladder. In rats and mice, increased incidence of pulmonary adenomas, papillomas, and adenomatoid lesions has been produced by arsenic trioxide. The use of animal models to study arsenic-induced cancer was for years confounded by the inability to elicit malignancies in laboratory animals. Postulated explanations for the difference in susceptibility to arsenic-induced neoplasia include species differences in arsenic metabolism (humans excrete significantly more methylated arsenic metabolites than most other species), differences in distribution and storage of arsenic within the body, and the possibility that arsenic acts more as a cocarcinogen rather than a primary carcinogen (Tokar et al., 2010). However, more recently a few animal models of arsenic-induced cancer have been developed. Malignant neoplasms associated with arsenic in laboratory animals include gastric adenocarcinomas (arsenic trioxide, rats), pulmonary adenocarcinoma (calcium arsenate, rats), pulmonary carcinomas (arsenic trioxide, hamsters), and lymphocytic leukemia and lymphoma (mice, sodium arsenate). Attempts to induce cancer in other

laboratory animal species, including dogs and rabbits, have been unsuccessful.

3.2. Organic Arsenic Sources and Exposure Organic arsenicals include phenylarsonics used pharmaceutically and organoarsenicals present in seafood including arsenobetaine, the methylated arsenicals monomethylarsonous acid and dimethylarsinous acid, arsenosugars, arsenocholine, and arsenolipids. In general, arsenicals of seafood origin are considered to be of low toxicity, although recent studies suggest that toxic metabolites may be generated during biotransformation and elimination of these compounds, albeit at very low levels (Luvonga et al., 2020). Phenylarsonics, including arsanilic acid, 3-nitro4-hydroxyphenylarsonic acid, 4-nitro-phenylarsonic acid, and p-ureidobenzenearsonic acid, are used in animal feeds as growth promoters and to control blood parasites and other infectious diseases. Significant human exposures are rare, and phenylarsonic toxicosis in animals usually occurs when mixing errors result in overdosage or when these products are fed to dehydrated animals (dehydration appears to lower the resistance to toxicity). Toxicology Phenylarsonics are well absorbed through the skin and gastrointestinal tract and are excreted in the urine largely unchanged. Some storage occurs within nervous tissues. Phenylarsonics are not used in ruminant animals, as rumen fermentation may break them down, releasing inorganic arsenic and resulting in acute toxicosis (see previous discussion). The proposed mechanism of action of phenylarsonic toxicosis is through impairment of B Vitamin function resulting in neuropathy rather than binding of sulfhydryl groups as with inorganic arsenic toxicosis. Manifestations of Toxicosis Signs and lesions of acute phenylarsonic toxicosis do not develop for at least 10 days following overexposure. In swine, the most commonly affected species, the syndromes of toxicosis differ depending on the type of phenylarsonic involved (Kennedy et al., 1984). Arsanilic acid toxicosis

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3. ARSENIC

presents as acute dermal erythema, hyperesthesia, vestibular dysfunction, blindness, muscle weakness, and severe ataxia progressing to paraparesis or paralysis. Histologically, there is edema of the white matter of the brain and spinal cord. Neuronal degeneration is present within the brainstem. Wallerian degeneration and demyelination of axons of the optic and peripheral nerves as well as necrosis of myelin supporting cells may be present. In contrast, 3-nitro-4hydroxyphenylarsonic acid toxicosis in swine is manifested as exercise-induced tremors and seizures, mild paraparesis or paralysis, and involuntary urination and defecation. Unlike arsanilic acid toxicosis, blindness is not a feature of 3-nitro-4-hydroxyphenylarsonic acid toxicosis. Histologic lesions are present within the dorsal proprioceptive and spinocerebellar tracts of the cervical spinal cord and the peripheral regions of the ventral and lateral funiculi of the posterior spinal cord; lesions consist of Wallerian degeneration of axons with mild myelin changes. Mild optic and peripheral nerve Wallerian degeneration may be present but is not a consistent finding. Chronic overdoses of phenylarsonics may manifest as gradual onset of ataxia, proprioceptive deficits, and poor growth. Lesions are similar to those seen in the acute disease. Increased incidence of pancreatic tumors was noted in male rats ingesting 4 mg/kg/day of roxarsone; other carcinogenic effects for phenylarsonics have not been found.

3.3. Arsine Arsine is a highly toxic gas that is generated upon exposure of arsenic-containing ores to acids, and it is a by-product of refining of nonferrous metals. Arsine is used extensively in the manufacture of microchips. Exposure to arsine gas results in severe depletion of erythrocyte glutathione, causing red blood cell membrane instability and rapid, massive intravascular hemolysis. Onset of signs occurs within 30– 60 min of exposure; signs include severe abdominal pain, vomiting, dyspnea, icterus, hemoglobinuria, hematuria, anemia, and death which occur due to rapid intravascular hemolysis. Those surviving the acute episode may develop renal failure, bone marrow depression, or conditions associated with chronic arsenic exposure (Sattar et al., 2016). Exposure to arsine in the

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form of Lewisite (dichloro[2-chlorovinyl] arsine) has been associated with the development of intraepidermal squamous cell carcinoma in humans; animal studies of the carcinogenicity of arsine are lacking.

3.4. Diagnosis and Treatment Blood levels of arsenic drop rapidly following absorption, making this matrix useful for diagnostic testing only in the first few hours after exposure (ATSDR, 2007). Chronic exposure to low levels of arsenic is not likely to be detected if blood is used as the only diagnostic sample, so blood is not considered a reliable means to monitor populations for arsenic exposure. Urine is a more reliable matrix for arsenic measurement, as arsenic can be detected in the urine for 1–2 days following exposure, and a linear relationship exists between arsenic exposure and urinary arsenic levels. However, interpretation of urine arsenic levels must be done with care, as recent ingestion of some seafoods can result in elevated urine arsenic measurements due to the presence of arsenobetaine, and most initial screening tests do not distinguish between the various forms of arsenic. Suspect samples should subsequently be analyzed using a method that can distinguish between organic and inorganic arsenic. Arsenic concentrations in human hair or nails may be useful for detecting past or chronic exposure to arsenic, but are not of value for diagnosis of peracute, acute, or subacute exposures. Contamination of hair or nails by exogenous arsenic can be a confounding factor in interpretation of analysis, so careful processing of samples is necessary in order to remove any external arsenic. Historically, treatment of arsenic toxicosis entailed administration of BAL (British antiLewisite, dimercaprol), a dithiol compound that competes with endogenous thiol groups to chelate arsenic thereby removing it from tissues; the chelate complex is eliminated via the urine (Kosnett, 2013; Kim et al., 2019; Nurchi et al., 2020; Tokar et al., 2013). More recently chelation therapy with DMPS (dimercaptopropane sulfonate) has largely replaced the use of BAL in arsenic toxicosis due to its more favorable pharmacologic profile (better efficacy, lower toxicity), although in severe cases of acute arsenic toxicosis DMPS and BAL are often utilized in

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combination. Dimercaptosuccinic acid (DMSA) and penicillamine are other chelators that have been used to treat chronic arsenic toxicosis. Chelation therapy does not guarantee a successful outcome because of the extensive organ injury that can occur in arsenic toxicosis; extensive symptomatic and supportive care will be required, and long-term sequelae are possible.

3.5. Chemical Warfare Considerations Arsenicals have been investigated or used as chemical warfare agents, with reports of arsenic-based compounds being used as weapons as early as the 4th century BC. Arsine gas would initially appear to be an ideal chemical warfare agent, as it can quickly result in lifethreatening hemolytic anemia. However, difficulties in arsine generation and inability to obtain sufficiently toxic concentrations under field conditions resulted in the development of a variety of organoarsine compounds, such as Lewisite, which are potent vesicants at lower concentrations than those required to cause acute hemolysis (Srivastava and Flora, 2020). Although banned by most countries and international treaties, because arsenic-based chemical weapons are relatively easily manufactured and can produce significant morbidity and mortality, they still pose a significant potential hazard in the 21st century.

4. BERYLLIUM

population, especially if appropriate handling guidelines are not followed. Environmental sources, primarily through exposure to combustion products from coal and oil, account for the bulk of nonoccupational exposure.

4.2. Toxicology Beryllium is poorly absorbed by oral exposure, and although cutaneous exposure may result in dermatitis, no significant dermal absorption occurs. The most significant route of exposure is via inhalation of beryllium fumes or dusts (Tokar et al., 2013). Particulate size determines the degree of penetration of inhaled beryllium into the lung – particles with diameters of less than 1–2 mm are deposited deep within the lung. Many of these particles are cleared by phagocytosis and transported either to the airways, where they are coughed up, swallowed, and eliminated via the feces, or to the pulmonary interstitium or lymph nodes, where immunologic responses are triggered. Beryllium that is absorbed into the blood is carried on plasma proteins and distributes to bone, liver, and kidney. Approximately 50% of inhaled beryllium is cleared within 2 weeks via the pulmonary mucociliary escalator; the remainder is largely sequestered within fibrotic granulomata or translocated to tracheobronchial lymph nodes, where it persists. Excretion of solubilized beryllium in the blood is via tubular secretion into the urine.

4.1. Sources and Exposure

4.3. Manifestations of Toxicosis

As beryllium (Be, atomic number 4) has become of increasing industrial importance in the past 60 years, its effects on human health have become recognized to the point that “beryllium disease” is a well-described entity resulting from occupational exposure to beryllium compounds (ATSDR, 2002). Beryllium is a byproduct of coal combustion, and it is incorporated into alloys used in a variety of manufacturing industries, including aerospace, electronics, computers, nuclear weapons, dental, welding, plating, and lighting manufacturing. Dental technicians working with berylliumcontaining dental alloys may be exposed to higher levels of beryllium than the general

Although beryllium can be directly cytotoxic to tissues, the primary lesions of acute beryllium toxicosis are usually related to the intense inflammatory response that the metal induces (ATSDR, 2002; Tokar et al., 2013). Acute oral exposure to beryllium causes mild to moderate gastrointestinal irritation and inflammation. Cutaneous exposure may cause dermatitis, ulceration, and delayed wound healing. Ocular exposure may result in conjunctivitis. Acute inhalation of beryllium salts or fumes may result in a doserelated inflammation of the respiratory tract from the nares to the lungs. Signs associated with acute berylliosis include dyspnea, productive cough, hemoptysis, fever, tachycardia,

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4. BERYLLIUM

tachypnea, rales, and cyanosis. Severe acute fulminating pneumonitis may occur at higher levels of exposure; in humans this process has occasionally been fatal, largely due to severe pulmonary edema. Lesions of acute pulmonary berylliosis include severe bronchitis and alveolitis with acute intra-alveolar inflammation and edema. Radiographic examination may show diffuse alveolar infiltrates and/or pulmonary edema. Bronchiectasis has been reported in humans as a sequela of acute berylliosis. Nonlethal cases resolve over periods of weeks or months, and approximately 17% of humans surviving the acute episode will develop chronic beryllium disease, causing some to suggest that the mechanisms behind acute and chronic beryllium disease are the same, and that these two manifestations represent a continuum of disease (Cummings et al., 2009). Chronic beryllium disease has been recognized in humans since the early 20th century. Unlike acute berylliosis, the ultimate severity of the chronic form does not appear to be dependent upon the magnitude of exposure. In humans, there is a variable latency period between initial beryllium exposure and onset of

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clinical disease; the latent period ranges from several months to 30 years or more, averaging 6–10 years. Chronic beryllium disease is a Type IV hypersensitivity, with beryllium acting as an antigen or hapten (Figure 10.1). The susceptibility to and the extent and nature of the chronic response to beryllium are species- and individual-specific. A specific major histocompatibility antigen MHC gene mutation has been associated with chronic beryllium disease in humans. The human leukocyte MHC II marker HLA-DPB1 plays a direct role in the immunopathogenesis of chronic berylliosis by altering the shape of the HLA-binding peptide pocket, thereby altering antigen specificity (Greaves et al., 2020). In the lung, beryllium combines with tissue proteins, and the beryllium–protein complexes are phagocytosed and processed by antigenpresenting cells. Presentation of beryllium antigen to T lymphocytes results in their stimulation, mediated by cytokines such as interleukin-1 and interleukin-6, which serve to amplify the immune response. T-helper (CD4þ) cells proliferate in the presence of processed beryllium; this response is further amplified by cytokines FIGURE 10.1 Cellular events in the development of beryllium granuloma in humans. Beryllium (Be) binds to cellular proteins, forming haptens. Phagocytosis, antigen processing, and presentation of antigen to T lymphocytes by antigen-presenting cells (APCs) stimulate the proliferation of CD4þ cells. Recruitment of additional lymphocytes, macrophages, and giant cells results in the formation of granulomas containing a preponderance of CD4þ lymphocytes. Lymphocyte stimulation and proliferation are mediated by cytokines such as interleukins (IL-1, IL2, IL-4, and IL-6) and interferon-g (IFN). Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013 Figure 41.7, p 1343, with permission.

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such as interleukin-2, interleukin-4, interleukin6, and g-interferon. The end result is a massive mononuclear cell infiltration that leads to the formation of granulomata containing a preponderance of CD4þ cells. In contrast to the pathologic effects of T-cells in beryllium disease, B-cell recruitment into the lung in chronic beryllium disease appears to play a protective role by an as yet unelucidated mechanism (Greaves et al., 2020). Lesions of chronic beryllium disease include granulomata and fibrogranulomata within the lungs and often in other organs such as liver, spleen, lymph nodes, myocardium, kidney, salivary gland, skeletal muscle, bone, and skin. Cutaneous granulomata may form due to direct contact but can also occur via inhalation exposure. Lesions range from diffuse mononuclear cell infiltration of the pulmonary interstitium to multifocal, noncaseating lymphogranulomata. The granulomata induced by beryllium in humans differ from those formed in rats; the latter are more typical of foreign-body reactions, containing large numbers of macrophages and monocytes and small populations of lymphocytes. In contrast, the human beryllium granuloma is characterized by large numbers of beryllium-specific CD4þ lymphocytes with smaller numbers of macrophages and monocytes. Mice exposed to inhaled beryllium develop pulmonary granulomata similar in appearance to those in humans, but these lesions tend to take longer to develop and usually resolve following cessation of beryllium exposure; additionally, the mouse granulomata tend to have significant numbers of B lymphocytes. Guinea pigs exposed to intratracheal injections of beryllium oxide have developed pulmonary granulomata, although attempts to reproduce the lesions under inhalation exposure have not been successful. Dogs exposed to inhaled beryllium develop granulomata similar to those seen in humans. Oral exposure to beryllium has resulted in “beryllium rickets” in rats, thought to be due to beryllium-induced interference with phosphorus absorption from the gastrointestinal tract and resulting in altered systemic calcium:phosphorus ratios (ATSDR, 2002). Oral dosing of beryllium in dogs has resulted in bone marrow erythroid hypoplasia, and erosive and inflammatory lesions of the gastrointestinal tract. The

gastrointestinal lesions are most severe in the small intestine, and include desquamation and necrosis of mucosal epithelium, focal ulceration, mucosal and submucosal edema, fibrin thrombi in submucosal capillaries, infiltration of neutrophils (early), and mononuclear cells (late). Rat and mouse studies using the same oral beryllium dose as in dogs failed to produce significant gastrointestinal lesions. No information is available on the reproductive or developmental effects of beryllium in humans or animals following inhalation or oral exposure. Intratracheal instillation or inhalation of beryllium has been shown to cause lung cancers (adenocarcinomas, bronchiolar alveolar cell tumors, anaplastic sarcomas, squamous cell carcinomas, and malignant lymphomas) in rats and monkeys (Macaca mulatta). Intramedullary and intravenous administration of beryllium has produced osteosarcomas in rabbits. Beryllium and beryllium compounds are listed as known human carcinogens. Human epidemiologic studies have demonstrated an increased risk of lung cancer in humans exposed to inhaled beryllium and beryllium compounds (ATSDR, 2002). Human studies investigating the carcinogenic potential of ingested beryllium are lacking, and studies in dogs, mice, and rats have not revealed carcinogenic potential for ingested beryllium. The National Toxicology Program lists beryllium and multiple beryllium compounds (alloys, salts, etc.) as human carcinogen and the IARC has classified beryllium and beryllium compounds as Group 1 carcinogens in humans; conversely, the US Environmental Protection Agency lists inhaled beryllium as a Group B1 probable human carcinogen.

4.4. Diagnosis and Treatment Diagnosis of acute berylliosis is generally based on history of exposure along with appropriate signs of respiratory irritation and inflammation; detection of beryllium in respiratory secretions or bronchoalveolar lavage fluid can confirm the diagnosis (ATSDR, 2002). Blood and urine levels do not reflect exposure levels and are rarely performed. Bronchoalveolar lavage can also aid in detecting granulomatous lung disease but may not differentiate beryllium granulomata from other granulomatous conditions. However, beryllium hypersensitivity is

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5. BISMUTH

a key feature in chronic beryllium disease, and this can be detected via several different assays. The most common assay for beryllium hypersensitivity is the beryllium lymphocyte proliferation test (BeLPT, formerly lymphocyte blast transformation test). This assay can use either peripheral blood lymphocytes or bronchioalveolar lavage lymphocytes and measures cell proliferation in the lymphocytes in the presence or absence of beryllium. The relatively low sensitivity (68%) and high specificity (97%) make the BeLPT a useful screening tool, but further evaluation is necessary when positive results occur (Middleton and Kowalski, 2010). Treatment of acute beryllium entails cessation of beryllium exposure and symptomatic medical management which may include antitussives, antibiotics, bronchodilators, and antiinflammatory medications as well as breathing support, depending on severity (Cummings et al., 2009). Follow-up evaluations are recommended in an attempt to detect the development of chronic beryllium disease as early as possible. Treatment of chronic beryllium disease primarily entails the use of corticosteroids as well as removal from the beryllium source; adjuvant therapies such as bronchodilators and diuretics may be indicated in some cases (Sood, 2009). Immunomodulating drugs such as azathioprine and methotrexate have been used as corticosteroid-sparing agents as well as in cases where corticosteroids are poorly effective, and the use of infliximab has been investigated (Maier et al., 2012).

5. BISMUTH 5.1. Sources and Exposure Bismuth salts, both organic and inorganic, have been used medicinally to treat a variety of infectious diseases, gastrointestinal disorders, and dermatological conditions. Bismuth-based radiocontrast media are used in diagnostic medicine. Bismuth is also used in alloys in the manufacture of dental devices, steel, and some forms of shot used in hunting game. Some bismuth salts have been used in cosmetic preparations such as dusting powders and hair dyes. Because many bismuth compounds are poorly soluble and poorly absorbed, significant exposure is

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primarily through the pharmaceutical use of bismuth-based formulations.

5.2. Toxicology Bismuth is not appreciably absorbed via inhalation or by cutaneous exposure and minimal absorption of bismuth occurs from bismuth–tin shot embedded in muscles of waterfowl (Sanderson et al., 1998; Slikkerveer and de Wolff, 1989). However, absorption of bismuth from wounds covered or packed with bismuth-impregnated dressings and paraffin has resulted in toxicosis in humans (Bridgeman and Smith, 1994; Ovaska et al., 2008; Saini et al., 2019). Gastrointestinal absorption is relatively poor, with the total absorption of ingested bismuth being approximately 1%, although considerable interindividual variation occurs. Absorbed bismuth is transported in the blood bound to a plasma metallothionein (MW 50,000) and distributes widely throughout tissues (Slikkerveer and de Wolff, 1989). Bismuth accumulates in kidney, liver, spleen, bone (metaphysis), lung, heart, and muscle. Bismuth in bone is very slowly turned over, with a half-life of months to years. Protein-bound bismuth also concentrates in the placenta and can cross the placenta into the amniotic fluid and the fetus. Bismuth is excreted primarily by the kidney, which contains the highest bismuth concentrations; lesser amounts of bismuth are excreted via saliva, milk, and bile. Bismuth has an affinity for epithelial cells, and many of the manifestations of bismuth toxicosis are the result of alterations of fluid and electrolyte transport within epithelial tissues. Bismuth also interferes with thiol-containing enzymes, resulting in altered cellular oxidation and metabolism. In humans, the main syndromes described in bismuth intoxication involve the kidney, alimentary tract, brain, and bone; natural bismuth toxicosis in animals is rare.

5.3. Manifestations of Toxicosis Acute renal failure may result from either acute or chronic bismuth toxicosis (Slikkerveer and de Wolff, 1989). Concentration of bismuth within the epithelium of proximal tubules causes epithelial swelling, necrosis, and mineralization. Affected epithelial cells may contain

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intranuclear inclusion bodies resembling those seen in lead toxicosis. The inclusions are composed of bismuth bound to nonhistone nuclear proteins. In addition to the renal tubular changes, bismuth may cause damage to glomeruli, resulting in proteinuria. The glomerular injury is secondary to capillary endothelial damage and microthrombosis. Oral ulcers, anorexia, vomiting, hypersalivation, colic, and diarrhea may occur in acute bismuth toxicosis, although massive oral overdosage is required to produce acute poisoning. Gastrointestinal signs and lesions are more commonly seen with chronic oral overdosage of bismuth compounds. Deposition of bismuth in the mucosa of the oral cavity, colon, and vagina results in blue-black pigmentation to these areas; the lesions in the oral cavity tend to occur on the gums and are termed “metal lines.” Lesions associated with bismuth toxicosis include ulcerative stomatitis, ulcerative colitis, and cervicovaginitis, all with blue-black mucosal pigmentation. In addition to the alimentary syndrome, bismuth toxicosis has been associated with hepatic failure, icterus, and clotting disorders secondary to multifocal hepatocellular degeneration and necrosis. Incidents of bismuth-induced encephalopathy have been reported in association with ingestion of bismuth-containing medications as well as absorption of bismuth from wound dressings and packings (Bridgeman and Smith, 1994; Ovaska et al., 2008; Saini et al., 2019; Slikkerveer and de Wolff, 1989). The syndrome is characterized by postural instability, ataxia, confusion, sleep disorders, irritability, memory disorders, multifocal limb myoclonus and hyperreflexia, coarse postural tremors, lethargy, coma, and rare deaths. A similar syndrome has been produced in mice given large intraperitoneal doses of bismuth subnitrate. The lesions of murine bismuth encephalopathy include hydrocephalus and swelling of axons within the spinal cord. Both the human and murine encephalopathies are reversible, and cessation of bismuth administration generally results in complete recovery. Storage of bismuth in the bone may lead to osteoporosis and osteomalacia, manifested clinically as bony deformities, increased bone fragility, and bone pain (Slikkerveer and de Wolff, 1989). Osteoarthropathy has also been

reported in humans with bismuth-induced encephalopathy; the skeletal abnormalities in these patients were suspected of being secondary to microfractures that occurred during repetitive episodes of severe myoclonus. An exfoliative dermatitis has been reported in humans with bismuth toxicosis. Bismuth subnitrate is directly toxic to testicular Leydig cells and can adversely affect male reproduction. Reproduction studies during pregnancy have not been performed; there are several case reports of pregnant women (9–35 weeks of pregnancy) who developed and were treated for bismuth toxicosis that subsequently gave birth to healthy babies (Celebi-Tayfur et al., 2019). Bismuth is not considered to be teratogenic, and although some mutagenic assays have reported DNA damage from certain bismuth compounds, bismuth is not currently classified as a carcinogen.

5.4. Diagnosis and Treatment Diagnosis of bismuth toxicosis can be confirmed by measuring blood bismuth concentrations, but severity of clinical effects does not necessarily correlate with blood bismuth levels (Slikkerveer and de Wolff, 1989). Urine concentrations of bismuth >20 mg/L and blood concentrations >50 mg/L are suggestive of excessive exposure to bismuth (Ovaska et al., 2008; Slikkerveer and de Wolff, 1989). Treatment of bismuth toxicosis entails removal of the bismuth source when possible along with symptomatic care based on the signs observed (e.g., sedatives for encephalopathy, intravenous fluid therapy for renal insufficiency). Chelation therapy with DMPS has shortened the duration of clinical signs some cases of bismuth encephalopathy, but results have not been uniformly successful (Akinci et al., 2015; Ovaska et al., 2008).

6. CADMIUM 6.1. Sources and Exposure Cadmium (Cd, atomic number 48) has become of increasing industrial importance since the early 1900s. The largest use of cadmium is in the electroplating and galvanizing industries, although it also has wide use in color pigments

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for paints, pigments and stabilizers for plastics, cathode materials for nickel-cadmium batteries, and as alloys that lend temperature and pressure stability to industrial equipment. Cadmium is a by-product of zinc and lead mining, refining, and smelting, and it is sometimes present in large quantities in sewage sludge used as fertilizer. Cadmium has been used in the past as a fungicide for turf, although cadmium-based fungicides have largely been replaced by less toxic products. The detection of cadmium in inexpensive jewelry items resulted in massive recalls of these items from specialty and department stores in 2010 (Weidenhamer et al., 2011). The main sources of exposure to cadmium are through contamination of food, water, and air. Ingestion of contaminated food serves as the most significant nonoccupational source of cadmium in humans and animals. Cadmium in soil is taken up by plants, which tend to contain higher levels than animal food products, and some plants have the ability to selectively incorporate cadmium into their tissues. Animals grazing cadmium-contaminated plants will concentrate the element in the liver and kidney, and shellfish from contaminated water may also accumulate high levels of cadmium. Inhalational exposure sources for cadmium include industry, fossil fuel combustion, and cigarette smoking. Because tobacco plants (Nicotiana spp.) concentrate cadmium independently of soil cadmium content, cigarette smoke serves as one of the major nonoccupational sources of inhaled cadmium in humans; an estimated 1– 3 mg of cadmium is absorbed per day in a onepack-a-day smoker.

6.2. Toxicology Ingested cadmium salts are poorly absorbed from the gastrointestinal tract, with the majority being excreted in the feces without being systemically absorbed. The degree of absorption of ingested cadmium varies with species, with 0.5%–3% absorption in monkeys, 1%–2% in rodents, 5% in humans, pigs, lambs, and goats, and 16% in cattle (ATSDR, 2012a). Highly soluble salts (e.g., cadmium acetate, cadmium chloride, cadmium nitrate, cadmium sulfate) are more readily absorbed than less soluble salts (e.g., cadmium sulfide, cadmium sulfoselenide), and metallothionein-bound cadmium in animal

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tissues is less well absorbed than unbound cadmium salts. Cadmium is absorbed primarily in the duodenum and proximal jejunum via enterocyte membrane transporters for essential nutrients such as iron, zinc, manganese, and cysteine. Cadmium absorption is enhanced by poor nutritional status, including dietary deficiencies of calcium, zinc, iron, or protein. Young children and animals have a higher capacity to absorb cadmium than do adults. Absorption of inhaled cadmium is dependent on the particulate size of the inhaled agent; the small particle size of cadmium in cigarette smoke results in enhanced absorption compared to other sources of airborne cadmium exposure. Particles greater than 2 mm will be deposited in the upper airways and removed through the mucociliary escalator, while those < 1–2 mm penetrated deep into the airways and can be systemically absorbed. Up to 30% of inhaled cadmium may reach the lungs, where it readily enters the bloodstream and binds to red blood cells, albumin, or metallothionein, and from there accumulates primarily in the kidney. Within the enterocyte, a fraction of absorbed cadmium binds with metallothionein, an approximately 6800 molecular weight, cysteine-rich protein (Klaasen and Liu, 1997). Metallothionein is present in most tissues, but liver and kidney cells have the largest capacity for metallothionein synthesis. Hence, up to 75% of the total body burden of cadmium ultimately accumulates in the liver and kidneys. Zinc exposure can induce metallothionein synthesis and thus increase cellular tolerance for cadmium. Conversely, zinc deficiency may alter the distribution of cadmium within the body and enhance the toxicity of cadmium. Much of the metallothionein-bound cadmium within enterocytes is eliminated in the feces as the enterocytes are desquamated during the normal process of intestinal mucosal cell turnover. Absorbed cadmium is transported within the bloodstream bound to plasma albumin, red blood cells, or metallothionein, and enters the liver via the portal vein. In the liver, cadmium binds to and induces the further production of metallothionein by hepatocytes. The cadmium– metallothionein complexes are excreted into the bile and eliminated via the feces, but some of the complexes are released by the liver into the general circulation, where they distribute widely

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throughout the tissues. Circulating complexes are filtered through the renal glomerulus and taken up by the proximal tubular cells, resulting in accumulation within renal tubular epithelial cells. Although the liver may transiently have high levels of cadmium following an acute exposure, the kidneys ultimately accumulate the highest concentrations. Similarly, while blood cadmium levels may transiently elevate following acute exposure, urine cadmium levels are more indicative of total body burden (ATSDR, 2012a). Besides liver and kidney, the testes, pancreas, and spleen contain the highest levels of cadmium in the body; muscle and bone do not accumulate significant levels of cadmium, and cadmium does not readily cross physiological barriers such as the blood–brain barrier or placenta (Rani et al., 2014). In the proximal renal tubular epithelium, the cadmium–metallothionein complex is broken down by lysosomes to release free cadmium (Klaasen and Liu, 1997). This free cadmium stimulates the de novo synthesis of additional metallothionein within the cell. If the rate of cadmium release from metallothionein exceeds the cell’s ability to produce metallothionein, cadmium attaches to other proteins within the cell, interfering with cellular metabolism and damaging organelle membranes. Cadmium is slowly eliminated from the body, with daily losses of approximately 0.009% of the total body burden in the urine and 0.007% in the feces. Once in tissues, cadmium has an extremely long biological half-life – estimates of the half-life of cadmium range from 1 to 2 years in rodents and dogs to 15–30 years in humans. Because of slow elimination of cadmium, aged animals tend to have higher body burdens than younger animals, and species with longer lifespans tend to accumulate higher body cadmium burdens than those with shorter lifespans. The potential for cumulative toxicosis following years of exposure makes even relatively low levels of exposures in children a concern. Although cadmium is slowly eliminated through the urine under normal circumstances, in cases of cadmiuminduced nephropathy elimination of cadmium through the urine will be increased. The mechanism of cadmium toxicity is due to a variety of activities including (1) oxidative stress due to production of reactive oxygen

species, (2) replacement of and competition with zinc in biologic systems, resulting in disruption of intracellular metallo-enzyme systems, (3) induction of epigenetic changes in DNA expression, (4) alteration of cellular transport and signal transduction pathways, particularly in the proximal S1 segment of renal tubules, (5) inhibition of heme synthesis, (6) inhibition of DNA damage repair, and (7) impairment of mitochondrial function, which may trigger apoptosis (ATSDR, 2012a; Bernhoft, 2013; Hartwig, 2010; Matovic et al., 2011; Rani et al., 2014). There are marked differences in susceptibility to cadmium-induced tissue injury, both within and between species. Compared to other domestic animal species, horses appear to be able to accumulate higher tissue cadmium levels without showing signs of toxicosis (Hooser, 2018). Inbred mouse strains vary in their susceptibility to cadmium toxicosis, and in some cases there are significant sexrelated differences as well (Table 10.2). Among humans, diabetics have been identified as a population that is more susceptible to the toxic effects of cadmium (Weidenhamer et al., 2011). The increased susceptibility of transgenic metallothionein-null mice to cadmium-induced nephrotoxicity demonstrates the importance of the cadmium–metallothionein complex in protecting the renal tubular epithelium against cadmium-associated injury (Liu et al., 2000; Tokar et al., 2013). However, other cadmiumsensitive strains of mice develop cadmiuminduced injury in the presence of tissue metallothionein levels similar to those of more resistant strains, suggesting that other factors are involved in cytoprotection from cadmiuminduced nephrotoxicity. Protective effects of metallothionein against cadmium-induced hepatotoxicity and bone injury have also been demonstrated. Cadmium-sensitive 129/J mice accumulate significantly more cadmium within the testicle, brain, and epididymis following parenteral cadmium chloride administration than do A/J mice (a cadmium-resistant strain) (King et al., 1999). This difference is attributed to a transport system that regulates the passage of cadmium across vascular barriers and that appears to be attenuated in cadmium-resistant mouse strains. Other factors, such as differences in inflammatory response to injury and differences in

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TABLE 10.2

Species and Strain Differences in Susceptibility to Cadmium-Induced Injury Relative susceptibility

Toxicologic effects

Sensitive

Resistant

Hepatotoxicity

C3H/HEJ mice Fischer 344 rats

DBA/2J mice SpragueeDawley rats

Nephrotoxicity

MT-null mice

MT competent mice

Testicular necrosis

129/J mice

A/J mice

Ectrodactyly (teratogenic)

C57BL/6N mice

SWV mice

Lung cancer

WistareFurth rats

Mice (most strains) Syrian hamsters

Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table I, p 702, with permission.

antioxidant systems, have been demonstrated between cadmium-sensitive and cadmiumresistant animals.

6.3. Manifestations of Toxicosis The primary target organs for acute cadmium toxicosis are dependent upon route of exposure (Table 10.3). In general, acute oral exposure leads to gastrointestinal disturbances, and acute cadmium inhalation results in pulmonary dysfunction (ATSDR, 2012a; Tokar et al., 2013). Oral exposure to high levels of cadmium causes vomiting, diarrhea, abdominal pain, and tenesmus. Depending on the dose ingested, effects of acute oral cadmium toxicosis may range from mild, self-limiting gastritis to severe, fulminating hemorrhagic gastroenteritis. Sequelae such as renal necrosis, hepatic necrosis, cardiomyopathy, and metabolic acidosis may occur in a dose-dependent manner. Microscopic gastrointestinal lesions of acute cadmium toxicosis range from broadening of intestinal villi with mild to moderate submucosal edema and inflammation to extensive necrosis and denudation of absorptive cells with submucosal hemorrhage, inflammation, and vascular thrombosis. In severe cases hepatotoxicosis occurs and is the major cause of acute cadmium-induced lethality. Acute cadmium toxicosis can also result in testicular injury characterized by testicular congestion, edema, hemorrhage, and necrosis within 24 h of exposure.

Acute exposure to significant levels of cadmium by inhalation may result in acute tracheobronchitis, pneumonitis, and/or pulmonary edema (ATSDR, 2012a; Tokar et al., 2013). The primary lesions are necrosis of type I pneumocytes with denudation of the alveolar basement membranes, type II pneumocyte proliferation, and vascular endothelial degeneration and necrosis. Alveoli may contain varying amounts of edema fluid and fibrin, and alveolar macrophages are increased in number. The inflammatory cell response to pneumocyte necrosis may lead to further oxidative damage to alveoli and bronchioles and, ultimately, fibrosis of pulmonary interstitium. In the human lung, subacute to chronic exposure to inhaled cadmium results in bronchitis with progressive alveolar injury and lower airway fibrosis secondary to emphysema. The level of damage correlates to the duration and degree of cadmium exposure. Much of the damage is due to necrosis of alveolar macrophages, with release of lysosomal enzymes causing alveolar damage and fibrosis. In early lesions, fibrosis is restricted to the alveolar interstitium. More progressive lesions include peribronchiolar fibrosis and bronchiectasis, alveolar interstitial and intraluminal fibrosis, and emphysema. Similar pulmonary lesions have been produced experimentally in rats injected with cadmium sulfide and in mice and rats exposed to inhaled cadmium over periods of several weeks. Cadmium-induced nephropathy is one of the more common manifestations of chronic

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TABLE 10.3 Syndromes and Lesions Associated With Cadmium Toxicosis in Humans and Animals Organ/System

Syndrome

Lesions

Gastrointestinal tract

Corrosive gastroenteritis

Diffuse gastrointestinal irritation, ulceration, inflammation, and hemorrhage; submucosal edema, epithelial necrosis, and sloughing

Respiratory tract

Respiratory tract irritation

Tracheobronchitis, pneumonitis, pulmonary edema; type I pneumocyte and alveolar macrophage necrosis

Proximal tubular nephropathy

Tubular dilation, epithelial degeneration, and necrosis;  epithelial cell regeneration; tubular atrophy, interstitial inflammation, and fibrosis

Glomerulopathy

Glomerular basement membrane thickening

Skeletal

Osteopathy

Osteoporosis, osteomalacia, pathologic fractures; increased bone resorptive surfaces, increased immature osteoid, decreased mineralized osteoid

Hepatic

Hepatopathy

Multifocal hepatocellular necrosis, diffuse fatty change

Cardiac

Myocardial damage

Focal to multifocal myocardial edema, degeneration, and necrosis with fibrosis

ACUTE TOXICITY

CHRONIC TOXICITY

Renal

(Continued)

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TABLE 10.3 Syndromes and Lesions Associated With Cadmium Toxicosis in Humans and Animalsdcont’d Organ/System

Syndrome

Lesions

Reproductive

Testicular ischemic necrosis and germ cell apoptosis

Apoptosis of spermatogenic cells; testicular edema, hemorrhage, necrosis, fibrosis, and mineralization

Reproductive dysfunction in females, ovarian necrosis

Hemorrhagic ovarian necrosis

Teratogenesis

Neural tube defects, delayed bone mineralization, limb formation abnormalities

Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table I, p 702, with permission.

cadmium toxicosis. Renal damage from cadmium exposure occurs when the levels of cadmium within the proximal tubular epithelium, primarily the S1 segment, exceed the ability of the kidney to synthesize metallothionein, resulting in free intracytoplasmic cadmium (ATSDR, 2012a; Bernhoft, 2013; Tokar et al., 2013). The “threshold” level of cadmium above which renal damage may occur is generally considered to be 150–200 mg/g of renal cortex, although some animal models have demonstrated nephrotoxicosis at lower levels of cadmium. Lysosomes may be particularly vulnerable to cadmiuminduced damage, resulting in decreased degradation of reabsorbed low molecular weight proteins; diminished resorptive ability of cadmium-damaged tubular epithelium contributes to the proteinuria. Cadmium-induced nephropathy is characterized by low molecular weight proteinuria (particularly b2 microglobulinuria), aminoaciduria, calciuria, glucosuria, and increased urinary excretion of N-acetyl-b-D-glucosaminidase, a high molecular weight lysosomal enzyme present in high concentrations in proximal renal tubules. There is also diminished capacity for renal concentration of urine. Renal lesions consist of proximal tubular cell degeneration and necrosis. Granular and proteinaceous casts may be present within

dilated tubules, which may develop evidence of cellular regeneration over time. More chronic lesions include tubular atrophy, interstitial inflammation, and interstitial fibrosis. Chronic exposure of Beagles to cadmium resulted in grossly discernible renal cortical atrophy, presumably due to renal cell necrosis exceeding the regenerative capacity of tubules. Other chronic renal lesions include fatty change in pars recta tubular epithelium, nephrocalcinosis, and glomerular disease resembling immune complex glomerulonephritis. The latter lesion has been associated with chronic cadmium exposure in rats; large molecular weight proteinuria in humans with chronic cadmium toxicosis suggests primary glomerular injury, but no specific glomerular lesions resembling those in rats have been described in humans. Effects of cadmium on the skeletal system are due to chronic alterations in calcium and Vitamin D metabolism and homeostasis (ATSDR, 2012a; Hooser, 2018; Tokar et al., 2013). Cadmium blocks the renal synthesis of Vitamin D, interferes with calcium and phosphorus absorption, induces active bone resorption, increases calcium excretion, and impairs osteoblast function, leading to poor skeletal growth and decreased bone mineralization. In the 1940s, a condition termed Itai-Itai (“ouch-ouch”)

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disease was described in a group of Japanese women who were chronically exposed to cadmium-contaminated rice. Postmenopausal and multiparous women had increased incidence of disease. The primary lesions described were osteomalacia and osteoporosis, manifested clinically as bone pain, increased bone fragility, and bone deformities. Histologic lesions in bone included increased resorptive surfaces, decreased osteoblast surfaces, increased immature osteoid, and decreased mineralized osteoid. Additionally, many Itai-Itai patients developed normocytic-normochromic anemias, calciuria, and nephropathy. Osteoporosis has also been reported in horses grazed on cadmiumcontaminated pastures; however, an 8-year study of the effects of chronic cadmium administration in Beagles yielded no evidence of skeletal abnormalities (Gunson et al., 1982; Hamada et al., 1991). Multifocal hepatocellular necrosis has been reported with cadmium toxicosis (ATSDR, 2012a; Tokar et al., 2013). Although the mechanism of hepatocellular injury from cadmium may be similar to that seen in the kidney, the ability to minimize hepatic damage by suppressing Kupffer cell function suggests that the hepatic lesion is due at least in part to the actions of the inflammatory response of Kupffer cells rather than a direct toxic effect on hepatocytes. Enhanced sensitivity of the sinusoidal endothelium to cadmium-induced injury occurs in C3H/HeJ mice, a strain susceptible to cadmium-induced hepatotoxicosis, when compared to DBA/2J mice, a cadmiumresistant strain, suggesting that damage to the hepatic microvasculature may be a contributing factor to cadmium-induced hepatic damage (Liu et al., 1992). In Fischer 344 rats, the sensitivity of the liver to cadmium-induced injury declines with advancing age, a phenomenon that is independent of hepatocyte metallothionein and glutathione levels but may be related to impaired Kupffer cell activation in aged animals, resulting in decreased recruitment of inflammatory cells (Yamano et al., 2000). Histopathologic lesions of cadmium-induced hepatopathy include intralobular fibrosis, cirrhosis, multifocal mononuclear cell infiltration, and smooth endoplasmic reticulum proliferation. Cardiovascular lesions in cadmium toxicosis include myocardial damage, hypertension, and

endothelial injury (ATSDR, 2012a; Tokar et al., 2013). Myocardial lesions associated with cardiac impairment have been described in rats chronically exposed to cadmium in drinking water at levels below those that induce renal or hepatotoxicosis. Proposed mechanisms of cadmium-induced myocardial damage include interference with myocardial calcium metabolism, myocardial ischemia secondary to cadmium-induced vascular damage or vasoconstriction, and disruption of selenium-dependent antioxidant mechanisms, resulting in increased susceptibility to myocardial oxidative injury. Lesions consist of foci of myocardial edema, degeneration, and necrosis with fibrosis; similar lesions may also be found in skeletal muscle. Cadmium-induced hypertension has been reported in laboratory rats in a dose-dependent manner and is thought to be due to alterations of vascular smooth muscle calcium regulation (Gokalp et al., 2009). However, epidemiologic studies investigating the relationship of cadmium and hypertension in humans have had conflicting results, and the prevailing assessment is that cadmium does not contribute to hypertension in humans (ATSDR, 2012a; Tokar et al., 2013). Vascular endothelium is sensitive to cadmium-induced cytotoxicity, which leads to capillary leakage, hemorrhagic injury, and inflammation in multiple organs (Prozialeck et al., 2006). These lesions are especially prominent in the testes, liver, and lung and are thought to be largely due to stimulation of lipid peroxidation and oxidative damage to endothelial cells because antioxidant pretreatment can reduce cadmium-mediated endothelial damage. Cadmium may also disrupt endothelial cell–cell junctions, a change especially prominent in glomeruli. Testicular damage from cadmium toxicosis is due to the direct effects of cadmium on testicular zinc metabolism, resulting in apoptosis of testicular spermatogenic cells (Siu et al., 2009). Indirect testicular damage is due to cadmiuminduced endothelial necrosis resulting in edema, hemorrhage, thrombosis, and necrosis followed by fibrosis and mineralization of testicle, epididymis, and vas deferens. In severe cases, extensive fibrosis may result in testicular atrophy and aspermatogenesis. Exposure of females to cadmium may inhibit ovulation and result in uterine dysfunction; these

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effects have been reported in rats, mice, and cattle (Thompson and Bannigan, 2008). In Syrian hamsters, subcutaneous injection of cadmium chloride caused damage to follicular and interstitial stromal arterioles, resulting in hemorrhagic ovarian necrosis. Although the placenta serves as an effective barrier to protein-bound cadmium, free cadmium ions and salts can readily cross the placenta and enter the fetal circulation. In embryonic tissue, cadmium may disrupt normal intracellular signaling pathways or interfere with zinc metabolism. Cadmium exposure during pregnancy has resulted in decreased fetal weights and increased fetal death in experimental animals. Teratogenic effects of cadmium exposure in rats and mice include neural tube defects, axial skeleton dysgenesis, defects in limb formation, or delayed bone mineralization. Similar effects have not been reported in humans. The effect of cadmium on immune function is dose-dependent and is thought to be due to the ability of cadmium to displace or compete with calcium in the activity of calcium-binding proteins such as calmodulin (ATSDR, 2012a; Nath et al., 1984). Cadmium also may interfere with antioxidant systems, resulting in increases in intracellular lipid peroxidation. Humoral responses are increased at lower levels of dietary exposure to cadmium, but at higher levels (>300 ppm) inhibition of humoral responses occurs. Oral cadmium exposure increases the cell-mediated immune response of monkeys (Macaca mulatta), induces antinuclear antibody formation in mice, increases circulating leukocytes in female rats, and causes timedependent inhibitory and stimulatory effects on natural killer cell activity in rats. Cadmium may play a role in the development of diabetes (ATSDR, 2012a). Epidemiological studies have linked exposure to cadmium with hyperglycemia in humans, and cadmium has been demonstrated to impair glucose tolerance with rats. Although accumulation of cadmium within pancreatic islets has been associated with degeneration, necrosis, and weak degranulation of pancreatic b-cells, the full role of cadmium in the development of diabetes remains to be established. Cadmium is a mutagen and causes transformation of cells through a variety of mechanisms including initiation of aberrant gene expression,

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inhibition of DNA repair mechanisms, induction of oxidative stress, inactivation of negative growth stimuli, and inhibition of apoptosis (ATSDR, 2012a; Hartwig, 2010; Tokar et al., 2013). Experimentally, cadmium has been associated with increased incidence of lung, hematopoietic, liver, pancreatic, renal, adrenal, pituitary, prostatic, Leydig cell, and Sertoli cell tumors in laboratory animals. Injection-site sarcomas are common in rats and mice administered cadmium compounds subcutaneously, with multiple injections resulting in development of more aggressive tumors. Attempts to extrapolate the results of rodent carcinogenicity studies for human risk assessment have been hampered by the intra- and interspecies differences in susceptibility to cadmium-induced tumors. In humans, exposure to cadmium has been most clearly associated with the development of lung cancer from inhalational exposure, and there is growing epidemiological evidence of association between dietary cadmium and breast and renal cancers (Genchi et al., 2020; Grioni et al., 2019; Hartwig, 2010). The results of epidemiologic studies investigating the relationship between cadmium exposure and prostatic, pancreatic, and gastric cancers in humans have been contradictory.

6.4. Diagnosis and Treatment Diagnosis of cadmium toxicosis is confounded by the extensive tissue stores, which are difficult to measure using standard laboratory clinical pathological assays (ATSDR, 2012a). Blood cadmium levels are of use primarily for identifying recent exposure and do not reflect the total body burden of cadmium. Urine cadmium concentrations are more reflective of total body burden than blood, and urine cadmium concentrations usually will transiently rise with recent acute exposure. Biokinetic models (e.g., Nordberg-Kjellstro¨m model) have been developed to estimate dietary or airborne cadmium levels based on cadmium concentrations in the urine. Because ingested cadmium is poorly absorbed from the gastrointestinal tract, fecal cadmium concentrations will correlate with dietary cadmium intake. Cadmium concentrates preferentially in liver and kidney and levels in these tissues can be measured by noninvasive in vivo neutron activation or X-ray fluorescence;

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however, these techniques are expensive and pose radiation hazards. Kidney or liver biopsy will give an accurate estimate of body burden, but biopsy is an invasive procedure and is not routinely recommended or performed. Hair cadmium levels appear to be more reliable in cases of high levels of exposure, but lack the sensitivity required to detect lower exposure levels. Urinary N-acetyl-b-D-glucosaminidase (NAG) activity is a sensitive early marker of renal tubular dysfunction that has good correlation with cadmium exposures (ATSDR, 2012a). Urinary NAG activity is useful as for screening at-risk populations, but lacks high specificity as NAG activity can increase due to effects other than nephrotoxicity. Urinary b2-microglobulin levels are sometimes used to screen for cadmium-induced renal tubular dysfunction, as these levels are consistently elevated in patients with cadmium-induced tubular injury. However, urinary b2-microglobulin levels lack specificity as they can be elevated with renal dysfunction from a variety of other causes; additionally, urinary b2-microglobulin levels will normally rise with age, which can make interpretation difficult. Similarly, measurement of retinolbinding protein, other proteins, amino acids, and other solutes in the urine are biomarkers of tubular injury or dysfunction but are not specific for cadmium nephropathy. There is currently no widely recognized, effective treatment for cadmium toxicosis (Bernhoft, 2013; Tokar et al., 2013). Whenever possible, the source of cadmium should be identified and removed from the environment, and symptomatic care for the clinical effects should be provided. The use of chelation therapy is controversial, as chelation during acute toxicosis increases the risk of nephropathy and chelation during chronic toxicosis risks pulling excessive cadmium from tissue depots, precipitating acute toxicosis with severe adverse effects. Chelation with DMPS, dimercaptosuccinic acid (DMSA), and ethylenediaminetetraacetic acid (EDTA) has been used experimentally, with EDTA showing the best efficacy at mobilizing intracellular cadmium (Bernhoft, 2013; Kim et al., 2019). Efficacy of EDTA is said to be improved with concomitant use of glutathione, other antioxidants, mannitol, thiamine, methionine, and zinc. Chelation therapy using a combination of

deferasirox and deferiprone reduced body burden of cadmium and improved clinical signs of cadmium toxicosis in rats (Fatemi et al., 2011).

7. CHROMIUM 7.1. Sources and Exposure Chromium (Cr, atomic number 24) is a naturally occurring metal in the earth’s crust that is released into the environment via natural and anthropogenic activity; the largest release is due to industrial activity (ATSDR, 2012b; Tchounwou et al., 2012). Chromium is used in a large number of industries including metal processing, welding, chrome and chromate production, leather tanning, and wood preservation. Chromium exists in several valence states, with trivalent (chromium(III)) and hexavalent (chromium(VI)) being of toxicological importance. Naturally occurring chromium is primarily in the trivalent state, while hexavalent chromium compounds are produced through anthropogenic oxidation of chromium(III) compounds. Chromium(III) is an essential nutrient in animals, as it potentiates the action of insulin thereby playing a role in fat, protein and glucose metabolism. Chromium(III) has a much lower level of toxicity compared to chromium(VI). Besides exposure through the many industrial uses of chromium, nonoccupational exposure to chromium can occur via ingestion of chromium-contaminated food or water and by inhalation of chromium in the air, with inhalation being the most common route of exposure. The intentional release of chromium(VI)contaminated wastewater into the environment from 1952 to 1966 by Pacific Gas & Electric Company in Hinkley, California, and the accidental release of chromium(VI)-contaminated effluent into the atmosphere on Australia’s Kooragang Island in 2011 gained worldwide attention to the hazards of chromium(VI) compounds, although to date, definitive evidence of a direct correlation of these events to any human illness or cancer has not been found (Langard and Costa, 2007; NSW Government Health, 2017; Pellerin and Booker, 2000).

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7.2. Toxicology The toxicokinetics of chromium compounds vary based on the valence state of the individual compound (ATSDR, 2012b; Tchounwou et al., 2012). Inhalation of chromium results in larger particles (>10 mm diameter) being deposited in the nasopharynx, with smaller particulates entering the deeper airways. Soluble forms of chromium reaching the alveoli are more likely to be absorbed than poorly soluble chromium compounds. Less than 10% of ingested chromium will be absorbed from the gastrointestinal tract, with soluble chromium(VI) being better absorbed than soluble chromium(III). Over 90% of ingested chromium is not absorbed and is eliminated via feces. In the stomach, chromium(VI) is reduced to chromium(III), effectively lowering the overall bioavailability of chromium(VI). Absorption of chromium(III) occurs in the upper small intestine and is dependent upon the dietary intake level; at lower dietary intake of chromium(III), the percent that is absorbed is increased compared to higher intake levels. Chromium has some ability to be absorbed across skin, with increase topical absorption occurring when the epidermal layer is compromised. Once absorbed, chromium is widely distributed throughout the body with highest concentrations in the liver and kidney; bone also accumulates chromium and may serve as a long-term storage depot. In the tissues, chromium(VI) is unstable and is sequentially reduced to chromium(V), chromium(IV), and chromium(III) by a variety of compounds including glutathione and ascorbate; these processes can result in generation of reactive intermediates, protein and DNA adducts, and secondary free radicals. Within erythrocytes, reduction of chromium(VI) can result in complexes with proteins and hemoglobin that are retained in the cell for it lifetime. Absorbed chromium crosses the placenta and is excreted in milk. Although absorbed chromium is eliminated primarily via the urine, some chromium is secreted in the bile. Small amounts are eliminated in the hair and nails. Elimination half-lives of chromium vary from 10 h for chromium(III) to 40 h for chromium(VI). Chromium(VI) is more toxic than chromium(III) due to its higher redox potential and its

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ability to more readily enter cells (ATSDR, 2012b; Chen et al., 2019). Chromium(VI) exists as a tetrahedral anion at physiological pH, resembling other natural anions and can pass through nonselective membrane channels, whereas chromium(III) is an octahedral complex that cannot easily enter cell membrane channels. Reduction of chromium(VI) to chromium(III) generates reactive intermediates which trigger oxidative stress, lipid peroxidation, cytoskeletal alteration, and alteration of cellular signaling pathways. The degree of cellular damage is dependent upon the concentration of chromium(VI), redox potential of the cell, and availability of antioxidants to mitigate effects of the reactive intermediates. Chromium(VI)mediated oxidative stress results in induction or inhibition of transcription factors, activation of p53, activation of hypoxia-inducible factor, cell-cycle arrest, and p53-mediated apoptosis. Direct damage to DNA as well as to DNA repair mechanisms can result in formation and persistence of chromium-DNA adducts, DNA interstrand cross-links, and chromosomal aberrations. Inhibition of DNA and RNA polymerases, mutagenesis, and altered gene expression may also occur. Chromium also can have epigenetic effects by altering histones and DNA methylation.

7.3. Manifestations of Toxicosis Inhalation of soluble chromium(VI) (e.g., chromium(VI) trioxide) at high levels can cause direct erythema, irritation, and erosion of the nasal mucosa, including nasal septum perforation, whereas inhalation of insoluble chromium(VI) compounds results in damage to the lower respiratory tract (ATSDR, 2012b; Tchounwou et al., 2012). Sensitized individuals may develop coughing, dyspnea, wheezing, and signs of asthma or respiratory distress with exposure to any form of chromium. Chronic exposure to chromium(VI) has been associated epidemiologically with increased risk of chronic, progressive, noncancer respiratory disease in humans. Ingestion of chromium(VI) compounds is associated with irritation, ulceration, and corrosive injury to the stomach and small intestine, gastrointestinal hemorrhage, abdominal pain, diarrhea,

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labored breathing, intravascular hemolysis, anemia, and severe liver and kidney damage. Hepatic lesions include congestion, necrosis, and fatty degeneration, and kidney lesions include acute tubular necrosis. Cardiovascular lesions include myocardial hypoxic changes in acute human cases and myocardial fibrosis, necrosis, vacuolization, and hemorrhage in chronic exposures in rats. Microcytic hypochromic anemia occurred in rats chronically exposed to high levels of chromium(III) in drinking water. Renal lesions associated with chromium(VI) exposure in rats included glomerular vacuolization, degeneration of the basement membrane of Bowman’s capsule, and renal tubular epithelial degeneration. Dermatological effects include epidermal ulcers and “blackjack dermatitis,” a manifestation of type I and type IV hypersensitivity reactions (Smith, 2013). Pulmonary edema and hemorrhage have occurred secondary to aspiration of gastric material. Chronic occupational inhalational exposure to chromium has been associated with decreased sperm counts and motility in men (ATSDR, 2012b). Reproductive abnormalities reported in laboratory animals (rats, mice, rabbits, monkeys) exposed orally to chromium(VI) compounds included decreased testes weight, disrupted spermatogenesis, decreased sperm counts in mice and rats but not monkeys. Female mice and rats exposed to chromium(VI) had decreased fertility and increased fetal resorptions; similar reproductive findings have not been identified in women with chronic cadmium exposure. Prenatal exposure of rodent fetuses to chromium(VI) resulted in subdermal hemorrhages, short, kinky tails, delay in ossification of bone (especially tail bones), and impaired postnatal development; some of the effects were thought to be attributable to maternal toxicity from chromium. No information on developmental effects of chromium in human fetuses was found. Increased risk of lung and stomach cancers has been found in epidemiologic studies of workers exposed to chromium(VI) compounds and in people exposed to high levels of chromium(VI) in drinking water (ATSDR, 2012b; Chen et al., 2019). Exposure of laboratory rodents to chromium(VI) has resulted in increased incidence of squamous epithelial tumors of the oral mucosa

and tongue, stomach tumors, and intestinal tumors. The IARC has classified chromium(VI) as a known human carcinogen, while chromium(III) is not classified as a human carcinogen.

7.4. Diagnosis and Treatment Diagnosis of chromium toxicosis is primarily based on clinical manifestations of disease, and exposure to excess chromium can be measured by evaluation of chromium concentrations of blood and urine (ATSDR, 2012b; Smith, 2013). In chronic exposures, steady-state plasma concentrations and urinary excretion rates occur approximately 7 days after initial exposure; after this time, urinary excretion concentrations will reflect the daily amount of chromium that is absorbed. However, declines in plasma and urine levels after cessation will occur within 7 days of the last exposure, so elevated blood and urine chromium levels are indicative of recent exposure and do not reflect the total body burden or severity of clinical effects. Effective treatment strategies for managing chromium(VI) exposures are lacking (Smith, 2013). Hemodialysis and exchange transfusion have been attempted in humans, with mixed results. Chelation therapy for chromium(VI) exposures has been uniformly unsuccessful using standard chelators such as BAL, DMPS, EDTA, and N-acetylcysteine, although N-acetylcysteine may have some value as an antioxidant. Ascorbic acid administered as a nonspecific antioxidant has been shown to be beneficial only in the first few hours following exposure; after 3 h, administration of ascorbic acid was actually detrimental in animal experiments.

8. LEAD 8.1. Sources and Exposure Lead (Pb, atomic number 82) is one of the most ubiquitous and useful metals known to humans, and it has been widely utilized throughout history for a variety of purposes (ATSDR, 2020; Tokar et al., 2013). Historically, lead was used in everyday products such as food utensils and was also added to wine to smooth out the flavor and color. Because of the well-recognized health hazards with such uses, lead is now rarely used

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in products associated with food or food preparation. Lead and lead alloys are used in automotive and other batteries, solders, electrical and telephone line shieldings, and paints (use in residential paints was banned in the United States in 1977, but outdoor and agricultural use of leadbased paints still occurs). In children, ingestion of lead-based paint flakes and chips from older buildings remains a medical and political concern. Improperly glazed cooking bowls have on occasion been sources of lead toxicosis in humans. Lead contamination of drinking water can occur through contamination of groundwater or due to the leaching of lead from lead-containing municipal service lines by acidic water, as happened in Flint, Michigan, in 2014–15, leading to media and political backlash (ATSDR, 2020; Banner, 2018). Lead contamination of drinking water in Tasmania, Australia, was sourced back to high lead solder used in stainless steel rainwater tanks, leading to lead levels in the water that exceeded established lead limits for drinking water by 200-fold (Lodo et al., 2018). Some fish and shellfish may accumulate lead from contaminated water, and food producing animals exposed to areas with lead contaminated water, air, or soils may have elevated levels of lead in the muscle and milk (ATSDR, 2020). Lead contaminated nutritional supplements and herbal preparations have resulted in lead toxicosis in the United States, England, and New Zealand; one study of 252 Ayurvedic herbal preparations revealed that 65% contained detectable lead levels, with 36% of these containing up to several thousand times the daily recommended intake values (Mikulski et al., 2016). Tetraethyl lead used in gasolines has been associated with elevated lead levels in humans and animals in urban areas; however, with the banning of leaded gasolines a reduction in background lead levels due to urban exposure has been reported (Tokar et al., 2013). In cattle, lead toxicosis is most commonly associated with ingestion of discarded batteries, farm machinery grease or oil, lead-based paints, putties and caulks, or roofing felt (GwaltneyBrant 2004; Thompson, 2018). The curious nature of cattle coupled with the unfortunate habit of some people to use pastures as junkyards has made lead toxicosis in cattle a relatively common occurrence. Horses and sheep are most commonly exposed when grazing on

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pastures contaminated by airborne emissions from nearby smelters. Other sources of lead for livestock include water from lead-lined water pipes, lead in drinking or feeding utensils, and lead arsenate pesticides (although acute arsenic toxicosis is the more common manifestation). Swine are considered to be relatively resistant to the effects of lead and reports of lead toxicosis in swine are rare. Household pets may be exposed to lead through ingestion of leaded paints in old houses, leaded artist’s paints, linoleum, and lead toys, weights, fishing sinkers, or ornaments. Lead toxicosis in waterfowl due to ingestion of spent lead shot is a serious concern (as few as eight No. 6 lead shot BBs can cause fatal lead toxicosis in waterfowl) and has been partially alleviated through the requirement that steel shot be used when hunting waterfowl on public lands. However, the use of lead shot for upland game and for waterfowl on private lands has resulted in continued environmental contamination with lead. Lead intoxication is the most common poisoning reported in raptors in the United States, due primarily to scavenging on carcasses containing spent lead ammunition (Redig and Arent, 2008).

8.2. Toxicology Lead fumes or fine particles less than 0.5 mm are readily absorbed across the lungs; larger particles may be coughed up and swallowed, causing oral exposure (ATSDR, 2020; Thompson, 2018; Tokar et al., 2013). Ingested lead is poorly absorbed in adults, but the young can absorb up to 50% of ingested lead, making them more susceptible to lead toxicosis. Calcium, zinc, or iron deficiency can enhance the gastrointestinal absorption of ingested lead. Due to the poor bioavailability of nonionized lead, lead embedded in soft tissues is not appreciably absorbed and does not cause toxicosis, although lead from embedded objects that elicit local inflammation (e.g., lead shot in a joint cavity) may become ionized by the inflammatory process, resulting in lead absorption. As with many other metals, while inorganic lead is poorly absorbed via epidermal exposure, organolead compounds such as tetraethyl lead are readily absorbed through the skin (ATSDR, 2020; Thompson, 2018).

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Absorbed lead is bound to red blood cells and distributed widely throughout tissues, with highest concentrations in bone, teeth, liver, lung, kidney, brain, and spleen (ATSDR, 2020; Thompson, 2018; Tokar et al., 2013). Lead crosses the blood–brain barrier and concentrates in the gray matter of the brain. Lead readily crosses the placenta, and alterations in calcium metabolism during pregnancy may result in significant amounts of lead being released from maternal bone and transferred to the developing fetus. Over time, most absorbed lead will be found in the bone, where it is substituted for calcium in the bone matrix. Bone serves as a long-term storage depot for lead, allowing the accumulation of large bone stores that may result in continued toxicosis long after exposure has ceased. Metabolic processes that enhance bone remodeling (e.g., fracture repair, lactation) may release stored lead and precipitate signs of toxicosis. Absorbed lead is slowly excreted through the kidneys, primarily through glomerular filtration, and significant loss of lead can occur through sloughing of renal tubular epithelium, where lead tends to concentrate. Chelation therapy can greatly enhance the urinary excretion of lead. Smaller quantities of lead are eliminated in sweat and other body secretions. Lead has a triphasic halflife in many species, with blood half-life of days to months, soft tissue half-life of weeks to years, and bone half-life of up to decades (ATSDR, 2020; Gwaltney-Brant, 2004). Lead exerts its effects through binding of sulfhydryl groups, competition with calcium ions, inhibition of membrane-associated enzymes, altered metabolism of Vitamin D, and generation of reactive oxygen species (ATSDR, 2020; Gwaltney-Brant, 2004; Thompson, 2018; Tokar et al., 2013). Lead has high affinity for sulfhydryl groups and can thereby inactivate enzymes, especially those involved in heme synthesis such as d-aminolevulinic acid dehydratase and ferrochelatase (Figure 10.2). By competing with calcium, lead becomes stored in bone, alters nerve and muscle transmission, disrupts physiological barriers (e.g., blood–brain barrier), and displaces calcium in the activity of essential calcium-binding proteins such as calmodulin. Lead inhibits membrane-associated enzymes such as sodium–potassium pumps, causing increased red blood cell fragility, renal tubular damage, and, in humans, hypertension.

Interference with Vitamin D metabolism results in derangements in calcium absorption. Lead may also interfere with zinc in some enzymes, and at high concentrations may interfere with GABA production or activity in the CNS. Generation of reactive oxygen species can occur in lead toxicosis leading to exhaustion of antioxidant stores, lipid peroxidation, interference with enzyme systems, nucleic acid damage, and inhibition of DNA repair processes (Sachdeva et al., 2018). Binding of lead to low molecular weight proteins such as metallothionein essentially sequesters lead and inhibits its toxic effects (de Sousa et al., 2018). Metallothionein also acts as a free radical acceptor that can help mitigate lead-induced oxidative stress. Glucoseregulated protein 78 (GRP78) is an endoplasmic reticulum chaperone protein that effectively binds lead and whose synthesis is increased

FIGURE 10.2 Effect of lead on heme synthesis. Lead inhibits d-aminolevulinic acid synthetase, resulting in accumulation of d-aminolevulinic acid and decreased formation of porphobilinogen. Lead-mediated inhibition of ferrochelatase decreases the incorporation of iron into protoporphyrin, which leads to decreased formation of heme. Lead also stimulates the activations of coproporphyrinogen to coproporphyrin. Red arrows designate lead-mediated inhibition, whereas green arrows designate lead-mediated stimulation. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013 Figure 41.3, p 1328, with permission.

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during exposure of glial cells to lead. Other leadbinding proteins include a2-microglobulin, acylCoA-binding protein, and thymosin b4.

8.3. Manifestations of Toxicosis in Animals Acute lead toxicosis in animals occurs most often in cattle and is usually manifested by neurological signs (Gwaltney-Brant, 2004; Redig and Arent, 2008; Thompson, 2018). Affected cattle develop hyperesthesia, ataxia, and muscle tremors followed quickly by recumbency and intermittent convulsions. Death generally occurs within 12–24 h. Adult cattle may show a lesser tendency to become recumbent and instead may develop dementia, head pressing, and blindness prior to terminal convulsions. Those animals surviving 4–5 days become apathetic and anorexic, and may appear blind. Cattle may display salivation, hyperesthesia, tenesmus, and severe depression. Sheep display similar signs as cattle. Horses may develop acute paralytic disease upon exposure to large amounts of lead. Signs begin as depression and progress to general paralysis, sometimes with clonic convulsions and abdominal pain. Acute lead poisoning in dogs and cats is neurologic in nature, with the signs including anorexia, agitation, muscle tremors, ataxia, and intermittent convulsions. Waterfowl may die acutely without prior signs, or they may display weakness, ataxia, and lethargy prior to death. Raptors with acute lead toxicosis may demonstrate weakness, dell mentation, visual impairment, and biliverdinuria. Specific lesions may be absent in animals dying of acute lead intoxication. Intranuclear inclusions in liver and kidney cells have been reported in dogs with lead toxicosis, although these are more commonly seen with chronic toxicosis. Chronic lead toxicosis in cattle generally presents with nervous signs similar to those of acute toxicosis, but of lesser severity and longer duration, accompanied by decreased rumen motility, emaciation, diarrhea, colic, and anorexia (Gwaltney-Brant, 2004; Thompson, 2018). Significant microscopic lesions include laminar cortical necrosis with swelling of cerebral capillary endothelium (Figure 10.3). Some cattle may have evidence of renal tubular epithelial cell degeneration, with or without fibrosis; these lesions are more commonly seen in calves.

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Basophilic stippling of erythrocytes, considered a diagnostic aid in humans and dogs, is not diagnostic of lead toxicosis in cattle, which may normally have stippled erythrocytes. In horses, chronic lead toxicosis manifests as “roaring,” an abnormal breathing noise secondary to laryngeal and pharyngeal paralysis; other cranial nerve deficits may also be present. The lesions include segmental myelin and axonal degeneration of motor neurons and are similar to those seen in the peripheral neuropathy in humans. In dog and cats, chronic

FIGURE 10.3 Polioencephalomalacia in the cerebrum of a cow. At low magnification, a laminar pattern of edema is visible in the middle to deep gray lamina of the cerebral cortex. H&E stain. Inset: At greater magnification, eosinophilic neurons (arrows) with pyknotic nuclei (ischemic cell change) can be seen in the left half of the field, in addition to edema (right field of view). H&E stain. Figure reproduced from Haschek WM, Rousseaux, CG, Wallig, MA, editors: Fundamentals of toxicologic pathology, ed 2, Academic Press, 2012, Figure 13.18, p 401, with permission.

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lead toxicosis often presents as a combination of vague signs of gastrointestinal upset and neurological dysfunction, including personality changes, seizures, lethargy, and ataxia. Prolonged anorexia, vomiting, and diarrhea may result in severe emaciation. Significant lesions are found in the nervous tissue and include edema of white matter of the brain and spinal cord, myelin degeneration within the cerebellum and cerebrum, and spongy degeneration in the subthalamus, head of the caudate nucleus, and deep cortical laminae. Vascular lesions similar to those seen in the nervous systems of cattle and human may be noted but are not consistently found. Mild astrogliosis may be present. The liver and kidney of dogs with lead toxicosis may show degenerative changes accompanied by intranuclear inclusions similar to those seen in the kidneys of humans with lead toxicosis (Papaioannou et al., 1998). Chronic lead toxicosis in waterfowl and other wildlife most often results in chronic weight loss, emaciation, and death (Figure 10.4) (Gwaltney-Brant, 2004; Thompson, 2018). Raptors with lead toxicosis are weak, emaciated, anemic, biliverdinuric, and depressed, with crop stasis. Lead shot may be present in the gizzard

of affected birds although, due to their habit of regurgitating (casting) indigestible ingesta, frequently raptors with lead intoxication have no identifiable lead in their digestive tracts at the time of diagnosis. In birds, atrophy of breast muscles results in a “razor keel” appearance. Pale streaks may be seen within the myocardium, corresponding to patchy areas of myocardial necrosis associated with fibrinoid necrosis of arterioles. Other microscopic lesions include hepatocellular necrosis, renal tubular degeneration and necrosis with occasional intranuclear inclusions in the proximal tubules, edema of brain and meninges, myelin degeneration in peripheral nerves, patchy necrosis of muscle of gizzard, and anemia with abnormalities in erythrocyte size and shape. Reproductive effects of lead include decreased fertility in males and females and increased fetal deaths (ATSDR, 2020). Abortions, premature births, low birth weights, and developmental delays have been associated with elevated blood lead levels in humans. Defects in cognitive ability in infants exposed to lead in utero have been reported, and aged monkeys (Macaca fascicularis) that had been exposed to lead as infants had increased presence of amyloid plaques in the cerebral cortex. Lead has been reported to cause renal tumors, specifically renal adenocarcinomas in rats and mice (ATSDR, 2020). Evidence for lead-induced tumors in humans is not conclusive, although inorganic lead is classified as a probable human carcinogen by the US Environmental Protection Agency; the IARC has classified inorganic lead compounds as probable carcinogens and organic lead as not classifiable as to carcinogenicity in humans.

8.4. Human Exposure and Disease FIGURE 10.4 Lead toxicosis, American alligator (Alligator mississippiensis). Extreme emaciation in an alligator from Florida that was found to have approximately 50 lead pellet fragments in its stomach. Hepatic lead levels were 70.8 ppm, more than 2000 times the reference value for alligators (27.7 ppb). Photograph courtesy Drs Jennifer Chilton and Darryl Heard, University of Florida. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013 Figure 41.6, p 1331, with permission.

Acute lead toxicosis in humans is less common than the chronic form, and it usually manifests as nephropathy characterized by acute renal failure, oliguria, aminoaciduria, glucosuria, and altered tubular ion transport (ATSDR, 2020; Diamond and Zalups, 1998; Tokar et al., 2013). Specific lesions may be absent in acute lead toxicosis, or renal lesions may be present. Degeneration and necrosis of tubular cells is often accompanied by the presence of dense, eosinophilic, and homogeneous intranuclear inclusion bodies

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FIGURE 10.5 Lead-induced nephrosis, rat. Acid-fast intranuclear inclusion bodies (arrow) present in the proximal convoluted tubular epithelium. Acid-fast stain with H&E counterstain. Photograph courtesy of Dr J. King, College of Veterinary Medicine, Cornell University. Figure reproduced from Newman et al: Urinary system. In McGavin M, Zachary JF, editors: Pathologic basis of veterinary disease, ed 4, 2007 Figure 11.41, p. 654, with permission. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013 Figure 41.4, p 1329, with permission.

that represent lead complexed with nonhistone nuclear proteins (Figure 10.5). The close association of metallothionein with intranuclear inclusion bodies in WT mice with lead-induced nephropathy suggests that metallothionein is mechanistically involved in formation of the inclusions (Waalkes et al., 2004). These inclusions are more commonly seen in chronic lead exposure and should not be considered pathognomonic, as similar inclusions can occur with exposure to other metals such as bismuth and neptunium. Other signs of acute lead toxicosis in humans include nausea, abdominal pain, vomiting, shock, paresthesia, and muscle weakness (ATSDR, 2020). Acute hemolysis resulting in anemia and hemoglobinuria has been reported. Death can occur within 1–2 days or the patient may survive to develop signs of chronic lead poisoning. Chronic lead toxicosis (plumbism) in humans is often divided into separate categories based on the organ system involved: gastrointestinal, neuromuscular, renal, and hematological, central nervous system (ATSDR, 2020; Sachdeva et al., 2018; Tokar et al., 2013). These syndromes may occur separately or in combination. In the United

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States, the central nervous system syndrome (lead encephalopathy) is more commonly seen in children and the gastrointestinal syndrome is more prevalent in adults. In children, lead encephalopathy is characterized by lethargy, mental dullness, vomiting, irritability, anorexia, and dizziness that may progress to ataxia, stupor, and possibly death. Sequelae in those recovering may include epilepsy, mental deficits, and optic neuropathy with blindness. In some cases, progressive decreases in cognitive function and increases in behavior disorders, especially aggression and hyperactivity, may be overlooked for many months or even years. Lesions include prominence of cerebral and cerebellar capillaries with endothelial cell swelling and necrosis, resulting in severe cerebral edema due to enhanced capillary leakage. There is gliosis and loss of neuronal cells most prominent in the middle to deep layers within the cerebral sulci. Degeneration and necrosis of cerebellar Purkinje cells may be present. Radiologic evaluation of growing bones may show heavy, multiple bands of increased density in the epiphyseal margins (“lead lines”). “Lead lines” may also be seen as blue-black discoloration of the gingiva in cases of lead encephalopathy. The gastrointestinal syndrome of lead toxicosis usually begins insidiously, with anorexia, myalgia, malaise, headache, and a metallic taste in the mouth (ATSDR, 2020; Sachdeva et al., 2018; Tokar et al., 2013). Early diarrhea may occur, but constipation eventually develops and may become quite severe. Severe abdominal cramping (“lead colic”) develops over time – attacks of colic are intermittent and severe. There are no specific lesions associated with the gastrointestinal syndrome. The neuromuscular syndrome or “lead palsy” is an uncommon manifestation of advanced lead toxicosis (ATSDR, 2020; Sachdeva et al., 2018; Tokar et al., 2013). Signs begin as muscle weakness and fatigue that may progress to paralysis. The muscles involved tend to be the most actively used muscles, especially the extensors of the forearm, wrist, and fingers, and extraocular muscles. The result is an inability to extend the hand or foot, resulting in “wrist drop” or “foot drop,” which, when it occurs, is considered almost pathognomonic for lead toxicosis in humans. Motor nerves appear to be selectively involved, as no sensory nerve deficits

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TABLE 10.4 Mechanisms of Lead-Induced Anemia Enzyme inhibited

Mechanism

Effect

d-Aminolevulinic acid dehydratase (ALA)

Decreased conversion of ALA to porphobilinogen

Accumulation of ALA, decreased heme synthesis

Ferrochelatase

Decreased incorporation of iron into protoporphyrin IX to form heme

Accumulation of protoporphyrin IX, decreased heme synthesis

RBC Naþ/Kþ ATPase

Alteration of cellular energy metabolism

Increased RBC membrane fragility

Pyrimidine-5nucleosidase

Accumulation of pyrimidine nucleotides within RBC or reticulocyte

RBC basophilic stippling

Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table I, p 716, with permission.

have been reported. Lesions include segmental demyelination and axonal degeneration of peripheral nerves. Hematologic manifestations of lead toxicosis relate to the interference of lead with normal heme synthesis and to increased erythrocyte membrane fragility due to lead-induced alterations in membrane-associated enzymes (see Table 10.4) (ATSDR, 2020; Sachdeva et al., 2018; Tokar et al., 2013). These abnormalities result in microcytic hypochromic anemia. Inhibition of pyrimidine-5-nucleosidase results in production of reticulocytes with basophilic stippling. The renal syndrome of chronic lead toxicosis is often subclinical, although renal function tests may detect asymptomatic renal azotemia and decreased glomerular filtration rate (ATSDR, 2020; Sachdeva et al., 2018; Tokar et al., 2013). Eventually, chronic renal failure develops. Lesions begin as tubular degeneration similar to that seen in the acute disease. Eventually, damage to tubular epithelium progresses to tubular atrophy and interstitial fibrosis. Intranuclear inclusions may be found in renal tubular epithelial cells and, occasionally, hepatocytes.

8.5. Diagnosis and Treatment Diagnosisofleadpoisoningisbasedonevaluation of blood lead concentrations in light of clinical signs, symptoms, clinical pathological abnormalities, and history (ATSDR, 2020; Sachdeva et al., 2018).

Elevated blood lead levels are indicative of recent exposure, but do not reflect total body burden. Radiography of the gastrointestinal tract may identify lead-containing foreign materials such as paint chips, lead sinkers, jewelry, etc. Clinical pathological abnormalities consistent with lead intoxication include normocytic to microcytic, hypochromic anemia, hemolytic anemia, basophilic stippling of erythrocytes, azotemia, elevations in urinary porphyrins, and proteinuria. Radiography of long bones may reveal “lead lines” in the metaphyseal areas in cases of chronic lead intoxication. Treatment of lead intoxication involves removal from the source of exposure, chelation therapy, and symptomatic treatment (Bjorklund et al., 2017; Kim et al., 2019; Sachdeva et al., 2018). Removal of lead objects from the gastrointestinal tract prior to chelation therapy is essential, as most chelators, with the exception of DMSA, will enhance the gastrointestinal absorption of lead. Chelators that have been used in lead poisoning include DMPS, DMSA, BAL, calcium EDTA, and penicillamine; of these, DMSA is considered the optimal chelator for moderate to severe lead poisoning due to its lower risk of side effects, superior efficacy, oral or rectal administration, and superior pharmacokinetic profile. Blood lead levels drop during chelation therapy, but it is common to see a rebound of blood lead concentration upon discontinuation of chelation due to

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redistribution of lead from soft tissue and bone stores (Bjorklund et al., 2017).

9. MERCURY 9.1. Sources and Exposure Mercury (Hg, atomic number 80) is a ubiquitous metal that is released into the environment through geologic activity such as volcanic eruptions, earthquakes, soil erosion, and leaching from the earth’s crust (ATSDR, 1999; Berlin et al., 2007; Magos and Clarkson, 2006; Tokar et al., 2013). Mercury is used in the manufacture of electrical instruments, thermometers, and batteries, and as a fungicide, algaecide, and seed treatment. Mercury is also used in the pharmaceutical and dental industries and has historically been used medicinally as a diuretic, antibiotic, and laxative. Mercury contamination of water, both fresh and salt water, through natural or anthropogenic sources, is of concern due to the ability of fish and seafood to accumulate mercury in their tissues. This bioaccumulation of mercury results from the ability of some marine organisms to form insoluble mercury–selenium complexes within cells, resulting in the development of relatively high tissue levels of mercury, which ultimately enter the food chain. Occupational exposure to mercury occurs in a variety of industries including mining operations, electrical instrumentation and fluorescent lamp manufacturing, and certain research facilities (e.g., medical, physics, dental research). Historically, mercury was used in taxidermy and the felt hat industry, potentially causing neurological disease in workers (thus the phrase “mad as a hatter”) (Strekopytov et al., 2017; Jackson, 2018). Nonoccupational exposure to mercury occurs primarily through food, especially methylmercury from predatory fish and from plants grown in areas with water and/or soil contamination, atmospheric contamination primarily from coal plant emissions, and absorption from mercury-containing dental amalgams. Mercury poisoning in humans has been associated with topical application of mercurycontaining skin lightening and antiwrinkle creams (Chan, 2011).

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9.2. Toxicology The mechanism of mercury toxicity has historically been attributed to binding of mercuric ions to sulfur, resulting in inhibition of thiol-containing enzymes in the mitochondria and microsomes, leading to disruption of normal cellular processes and cell death (ATSDR, 1999; Berlin et al., 2007). However, more recent study has suggested that mercury has a higher affinity for selenium than for thiol groups and that the primary molecular targets of mercury may be selenoproteins of enzyme systems such as thioredoxin reductase and glutathione reductase (Spiller, 2018). Mercuryinduced inhibition of these enzyme systems results in disruption of the intracellular redox environment, leading to proliferation of intracellular reactive oxygen species which in turn can trigger lipid peroxidation, impaired protein repair, mitochondrial injury, calcium dysregulation, and apoptosis. Mercury ions may also disrupt ion exchanges across voltage-gated and ligand-gated channels, interfering with normal membrane transport (Berlin et al., 2007; Tokar et al., 2013). Additionally, substitution of mercury for diatomic hydrogen to form mercaptides can further alter enzyme function, cellular respiration, and membrane transport. In the nervous system, mercury has been associated with increased production of amyloid-beta peptides and abnormal tau proteins, contributing to development of amyloid plaques and neurofibrillary tangles (Fonseca Arrifano et al., 2018). Through these mechanisms, mercury alters the blood–brain barrier, suppresses synaptic transmission, and inhibits neuronal protein synthesis, resulting in neurologic dysfunction. Alteration of sodium–potassium pumps by mercury binding results in leaky membranes, endothelial swelling, and increased release of fluid into the interstitium. These effects are particularly evident within the nervous system. Mercury has complex chemical properties and a wide range of toxic effects on humans and animals (Table 10.5). From a toxicological point of view, it is more convenient to consider the different forms of mercury separately, as elemental mercury, inorganic mercury compounds, and organic mercurials.

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TABLE 10.5 Toxicologic Effects of Mercury Form

Organ/system

Effect

Lung

“Metal fume fever”: Bronchitis, bronchiolitis, pneumonitis, pyrexia, progressive pulmonary fibrosis

Kidney

Renal tubular necrosis

Gastrointestinal tract

Ulcerative colitis

Nervous system

Ataxia, behavior change, tremor

Gastrointestinal tract

Gingivitis, stomatitis

Kidney

Renal tubular necrosis, glomerulonephritis

Gastrointestinal tract

Corrosive stomatitis, esophagitis, gastroenteritis, colitis

Kidney

Renal tubular necrosis

Kidney

Renal tubular necrosis, glomerulonephritis

Nervous system

Ataxia, behavior change, dementia, tremor

Dermal

“Acrodynia”: Erythema, hyperkeratosis, dermatitis, edema

Nervous

Ataxia, behavior change, dementia, paresthesia, paralysis, tremor

Kidney

Renal tubular necrosis

ELEMENTALMERCURY

Acute toxicity

Chronic toxicity

INORGANIC MERCURY

Acute toxicity

Chronic toxicity

ORGANIC MERCURY

(Continued)

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TABLE 10.5 Toxicologic Effects of Mercurydcont’d Form

Organ/system

Effect

Cardiac

Purkinje fiber degeneration, myocarditis, myocardial mineralization

Reproductive

CNS and skeletal deformities

Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Table I, p 708, with permission.

9.3. Elemental Mercury Elemental mercury, or quicksilver, is a liquid that can vaporize at room temperature. The toxicity of elemental mercury is generally limited to inhalation of vapors, resulting in “metal fume fever” in humans (Berlin et al., 2007; Magos and Clarkson, 2006; Tokar et al., 2013). Gastrointestinal or cutaneous exposures result in minimal absorption of elemental mercury. However, because the monoatomic gas is highly diffusible and lipid soluble, inhalation results in rapid and complete absorption through the alveoli. In the blood, most mercury enters red blood cells, but the free mercury in blood is rapidly transported throughout the body, tending to concentrate in the kidney, brain, and heart. Highest body concentrations occur within the kidney. As a monoatomic gas, mercury readily crosses the placenta and the blood–brain barrier, where highest levels occur within the brainstem, cerebellar nuclei, and spinal cord. Mercury in red blood cells and tissues is oxidized by catalase to form divalent inorganic mercury; catalase-mediated oxidation of mercury passing through the placenta takes place in fetal tissues. Fecal and urinary excretion occurs approximately equally. A small amount of solubilized mercury may pass back into the alveoli, where it is eliminated via expiration. Acute metal fume fever due to inhalation of mercury is rarely seen these days because of tighter regulations that reflect the increased knowledge of the toxic potential of mercury (ATSDR, 1999; Berlin et al., 2007; Magos and Clarkson, 2006; Tokar et al., 2013). Acute mercury inhalation causes severe bronchial irritation and coughing followed by fever, dyspnea, nausea, stomatitis, vomiting, diarrhea, confusion, dehydration, and shock. Death

may occur within a few hours of massive exposure. Those surviving the initial onset may develop progressive pulmonary dysfunction, ulcerative colitis, oliguria, or azotemia. Lesions include erosive bronchitis and bronchiolitis with acute pneumonitis and edema. Progressive fibrosis of the lungs has been reported in human exposure to mercury vapor. Renal damage is characterized by hydropic degeneration and necrosis of tubular epithelium, epithelial desquamation, and albuminous luminal casts; those surviving 7–10 days will have evidence of regeneration of tubular epithelial cells. Experimentally, signs and lesions consistent with metal fume fever have been reproduced in rabbits and rats exposed to moderate to high levels of mercury vapor. Chronic elemental mercury toxicosis, while still quite uncommon, is more frequently seen than acute toxicosis (ATSDR, 1999; Magos and Clarkson, 2006; Tokar et al., 2013). Gastrointestinal disturbances such as hypersalivation, gingivitis, and stomatitis occur, accompanied by neurological manifestations such as hyperexcitability, irritability, aggression, anxiety, and/or fine muscle tremors that worsen with emotional stress. Other signs include lethargy, weakness, diaphoresis, ataxia, hemiplegia, and lens opacities. Glomerulonephritis resembling that seen with inorganic mercury toxicosis (see below) has occasionally been described in humans but has not been reproduced in laboratory animals exposed to mercury fumes.

9.4. Inorganic Mercury Because of their use as topical and oral medications in the past, the monovalent and divalent salts of inorganic mercury have historical significance (Tokar et al., 2013). The use of mercury-

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based medicinals is currently banned in many countries, but some countries still find use for these products as medicinal agents. In the United States, the most common source of inorganic exposure is through occupational inhalation of elemental mercury with in vivo enzymatic conversion to inorganic mercury. Less than 15% of ingested inorganic mercury salts are absorbed by the gastrointestinal tract, although significant absorption of topically applied inorganic mercurial compounds may occur (ATSDR, 1999; Berlin et al., 2007; Magos and Clarkson, 2006; Tokar et al., 2013). Once absorbed, inorganic mercury is transported by plasma proteins and accumulates in the kidneys. Although the blood–brain barrier serves to keep most inorganic mercury from the nervous system, with chronic administration significant levels of mercury may accumulate in cerebellar and cerebral cortices. Poor passage of mercuric ions through the placenta results in little risk of fetotoxicity. Inorganic mercury is oxidized by catalase to divalent mercury, which localizes within lysosomes of renal tubular epithelium. Elimination of mercury is through the urine; however, renal excretion is inefficient, resulting in accumulation of mercury within the kidneys. Many inorganic mercurial salts are corrosive, and ingestion can lead to necrosis of oral, pharyngeal, esophageal, and gastrointestinal mucosa. Vomiting, oral pain, abdominal pain, and gastrointestinal bleeding may occur. In severe cases, gastrointestinal corrosion may lead to severe gastrointestinal hemorrhage, luminal pooling of fluids, hypovolemia, electrolyte imbalance, shock, and death. Acute renal failure may occur in those surviving beyond 24 h. Lesions include hemorrhage and ulcers throughout the gastrointestinal tract and necrosis of renal tubular epithelium, beginning in the pars recta and ultimately spreading throughout all regions of the renal tubules. Chronic inorganic mercury poisoning may result in renal failure, dementia, and acrodynia (“pink disease”) (ATSDR, 1999; Berlin et al., 2007; Magos and Clarkson, 2006; Tokar et al., 2013). Gross lesions described in rats, cattle, and pigs include grossly swollen, wet, pale kidneys. Microscopic renal lesions include hypertrophy and dilatation of proximal convoluted tubules, with epithelial degeneration and necrosis (Figure 10.6). Chronically, mononuclear

FIGURE 10.6 Inorganic mercury-induced nephrosis, mouse, 3 days after receiving an intraperitoneal injection of 3 mg/kg mercuric chloride. Tubular epithelial necrosis (arrowhead) and dilatation, intraluminal necrotic debris (ND), and hyaline casts (*). H&E stain. Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013 Figure 41.2, p 1326, with permission.

interstitial inflammation and fibrosis occur. Experimentally, autoimmune-mediated glomerulonephritis has been produced in mice and rats following administration of inorganic mercurial salts. Early antibasement membrane glomerulonephritis is followed by IgMmediated immune complex glomerulonephritis with transient elevations of circulating immune complexes. Eventually, an interstitial immune complex nephritis may develop. Dementia and tremors similar to those seen in elemental mercury toxicosis may occur with chronic inorganic mercury toxicosis. Acrodynia is described in humans as erythema and edema of the hands and feet, hyperkeratosis, skin rash, tachycardia, hypertension, photophobia, irritability, and decreased muscle tone. Splenomegaly and lymphadenopathy are described in children, who are more disposed to developing acrodynia. The mechanisms of acrodynia are unknown, but it is postulated to be a manifestation of a hypersensitivity reaction.

9.5. Organic Mercury Organic mercury toxicosis usually occurs through ingestion of contaminated food products, especially fish and seafood products due

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to their bioaccumulation of mercury from contaminated water (ATSDR, 1999; Berlin et al., 2007; Magos and Clarkson, 2006; Tokar et al., 2013). Minamata disease was a neurological syndrome described in humans, birds, and cats that were exposed to methylmercurycontaminated fish in the Minamata area of Japan in the 1960s. Organomercurials are also used as fungicides for seeds and plants. In humans, sporadic outbreaks of neurological diseases related to ingestion of flour made from ethylmercury-treated seed grain have been in Iraq, Pakistan, Guatemala, and Ghana (Ni et al., 2017). Organomercurials are well absorbed by inhalation or ingestion (ATSDR, 1999; Magos and Clarkson, 2006). In ruminants, methylmercury is demethylated to inorganic mercury, decreasing absorption. Dermal absorption, although slow, can be sufficient to cause toxicosis. Absorbed organomercurials are transported bound to red blood cells and are widely distributed throughout the body. Organomercurials readily cross the blood–brain barrier and placenta, where fetal blood levels can exceed maternal blood levels by up to 20%, which can result in significant damage to the fetal nervous system. Organomercurials also are secreted into milk. The biological half-life of organic mercury in humans is approximately 65 days. Alkylmercury compounds have stable mercury–carbon bonds that are slowly cleaved after absorption, making these products more persistent and more toxic. Aryl and alkoxyalkyl mercury compounds have mercury–carbon bonds that are more readily cleaved, and these compounds are subsequently catabolized to free mercury ions, which are then eliminated. Methylmercury is the most toxic of organomercurials, primarily due to its persistence (as demethylated inorganic mercury) within the central nervous system. Methylmercury that is not sequestered within the nervous system is demethylated and primarily excreted in the feces as inorganic mercury. The primary target organ for organomercurials is the nervous system (ATSDR, 1999; Magos and Clarkson, 2006; Tokar et al., 2013). Signs may begin days to weeks following exposure, depending on the amount and type of organic mercury involved. Neurological effects include visual disturbances, ataxia, paresthesia, hearing

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loss, dysarthria, mental deterioration, muscle tremor, sensory dysfunction, movement disorders, behavioral changes, and death. Pigs tend to develop a paralytic disease, while in cattle the syndrome resembles lead toxicosis. Rats and monkeys (M. fascicularis) develop ataxia, impaired motor function, and gait abnormalities upon exposure to dietary organomercurials. Neurologic lesions include cortical neuronal loss that may be manifested grossly as laminar cortical necrosis, with or without cerebellar or cerebral edema. Histopathologic nervous system lesions in organomercury toxicosis have been described in humans, cats, swine, laboratory rodents, mink, nonhuman primates, and several other species (Figure 10.7) (ATSDR, 1999; Gupta et al., 2018; Haschek et al., 2007). There is atrophy of folia in the sulci of the lateral lobes of the cerebellum, most evident in the granular cell layers. Purkinje cell degeneration may be quite pronounced in mice and cats. Bilateral cortical atrophy in the anterior calcarine fissure may be present. Areas of neuronal degeneration and necrosis with neuronophagia are present within the hypothalamus, thalamus, midbrain, and basal ganglia. Neuronal lesions are accompanied by multifocal to extensive areas of gliosis and spongiosis. Fibrinoid necrosis of cerebral arterioles has been reported in several species, and cattle and pigs may have fibrinoid necrosis of the leptomeningeal arteries. Axonal lesions include Wallerian degeneration and demyelination of sensory nerve fibers, dorsal nerve roots, and peripheral nerves; in humans, demyelination of the lateral columns of the spinal cord has been associated with toxicity to aryl mercury compounds. Nonneurologic lesions of organomercury toxicosis are less commonly reported (ATSDR, 1999; Gupta et al., 2018; Tokar et al., 2013). Renal lesions resemble those of inorganic mercury toxicosis (see above). In organomercurial toxicosis of cattle, pigs, rats, and humans, myocardial injury may result in cardiac arrhythmias and failure. Grossly, cardiac lesions are minimal, although hydropericardium has been described in pigs. Histologically, Purkinje fiber degeneration, myocarditis, and myocardial mineralization have been described; cardiac lesions may be minimal to absent in pigs. Gastrointestinal signs (vomiting, abdominal pain, and diarrhea)

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FIGURE 10.7 Mercury toxicosis, cerebrum, pig. Note the eosinophilic neurons (arrows) with pyknotic nuclei (ischemic cell change) located in the middle to deep lamina of the cerebral cortex. Hematoxylin and eosin stain. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Figure 13.9, p 394, with permission.

have occasionally been reported in humans, but specific gross or histopathologic gastrointestinal lesions are not reported. Organomercurials readily cross the placenta and may result in spontaneous abortions, severe fetal cerebellar and cerebral deformities, cleft palates, and limb deformities in various species (ATSDR, 1999; Gupta et al., 2018; Ni et al., 2017). Significant strain and species variation in sensitivity to organomercurial teratogenesis exists. In studies using guinea pigs, rats, mice, and monkeys, organomercurials administered during gestation frequently resulted in alterations in learning and behavioral abnormalities in offspring. Behavioral alterations were associated with decreased numbers of muscarinic receptors in the brains of young Sprague–Dawley rats exposed prenatally to methylmercury, although no specific neurologic histopathologic lesions were noted. Exposure of male Fischer 344 rats to inorganic mercuric chloride for 2 years resulted in increased incidence of forestomach squamous cell papillomas and thyroid follicular cell carcinomas. Wistar rats exposed to phenylmercuric acetate in the drinking water at 4.2 mg/kg/day for 2 years had significant increases in renal cell adenomas. Dietary exposure of ICR mice and B6C3F1 mice to dietary organomercurials resulted in development of renal epithelial cell adenomas and carcinomas. The International Agency for Research on Cancer (IARC) has

classified methylmercury as a possible human carcinogen, and the US Environmental Protection Agency categorizes mercury chloride and methylmercury as potential human carcinogens.

9.6. Diagnosis and Treatment Clinical diagnosis of mercury intoxication can be difficult due to the myriad of other diseases that may cause similar signs and symptoms (Bernhoft, 2012; Risher and Amler, 2005). As with most other metals, blood and urine mercury concentrations do not correlate with total body burden. Blood and urine mercury levels generally remain elevated for just a few days following exposure. Some governmental agencies have established reference values for mercury in urine, the fact that significant clinical signs of mercury poisoning can occur in individuals with urinary levels below these values demonstrates that measurement of urine mercury concentrations alone is not an ideal means of diagnosis. However, measurement of urine mercury concentrations in samples collected for 24 h before and 24 h after administration of a chelator can provide a relatively reliable estimate of the body burden of mercury; for this application, DMPS has been shown to be superior to DMSA and to induce fewer adverse effects than BAL (Bernhoft, 2012). Hair may be a useful matrix to track chronic exposure to

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organic mercury compounds but does not provide information on the body burden (Risher and Amler, 2005). There is no well-studied or uniformly approved treatment for mercury intoxication, although, as with other metals, removal of the source of exposure is a primary goal. The use of traditional chelating agents such as BAL, DMPS, DMSA, or EDTA in the management of mercury poisoning is controversial largely due to the legitimate concern that rapid mobilization of mercury from tissue stores may result in redistribution of mercury into the nervous system, resulting in more severe neurologic injury and potentially worsening clinical signs of toxicosis (Berlin et al., 2007; Bjorklund et al., 2017; Kim et al., 2019; Kosnett, 2013; Risher and Amler, 2005; Tokar et al., 2013). Chelation therapy also may worsen renal tubular injury as the chelated metal is eliminated via the urine. Of the chelators, BAL is associated with the most severe adverse effects when used to chelate mercury, and DMPS is a more effective chelator of inorganic mercury than is DMSA. Chelation therapy can decrease renal concentrations of elemental and inorganic mercury, but chelators vary in their ability to remove organomercurials. BAL can lower tissue levels of most forms of mercury but is not recommended for organomercurial chelation due to the potential to produce severe neurological adverse effects. DMSA can remove methylmercury from nervous tissue without the severe adverse effects seen with BAL. DMPS does not appear to effectively reduce brain levels of methyl- or phenylmercury; alkylmercury has been successfully chelated using a combination of oral DMPS and intravenous N-acetylcysteine or cysteine. Supplementation with selenium and vitamin E has been suggested, as these compounds have been shown to reduce the toxicity of inorganic mercury (Spiller, 2018; Tokar et al., 2013).

10. PLUTONIUM (See Also Volume 3, Chap 14, Radiation and Other Physical Agents) 10.1. Sources and Exposure Plutonium (Pu, atomic number 94) is an anthropogenic radioactive element that is a by-

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product of uranium use in nuclear reactors; it is an extremely hazardous compound due to intense emission of a-particles (ATSDR, 2010). It is estimated that approximately 10,000 kg of plutonium released from research facilities, nuclear weapons production and testing, nuclear waste disposal, and accidents are present in the environment, accounting for the majority of nonoccupational exposure. An estimated 70–75 metric tons of plutonium is produced from nuclear reactors worldwide each year. Trace amounts of plutonium occur worldwide due primarily to fallout from nuclear weapons testing and usage. Atmospheric plutonium deposits onto water and soil, where it can adsorb to soil and sediments or bioaccumulate in aquatic and terrestrial food chains. 239Pu, with a half-life of 24,390 years, has potential to cause chemical as well as radiation injury (see Vol 3, Chap 14; Radiation and Other Physical Agents). Most significant exposures to plutonium are restricted to occupational exposures of those working in the nuclear industry.

10.2. Toxicology Plutonium is poorly absorbed by inhalation, ingestion, or dermal exposure (ATSDR, 2010). Although trace amounts of plutonium may occur in water and food, resulting in ingestion, by far the most significant exposure to plutonium is via inhalation. The degree of absorption is determined by valence state and solubility of the plutonium compound, with hexavalent and chelated forms being more readily absorbed into the blood from alveoli. A significant amount of inhaled plutonium is taken up by pulmonary phagocytic cells and may remain in the pulmonary interstitium and lymph nodes for many years. Absorbed plutonium localizes largely to the bone (primarily in phagocytic cells of the bone marrow) and liver; strong binding to plasma proteins limits the ability of plutonium to freely distribute to other body compartments. Excretion of absorbed plutonium is slow, with less than 15% of an intravenously administered dose being eliminated through the urine and feces within 2 years. The remaining plutonium is retained within the body, primarily within phagocytic cells of lung, bone marrow, and, to a lesser extent, liver.

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Because it can cause both chemical and radiation injury, it can be difficult to distinguish between the chemical versus radiation pathologic effects of plutonium (ATSDR, 2010). The primary mechanism of plutonium-induced injury is attributable to short-range alpha radiation emissions, which result in typical radiation effects: injury to rapidly dividing cells, especially of the gastrointestinal tract and bone marrow, and DNA damage leading to various cancers. Because alpha radiation has limited ability to penetrate beyond its site of deposition, the primary tumors associated with plutonium exposure are related to the lungs (site of entry) and to the bone marrow and liver (sites of distribution) (Muggenburg et al., 1996). The mechanism of chemical toxicity of plutonium is thought to be due to binding of ionized plutonium molecules to cellular proteins and phospholipids causing membrane damage, enzyme disruption, and organelle derangement.

10.4. Diagnosis and Treatment In most cases, management of plutonium exposure is necessitated by the radiologic hazard posed by exposure rather than concern for chemical toxicosis (Fukuda, 2005; NCRP, 2008; Poudel et al., 2018). Management of plutonium exposure entails removal from the source of exposure and provision of supportive care. Removal from areas of airborne contamination and copious cleansing and rinsing is recommended for inhalational and topical exposures, respectively. Wounds containing plutonium should be cleansed and thoroughly irrigated with saline solution to prevent systemic absorption; in some cases, surgical excision of the wound to remove embedded material and/or contaminated tissues may be indicated. When used early following exposure, diethylenetriaminepentaacetic acid (DTPA) is an effective chelating agent that can enhance excretion of plutonium, and is FDA approved for such use.

10.3. Manifestations of Toxicosis The target organs for plutonium are the bone marrow and kidney (ATSDR, 2010). Proximal renal tubular and glomerular cells exposed to plutonium demonstrate severe mitochondrial injury, manifested by swelling and destruction of cristae; these changes are thought to be due more to the chemical effects of plutonium rather than radioactive effects. Similar ultrastructural lesions have been described in bone marrow and hepatocytes. Pulmonary lesions have been described in rats, hamsters, dogs, mice, and baboons exposed to long-term inhalation of plutonium; however, these lesions were more consistent with radiation pneumonitis with its progression to chronic fibrosis and neoplasia (Tannoo and Parquet, 1996). No reports of chemically induced lung disease due to chronic plutonium exposure in humans could be found, and epidemiologic studies on workers occupationally exposed to inhaled plutonium have shown no specific pulmonary lesions even after more than 35 years had elapsed. There are no reports of carcinogenic or teratogenic effects of plutonium in animals or humans other than those describing changes attributed to the radionuclide effects of the chemical (see Volume 2, Chap 31, Radiation and Physical Agents).

11. THALLIUM 11.1. Sources and Exposure Thallium (Tl, atomic number 81) is found in nature in potash, mineral ores, and fossil fuels, and is produced as a by-product of cadmium, lead, and zinc smeltering (ATSDR, 1992; Duan et al., 2020; Hoffman, 2003; Tokar et al., 2013). Naturally occurring high thallium concentrations in groundwater and soil may lead to assimilation by crops, such as that which has caused endemic toxicosis in human populations in Guizhou Province in China (Ning et al., 2021). Environmental exposures also occur from emissions of cement factories, coal-burning power plants, and smelters. Thallium is used in manufacture of electronics components, fireworks, dyes, optical lenses, jewelry, and superconductors. The use of thallium as a rodenticide and pesticide is restricted in many countries, including the United States, but still occurs in some developing countries. Due to its ease of acquisition and its colorless, odorless, and tasteless characteristics, thallium has notoriously been used as a means of poisoning and suicide for well over 100 years. Historically, thallium was used in treatment of diseases such as

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11. THALLIUM

syphilis and tuberculosis, and thallium radioisotopes are currently used as myocardial imaging agents in human medicine.

11.2. Toxicology Thallium is rapidly and completely absorbed via dermal, oral, and respiratory routes (ATSDR, 1992; Hoffman, 2003). The high degree of absorption, high bioavailability, and slow excretion contribute to the high toxicity of thallium. Once absorbed, thallium distributes widely, with highest levels in the renal medulla and significant levels present in heart, liver, stomach, and hair. Thallium can cross the placenta, but fetal levels tend to be much lower than maternal blood levels. In chronic exposures, accumulation in bone may occur. Thallium is slowly eliminated through the urine and bile. Because of the slow rate of excretion, cumulative thallium toxicosis is possible even at relatively low levels of exposure. In humans, the elimination half-life of thallium ranges from 1 to 30 days, likely dependent upon the initial absorbed dose. Toxic effects of thallium are due to alteration of potassium-dependent processes, interaction with sulfhydryl groups, interference with B Vitamins (especially riboflavin and thiamine), alteration of CNS neurotransmitters, and pertubation of intracellular calcium levels (ATSDR, 1992; Cvjetko et al., 2010; Hoffman, 2003). With an affinity for sodium–potassium ATPase pumps 10 times that of potassium, at low levels, thallium replaces potassium and is actively transported across membranes where it accumulates intracellularly. At high levels, thallium inhibits potassium-dependent pumps. Thallium also replaces potassium in some NADHmediated cellular systems, interfering with intracellular signaling and metabolism. Mitochondria are very dependent on potassiumdependent channels and are particularly susceptible to the effects of thallium. Inhibition of mitochondrial pyruvate dehydrogenase and succinate dehydrogenase complexes blocks the catabolism of carbohydrates and the entry of electrons into the electron transport chain, decreasing ATP generation. Thallium may also activate other intracellular potassiumdependent enzymes, disrupting normal cell metabolism. Substitution of thallium for potassium alters nerve cell excitability and muscle fiber contractility. Thallium further interferes

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with cellular respiration by binding sulfhydryl groups on mitochondrial enzymes such as pyruvate dehydrogenase and succinate dehydrogenase. Binding of thallium to sulfhydrylcontaining enzymes such as hydrolases, oxidoreductases, and transferases interferes with normal cellular metabolism. By binding sulfhydryl groups on cysteine molecules in cysteinerich keratin, thallium increases the solubility of keratin and decreases its resistance to stretching – these alterations are manifested clinically as skin, hair, and nail abnormalities. Thallium may also reduce glutathione levels, resulting in increases in free radical and peroxide-induced damage to cellular membranes.

11.3. Manifestations of Toxicosis In acute thallium toxicosis, onset of clinical signs may occur within hours of exposure, although a delay in onset of 24–48 h is not uncommon (ATSDR, 1992; Cvjetko et al., 2010; Hoffman, 2003). At low levels of exposure, onset may be delayed as much at 3 weeks. In humans, a triad of gastroenteritis, polyneuropathy, and progressive alopecia is considered the hallmark of thallium intoxication. Gastroenteritis precedes other signs, neurological signs follow in approximately 2–5 days, and alopecia may not develop for 1–3 weeks following exposure. The initial signs of vomiting and hypersalivation are followed in hours to days by severe colic, ulcerative stomatitis with brick-red mucous membranes, diarrhea, and anorexia. Death due to shock may occur rapidly following onset of gastrointestinal signs, or signs may persist for up to several weeks. Hemorrhagic gastroenteritis, and colitis, and colonic ulceration may occur in those surviving the initial signs. Vagal nerve involvement can cause decreased esophageal and intestinal motility and may result in esophageal dilatation with secondary ulcerative esophagitis. Microscopic gastrointestinal lesions include mucosal epithelial necrosis with focal ulceration, submucosal edema, erythema, and hemorrhage, and infiltration of submucosa by inflammatory cells. Focal suppurative pancreatitis has been described in animals with thallium toxicosis. Neurological signs consist of paresthesia and hyperesthesia of extremities, ataxia, weakness, tremors, and motor neuropathy that may progress to respiratory paralysis, visual defects, and

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behavioral abnormalities such as aggression, anxiety, lethargy, and disorientation (ATSDR, 1992; Osorio-Rico et al., 2017). Microscopic lesions of neural damage due to thallium include focal areas of necrosis within the mesencephalon, cerebral and cerebellar edema, perivascular cuffing of mononuclear cells, neuronal chromatolysis, and neuronophagia. Lesions in peripheral nerves include focal axonal swelling and fragmentation, swelling, and degeneration of myelin sheaths. Long peripheral nerve fibers, especially large sensory nerve tracts, appear to be preferentially affected. In animals, cataracts, iritis, and conjunctivitis have been reported. The cataracts appear early as radial striations in the anterior lens cortex between the sutures and equator that progress to subcapsular opacities. Microscopically, the subcapsular epithelium is disrupted and accumulations of a homogeneous to granular material are present axially to the lens fibers. Optic neuritis with atrophy of the optic nerve may also occur. Cutaneous lesions can be detected as early as 3–5 days following exposure and are originally manifested as black pigmentation of hair roots, representing deposition of thallium in hair shafts (ATSDR, 1992; Cvjetko et al., 2010; Hoffman, 2003). Further lesions develop over days to weeks and, in humans, include palmar erythema, acne, anhydrosis, hyperkeratosis, seborrhea, onychodystrophy with Mee’s lines, and enhanced epilation of hair. Alopecia tends to originate in areas of cutaneous friction. In dogs and cats, cutaneous lesions begin at the commissures of the lips, at the nasal cleft, and at the ear margins, and expand over the face and head. Cutaneous erythema, hyperkeratosis, alopecia, scaling, exudation, and secondary pyoderma may develop; in dogs, hyperkeratosis of footpads may result in the development of thick scales resembling the “hard pad” lesions described in canine distemper. Biopsy of affected skin in dogs reveals a preponderance of catagen and telogen follicles with degenerative changes in anagen follicles. These lesions suggest that the mechanism of thalliuminduced alopecia is due primarily to interference with energy metabolism in rapidly dividing matrix cells of anagen follicles rather than alterations in keratin formation. Other histologic lesions include parakeratotic hyperkeratosis of epidermal surface and external root sheath epithelium, follicular plugging, intraepithelial

microabscesses, hypogranulosis, dermal edema and hyperemia, dermal infiltration with neutrophils and mononuclear cells, and focal necrosis of sweat and sebaceous glands. Cutaneous lesions are largely reversible in those surviving the more severe gastrointestinal and neurological manifestations, although months may be required for complete resolution of signs. Alterations in heart rate and blood pressure have been reported in humans with thallium intoxication (ATSDR, 1992; Cvjetko et al., 2010; Hoffman, 2003). Proposed mechanisms for these alterations include interference in potassium channels in the myocardium, resulting in altered myocardial contractility and altered nervous stimulation due to vagus nerve damage. Multifocal necrosis of both myocardial and skeletal muscle fibers has been described in thallium intoxication in animals. Other lesions of thallium toxicosis include renal tubular degeneration and necrosis, pulmonary edema, reticuloendothelial hyperplasia, lymphoid depletion, and secondary bronchopneumonia. Hepatic necrosis and bone marrow suppression have been described in cats with thallium intoxication. Chronic thallotoxicosis may lead to progressive debilitation, secondary infections, and death. Adult male Wistar rats chronically exposed to 10 ppm of thallium sulfate in drinking water developed testicular lesions characterized by degenerative changes of Sertoli cells and seminiferous tubules (Leung and Ooi, 2000). Alopecia and nail abnormalities have been described in a human fetus exposed to thallium in the last trimester; epidemiologic studies on prenatal exposures to thallium have otherwise shown either normal development or a trend toward prematurity (Hoffman, 2003; Tokar et al., 2013). Prenatal exposure to thallium in humans has been associated with shortened neonatal telomere length and decreased neonatal mitochondrial copy numbers, although the clinical significance of these changes has not yet been elucidated (Wu et al., 2021; Wu et al., 2019). Thallium can induce DNA damage and is mutagenic in some in vitro assays (Cvjetko et al., 2010; Rodriquez-Mercado and AltamiranoLozano, 2013). Rats chronically exposed to thallium developed proliferative gastric lesions and gastric papillomas. However, due to insufficient data, thallium has not been classified as a carcinogen in animals or humans.

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12. URANIUM

11.4. Diagnosis and Treatment Suspicion of thallium toxicosis is usually triggered by the characteristic clinical findings, especially alopecia and peripheral neuropathy (Hoffman, 2003; Tokar et al., 2013). Other less specific symptoms and clinical laboratory results, such as proteinuria or ECG abnormalities, may help raise the index of suspicion sufficiently for more advanced testing to be instituted. Thallium can be detected in blood, urine, feces, saliva, hair, and nails. Blood and urine levels tend to remain elevated longer than other metals, but still will decline with time following an acute exposure, but chronic exposures generally display elevated thallium concentrations. Management of thallium toxicosis entails removal of the source of exposure (including gastrointestinal decontamination for recent exposure) (Cvjetko et al., 2010; Hoffman, 2003). Historically, the standard treatment for thallium toxicosis was potassium ferric ferrocyanide (potassium ferric hexacyanoferrate, Prussian blue, Paris blue), which preferentially binds thallium in the GI tract, altering the concentration gradient and interfering with enterohepatic recirculation and thereby reducing the body burden. Forced diuresis and repeated dosing with activated charcoal have been recommended along with Prussian blue administration. However, there have been no controlled trials on the efficacy of any of these modalities alone or in combination, and published data are limited to individual case reports or case series. Combination hemoperfusion/hemodialysis did appear to yield high thallium clearance from the blood in some cases, but this modality is poorly studied and should be considered only in cases of life-threatening toxicosis until further data are available.

12. URANIUM (See also Volume 3, Chap 14, Radiation and Other Physical Agents) 12.1. Sources and Exposure Uranium (U, atomic number 92) is found in nature as the isotopes 238U, 235U, and 234U (ATSDR, 2013; Dinocourt et al., 2015). 238U comprises over 99.2% of uranium found in

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nature, where it is admixed with 0.7% 235U and 0.01% 234U. “Enriched” uranium has been chemically manipulated to increase the concentration of 235U to either 2%–4% for poorly enriched uranium (used for nuclear power reactors) or to >90% for highly enriched uranium (used for research reactors, nuclear submarine reactor cores, and nuclear weapons). Depleted uranium is previously enriched uranium that has had its radioactivity largely spent and is less radioactive than natural uranium, being composed of 99.8% 238U, 0.2% 235 U, and 0.0006% 234U. Natural uranium and depleted uranium are a-emitters; depleted uranium is also a weak g-emitter. 238U has the longest half-life of the three isotopes (4.5 billion years), making it the least radioactive isotope and the isotope most likely to cause chemical toxicosis rather than radiation injury. 238U is used for photographic intensifiers, ceramic colorants, dental porcelain additives, radiation shielding, military armor, and armor-piercing bullets. The main source of exposure to uranium is from mining and manufacturing of uranium products. The use of depleted uranium products in military armor and ammunition has resulted in a significant number of military veterans who retain embedded fragments of depleted uranium in their soft tissues due to ammunition or shrapnel wounds; the risk to health of this type of exposure has remained a political hot-button for years. Nonoccupational exposure to uranium would be primarily from environmental, food, or water contamination.

12.2. Toxicology The solubility, physiochemical form, and entry route of uranium determine the target organs, toxic response, and method of elimination (ATSDR, 2013; Vincente-Vincente et al., 2010; Yue et al., 2018). The major route of exposure is via inhalation. Inhalation absorption is dependent on particulate size and solubility, with particles larger than 10 mm in diameter likely to be transported out of the tracheobronchial region by the mucociliary escalator and swallowed. Smaller particles may reach the alveoli, where they enter the blood at a rate corresponding with their solubility; highly soluble forms (e.g., uranyl fluoride) get into the blood

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within days whereas a poorly soluble form (e.g., uranium dioxide) may remain in the lungs and regional lymph nodes for years. Uranium has poor oral absorption, with absorption of ingested uranium being less than 5% in humans and less than 1% in laboratory animals. Based on animal studies, dermal absorption of soluble uranium compounds can be sufficient to cause systemic poisoning. Absorption of uranium embedded in soft tissues does occur, but neither the extent nor the rate of absorption has been well defined. Ocular exposure also has resulted in significant systemic absorption. Following intravenous injection, approximately 50% of depleted uranium is eliminated via urine, 1%– 2% is eliminated via feces, w25% distributes to bone, and the remainder enters soft tissues. Highest tissue levels accumulate in bone (66%), liver (16%), and kidney (8%), where uranium selectively isolates in the proximal renal tubules. In bone, uranium eventually diffuses into the mineralized matrix, where it may persist for years. Although uranium distributes to the liver, the liver is not a major storage organ. Oxidation of tetravalent uranium to the hexavalent form precedes conversion to uranyl ion (UOþ2 2 ) (ATSDR, 2013; Yue et al., 2018). Uranyl ion may complex with citrate, bicarbonate, or plasma proteins such as transferrin. The uranium–bicarbonate complex is filtered through the glomerulus and excreted in the urine in a pH-dependent fashion. Protein-bound uranium is poorly eliminated by glomerular filtration due to the size of the complex. Free uranyl ion binds local proteins, transferrin in the blood, and proteins or phospholipids in proximal tubular cells in the kidney. The majority of circulating uranium is rapidly excreted, while uranium bound in bone or sequestered in pulmonary lymph nodes may be slowly released to the plasma and eliminated over a period of months or years. In the kidneys, the uranium–bicarbonate complex is dissociated, and uranyl ion released from the complex causes tubular damage by binding phospholipids and proteins of the tubular epithelial cell membranes. Membrane damage results in interference with reabsorption of glucose, sodium, amino acids, and protein, and cell death results from suppression of cellular respiration.

12.3. Manifestation of Toxicosis Acute uranium toxicosis is rare and is most likely to occur from inhalation and absorption of soluble uranium compounds (Vincente-Vincente et al., 2010). There is considerable species variation in sensitivity to uranium toxicosis, and a proposed order of species sensitivity from most to least sensitive is: rabbit > rat > Guinea pig > pig > mouse > dog > cat > human. Sex, age, and body mass index differences in susceptibility to uranium toxicosis have also been identified. The target organ for acute uranium toxicosis is the kidney, where glomerular and tubular damage results in proteinuria, glucosuria, and aminoaciduria (ATSDR, 2013; VincenteVincente et al., 2010; Yue et al., 2018). Acute renal failure may occur with high levels of exposures. Chronically, kidneys may show a mosaic of degenerated and regenerated tubules and glomeruli. Histologic renal lesions include glomerular endothelial swelling, necrosis, and sclerosis; ultrastructurally, there is loss of cell foot processes and alterations in endothelial fenestrae. Tubular epithelium shows apical nuclear displacement and vesiculation, loss of the brush border, vacuolation, and necrosis. Interstitial fibrosis and mononuclear inflammation may occur in long-standing toxicoses. Upon cessation of uranium exposure, regeneration of tubular epithelium and glomerular endothelium may occur if fibrosis has not intervened. Because up to 66% of absorbed uranium is distributed to and stored in bone, the potential for uranium-induced bone abnormalities has been the subject of some limited studies in laboratory animals (ATSDR, 2013; Yue et al., 2018). Chronic exposure of rats to depleted uranium compounds resulted in decreases in bone formation and increases in bone resorption, suggesting that uranium may impair bone healing and may contribute to osteoporotic/osteopenic bone disorders. Although acute corrosive rhinitis, tracheitis, and pneumonitis have been described in humans and laboratory rodents exposed to inhaled uranium hexafluoride, these lesions were considered to have been secondary to the fluoride content rather than the uranium (ATSDR, 2013). Similarly, necrosis of type I and type II pneumocytes, interstitial pulmonary edema and

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13. SUMMARY AND CONCLUSIONS

inflammation with fibrosis, and hyperplastic or metaplastic epithelial changes that have been described in some chronic human exposures have been largely attributed to inhaled contaminants such as radium or radon. Animal studies of uranium inhalation have failed to produce significant pulmonary alterations. A single human case of intentional ingestion of uranium acetate resulted in acute renal failure, refractory anemia, rhabdomyolysis, myocarditis, liver dysfunction, and paralytic ileus. Although the patient survived the exposure, residual renal damage in the form of incomplete Fanconi syndrome was present 6 months following exposure. Although some experimental uranium exposures in rodents have been associated with neurobehavioral abnormalities, the evidence is contradictory and difficult to interpret in light of confounding factors present in the existing rodent studies (Dinocourt et al., 2015). The evidence that uranium can induce behavioral disturbances is stronger in studies where animals were exposed during cerebral development than in adult. Reproductive abnormalities identified with high doses of uranium in laboratory animals include testicular lesions, decreased sperm counts, ovarian dysfunction, fetal toxicity, and teratogenicity (ATSDR, 2013). Epidemiological studies in humans have not found an association between exposure to natural or depleted uranium and adverse effects on reproduction. Epidemiologic human studies and experiments with laboratory animals have not shown a clear connection between the metallic toxicity of uranium and cancer (ATSDR, 2013). Some studies have found increased risk of lung cancer, but it is unclear whether uranium is the causative agent and whether cancer may have been induced by chemical toxicity rather than radiotoxicity.

12.4. Diagnosis and Treatment The primary biomarker of exposure to uranium is the detection of total uranyl ions in the urine (ATSDR, 2013). Urinary uranium concentrations tend to correlate with airborne uranium exposures. Elevated uranium concentrations in urine and feces are indicative of recent exposure, whereas nail and hair samples can identify uranium exposure weeks or months

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prior to sampling. Unlike many other metals, hair and nail uranium levels have been found to correlate well with daily intake of uranium from drinking water (Karpas et al., 2005). Management of uranium exposure entails removal from the source of exposure and provision of supportive care. In many cases, removal of uranium from the body is necessitated by the radiologic hazard posed by uranium exposure rather than concern for chemical toxicosis. Although various chelating agents including DPTA, siderophores, bisphosphonates, calixarenes, and polyaminocarboxylic acids have been investigated as potential chelators for uranium, few have shown efficacy and safety, and there are no approved chelators currently available for management of uranium toxicosis (Fukuda, 2005; Smith, 2013; Yue et al., 2018). Intravenous sodium bicarbonate infusion has historically been used in uranium toxicosis to alkalinize urine and trap uranyl ions in the urine; however, sodium bicarbonate has relatively low efficacy and poses the potential risk of iatrogenic systemic acid–base imbalance.

13. SUMMARY AND CONCLUSIONS As should be apparent from this discussion, although our knowledge regarding the signs and lesions of heavy metal intoxication might be quite broad, in many cases the exact mechanism of action of the metal is still not fully elucidated. For example, lead is a metal that has been utilized for centuries, yet we are only now beginning to grasp the biochemical mechanisms surrounding the development of lesions of lead toxicosis, and it may be quite some time before the full nature of cellular damage induced by lead is comprehended. In other cases, such as chronic beryllium disease, much of the biochemistry has been determined, to the point that it is now possible to detect those people who possess the genetic mutation that puts them at risk for the hypersensitivity caused by beryllium exposure. Because metals will continue to be present in every aspect of human life, further research into mechanisms of metal-induced toxicosis may prove the key to reducing environmental contamination, minimizing the risk of toxicity, and improving the available treatment modalities.

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REFERENCES Akinci E, Koylu R, Yortanli M, et al.: Acute bismuth intoxications: acute renal failure, tonsillar ulceration and posterior reversible encephalopathy syndrome, Hong Kong J Emerg Med 22:121–125, 2015. ATSDR: Toxicological profile for antimony. Agency for Toxic Substances and Disease Registry (ATSDR), Atlanta, GA, 2019, US Department of Health and Human Services. Public Health Service. ATSDR: Toxicological profile for arsenic. Agency for Toxic Substances and Disease Registry (ATSDR), Atlanta, GA, 2007, US Department of Health and Human Services. Public Health Service. ATSDR: Toxicological profile for beryllium. Agency for Toxic Substances and Disease Registry (ATSDR), Atlanta, GA, 2002, US Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for cadmium, Atlanta, GA, 2012a, US Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for chromium, Atlanta, GA, 2012b, US Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for lead, Atlanta, GA, 2020, US Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for mercury, Atlanta, GA, 1999, US Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for plutonium, Atlanta, GA, 2010, US Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for thallium, Atlanta, GA, 1992, US Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for uranium, Atlanta, GA, 2013, US Department of Health and Human Services. Public Health Service. Banner W: “Toxicohistrionics”: Flint, Michigan and the lead crisis, J Pediat 195:15–16, 2018. Bernhoft RA: Mercury toxicity and treatment: a review of the literature, J Environ Public Health 2012(460508), 2012. Bernhoft RA: Cadmium toxicity and treatment, Sci World J 2013:394652, 2013. Belyaeva AP: The effect of antimony on reproduction, Gig Truda Prof Zabol 11:32, 1967. Berlin M, Zalups RK, Fowler BA: Mercury. In Nordberg GF, Fowler BA, Nordberg M, et al., editors: Handbook on the toxicology of metals, ed 3, Boston, 2007, Elsevier, pp 675–727.

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Slikkerveer A, de Wolff FA: Pharmacokinetics and toxicity of bismuth compounds, Med Toxicol Adverse Drug Exp 4:303– 323, 1989. Smith SW: The role of chelation in the treatment of other metal poisonings, J Med Toxicol 9:355–369, 2013. Sood A: Current treatment of chronic beryllium disease, J Occup Environ Hyg 5:762–765, 2009. Spiller HA: Rethinking mercury: the role of selenium in the pathophysiology of mercury toxicity, Clin Toxicol 56:313– 326, 2018. Srivastava S, Flora SJS: Arsenicals: toxicity, their use as chemical warfare agents, and possible remedial measures. In Gupta RC, editor: Handbook of toxicology of chemical warfare agents, ed 3, San Diego, 2020, Academic Press, pp 303–319. Strekopytov S, Brownscombe W, Lapinee C, et al.: Arsenic and mercury in bird feathers: identification and quantification of inorganic pesticide residues in natural history collections using multiple analytical and imaging techniques, Microchem J 130:301–309, 2017. Sundar S, Chakravarty J: Antimony toxicity, Int J Environ Res Publ Health 7:4267–4277, 2010. Tannoo DR, Paquet F: Early ultrastructural alterations in rats after administration of 239Pu-citrate, Cell Mol Biol 42:431– 438, 1996. Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ: Heavy metals toxicity and the environment, EXS 101:133–164, 2012. Thompson J, Bannigan J: Cadmium: toxic effects on the reproductive system and the embryo, Reprod Toxicol 25:304– 315, 2008. Thompson L: Lead. In Gupta RC, editor: Veterinary Toxicology: basic and clinical principles, ed 3, San Diego, 2018, Academic Press, pp 439–443. Tokar EJ, Benbrahim-Tallaa L, Ward JM, et al.: Cancer in experimental animals exposed to arsenic and arsenic compounds, Crit Rev Toxicol 40:912–927, 2010. Tokar EJ, Boyd WA, Freedman JH, et al.: Toxic effects of metals. In Klaassen CD, editor: Casarett & Doull’s toxicology: the basic science of poisons, ed 8, New York, 2013, McGrawHill, pp 981–1030. Vincente-Vincente L, Quiros Y, Perez-Barriocanal F, et al.: Nephrotoxicity of uranium: pathophysiological, diagnostic and therapeutic perspectives, Toxicol Sci 118:324– 347, 2010. Waalkes MP, Liu J, Goyer RA, et al.: Metallothionein-I/II double knockout mice are hypersensitive to lead-induced kidney carcinogenesis: role of inclusion body formation, Cancer Res 64:7766–7772, 2004. Weidenhamer JD, Miller J, Guinn D, et al.: Bioavailability of cadmium in inexpensive jewelry, Environ Health Perspect 119:1029–1033, 2011. Wu M, Shu Y, Song L, et al.: Prenatal exposure to thallium is associated with decreased mitochondrial DNA copy

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C H A P T E R

11 Agrochemicals Elizabeth F. McInnes1, Sabitha Papineni2, Matthias Rinke3, Frederic Schorsch4, Heike A. Marxfeld5 1 3

Syngenta, Berkshire, United Kingdom, 2Labcorp Early Development Laboratories Inc., Greenfield, IN, United States, Formerly of Bayer AG, Wuelfrath, Germany, 4Bayer S.A.S, R&D Crop Science, Sophia Antipolis, France, 5BASF SE, Ludwigshafen, Germany

O U T L I N E 1. Introduction

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2. Herbicides 2.1. Introduction 2.2. Inhibition of Cell Division and Growth 2.3. Activation of Reactive Oxygen Species 2.4. Inhibition of Cellular Metabolism

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3. Fungicides 3.1. Introduction 3.2. Triazole-Containing Azole Fungicides (Conazoles)/DMI-Fungicides (Demethylation Inhibitors)/C14-Demethylase Inhibitors 3.3. Succinate Dehydrogenase Inhibitor Fungicides 3.4. Strobilurins or Quinol Oxidation Site of Complex III Inhibitor Fungicides

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4. Insecticides 4.1. Introduction

Organophosphates and Carbamates Organochlorines Pyrethrins and Pyrethroids New Insecticides

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5. Rodenticides 5.1. Introduction 5.2. Anticoagulant Rodenticides 5.3. Cholecalciferol 5.4. Inorganic Compounds: Metal Phosphides 5.5. Alphachloralose 5.6. Bromethalin 5.7. Corn Cob 5.8. Strychnine

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1. INTRODUCTION The continuous improvement of agrochemicals is necessary to protect crops globally from weeds, plant diseases, and pests, thus enabling increased yields of food and fiber from available farmland in order to feed the earth’s projected population of 9 billion people by the middle of the 21st century. Challenges in the field of

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00011-3

4.2. 4.3. 4.4. 4.5.

agrochemicals include the loss of active ingredients due to the regulatory environment, increased resistance of fungi, weeds and insects to agrochemicals, increasing costs of research and development, pressure from environmental groups and the public to decrease the use of agrochemicals to protect the health of humans and animals, including pollinators, and changes in environmental conditions such as increasing

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temperatures, floods, and droughts due to climate change. The aim of this chapter is to give readers a broad overview of both old and new agrochemicals that are available. A previous chapter describes the safety assessment of these chemicals (see Agricultural and Bulk Chemicals, Vol 2, Chap 12). In addition, key aspects of the agrochemical mode of action, toxicity, and pathology findings in laboratory animals and, if relevant, in humans are examined.

2. HERBICIDES 2.1. Introduction Synthetic organic herbicides represent almost half of the global volume of pesticides used. Their phytotoxicity may be specific or broad-spectrum. Herbicides are classified based upon various aspects, such as mode of action, site of action, chemical families, time of application, selectivity, and translocation. Over the years, more than 200 active herbicidal ingredients have been used, representing more than 60 chemical classes and a number of herbicidal mechanisms of action (MOA); thus, this section only provides an overview of these

concepts. Many herbicides, such as the dinitrophenols, sulfuric acid, arsenicals, and petroleum oils, have now been withdrawn from the market because of nonselectivity, toxicity to mammals or the environment, or carcinogenicity. To mitigate resistance by target plant species, herbicides with different MOAs are often combined. With the low mammalian toxicity of most modern herbicides, real-world poisoning is relatively rare. Causes of acute poisoning include malicious or suicidal intent, accidental ingestion (such as when chemicals are decanted from their original containers and divided into single-use aliquots, often stored in beverage bottles), animals gaining direct access to undiluted chemicals, or improper use. Herbicides are broadly classified into three groups based on their herbicidal activity. These are activation of reactive oxygen species, inhibition of plant cell division and growth, and inhibition of plant cellular metabolism (Table 11.1). Each of these categories is again grouped into different categories based on their mode of action, which are further segmented into different chemical classes or families. An updated list of all the herbicidal mode of action groups and chemical families has been reported by the Herbicide Resistance Action Committee

TABLE 11.1 Herbicides Classified in Terms of Mode of Action (HRAC, 2020).

HRAC Classification Class Compound examples

Inhibition of Plant Cell Division and Growth

Activation of Reactive Oxygen Species

Inhibition of Plant Cellular Metabolism

Auxin mimics Phenoxy, benzoic acid, and pyridine chemical families

Pyridiniums Paraquat and diquat

Inhibition of enolpyruvyl shikimate phosphate synthase Glyphosate

Microtubule organization inhibitors Carbamates

Inhibition of glutamine synthetase Phosphinic acids Glufosinate

Inhibition of very long-chain fatty acid synthesis Thiocarbamates

Inhibition of photosynthesis Ureas and Thioureas Triazines Inhibitors of 4hydroxyphenylpyruvate dioxygenase (HPPD) bicyclopyrone and mesotrione

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(HRAC) (HRAC, 2020). This section will review the toxicological profiles of the most commonly used herbicide chemical families in agriculture and references the scientific opinions of the Environmental Protection Agency (EPA) in the United States (US EPA) and the European Food Safety Authority (EFSA) in the European Union, the two regulatory bodies that review the safety data for pesticide registrations in these regions, when discussing the toxicological effects of the herbicide groups below.

2.2. Inhibition of Cell Division and Growth The inhibition of cell division and growth category includes herbicides with different modes of action such as auxin mimics, auxin transport inhibitors, inhibitors of microtubule assembly and organization, and many others as described by the HRAC. The toxicological and pathological profile of some of these classes will be discussed further below. Auxin Mimics Auxin herbicides mimic the natural occurring auxin hormone called indole acetic acid (IAA) that leads to unregulated and uncontrolled growth of broadleaf plants (dicots) resulting in death. Therefore, these herbicides are used for weed control in grass cereal grains (monocots) such as rice and wheat. Auxin herbicides include the chlorophenoxy, benzoic acid, and pyridine chemical families. CHLOROPHENOXY HERBICIDES

Chlorophenoxy herbicides include 2,4-dichlor ophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophen oxy) butyric acid (2,4-DB), 2-methyl-4-chlorophe noxyacetic acid (MCPA), 2,4-MCPB, 4-(4-chloroo-tolyloxy) butyric acid (MCBA), and others. The generation of dioxin impurities during the manufacture of phenoxy herbicides has been raised as a concern over the years. Dioxins are considered a chemical class of concern and regulatory agencies have established strict limits for levels of dioxins as manufacturing impurities in technical grade chlorophenoxy herbicides (see Environmental Toxicologic Pathology and Human Health, Vol 3, Chap 1).

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In general, chlorophenoxy herbicides used today have low mammalian toxicity. Certain salt forms are moderately irritating to skin, eyes, and the respiratory and gastrointestinal tracts. They are well absorbed via the oral route. There is limited biotransformation (conjugation of the acids) of these compounds. They do not tend to bioaccumulate and are excreted mainly through the urine. Renal excretion of chlorophenoxy herbicides occurs via active transport, and toxic effects have only been observed at dose levels exceeding the threshold for saturation of renal clearance (TSRC). Nephrotoxicity and hepatotoxicity have been observed at high-dose levels (above TSRC) in laboratory animals (USEPA, 2013). While acute chlorophenoxy toxicity may occur in farm animals following direct consumption of large quantities of herbicide, toxic dose levels are not achieved by animals grazing on sprayed pastures. The primary target organs of chlorophenoxy herbicides include kidneys, liver, spleen, thyroid, and the hematological system. Compared to other species, the dog is the most sensitive to chlorophenoxy herbicide exposure, apparently because of its relatively poor ability to excrete organic acids compared to both rats and humans (Timchalk, 2004). Because the dog appears to be an outlier compared to other species, it is considered to be a poor surrogate for evaluation of human health. The clinical signs of chlorophenoxy toxicity, at doses that exceed renal excretory capacity, are most striking in dogs, which may develop myotonia, ataxia, posterior weakness, vomiting, diarrhea, and metabolic acidosis. Reproductive and developmental effects with chlorophenoxy herbicides were observed only in the presence of excessive parental toxicity (EFSA, 2014). HUMAN RISK Human poisoning associated with chlorophenoxy compounds has occurred because of deliberate suicidal ingestion of large quantities, resulting in severe metabolic acidosis. Most commonly reported symptoms due to intentional poisoning with chlorophenoxy herbicides include minimal to mild gastrointestinal symptoms with progression to renal failure or multiple organ failures in fatal cases (USEPA, 2013). Other nonspecific findings such as headache, generalized weakness, and dizziness have also been reported (USEPA, 2013).

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Microtubule Organization Inhibitors CARBAMATES

Commonly used carbamate herbicides include chloropropham, carbetamide, and barban. They may be formulated singly or in combination as selective pre- and early postemergent herbicides. Some, like chloropropham, serve as antisprouting agents on stored potatoes. In general, carbamate herbicides are absorbed through the root system and act slowly, taking 2 to 3 weeks for visible results. Carbetamide is used as an outdoor foliage spray against annual grasses and some broad-leaved weeds in oilseed rape. TOXICOLOGY, CLINICAL SIGNS, AND PATHOLOGY Herbicidal carbamates may inhibit dihy-

dropteroate synthetase or mitosis and disrupt microtubule organization. The oral absorption of chloropropham is 90%. It is widely distributed and predominantly excreted via the urine with no bioaccumulation. It has been shown to be of low acute toxicity, but is a mild eye and skin irritant. It did not show any toxicity via dermal exposure. The dog is the most sensitive species with hematotoxicity and histologic findings in the thyroid gland. In a 2-year rat study, anemia was observed with marked hematopoiesis in bone marrow, as well as splenic hemosiderosis. Hematotoxicity was observed in a rat reproductive study. Chloropropham is classified carcinogenic category 2 based on Leydig cell tumors in the 2-year chronic rat study as noted by EFSA (EFSA, 2017a,b). Endocrine-mediated mechanisms secondary to liver enzyme induction could not be excluded for the Leydig cell tumors in rats and thyroid findings in dogs (EFSA, 2017a,b). The US EPA has imposed a cancer potency factor against any potential risk from metabolite exposures. Human exposure is expected in occupational settings when chloropropham is applied as an aerosol or using forced air distribution (USEPA, 1996). The EPA requires use of a respirator and chemicalresistant gloves as personal protection equipment (PPE) for workers during application and ventilation of stored potatoes. Carbetamide has been shown to have a low acute toxicity in rats but is moderately toxic in mice via oral route. It is not a skin or eye irritant nor a sensitizer. Carbetamide is extensively and rapidly absorbed, with >80% oral absorption,

and excreted after oral administration. Target organs include liver in rats and dogs (increased organ weight with correlative hepatocyte hypertrophy) and thyroid in dogs (increased organ weight and hypertrophy). Other liver effects include hemosiderin deposition in Kupffer cells which is sometimes associated with decreases in red blood cell counts with evidence of hemolysis. In dogs, neurological signs have also been observed. Carbetamide was negative for genotoxicity potential. Hepatocellular carcinomas and adenomas and thyroid follicular adenomas were seen in the mouse chronic study at highdose levels that exceeded the maximum tolerated dose (MTD). Mechanistic studies in mice suggest carbetamide is a P450 enzyme inducer. No adverse effects on fertility and/or other reproductive parameters were observed. Developmental toxicity in rats was observed with complex malformations at doses that did not elicit maternal toxicity. Similarly in rabbits, skeletal abnormalities, delayed ossifications, and postimplantation losses were noted at doses that cause only minimal maternal toxicity. Carbetamide is labeled as a developmental toxicant with the risk phrase “Possible risk of harm to the unborn child.”

2.3. Activation of Reactive Oxygen Species This category includes herbicides with different modes of action such as inhibition of photosynthesis, glutamine synthetase, hydroxyphenylpyruvate dioxygenase, and others as described by the HRAC. The toxicological and pathological profile of some of these classes will be discussed further below. Pyridiniums: Paraquat and Diquat PYRIDINIUMS DEVELOPMENT AND USE Paraquat and diquat are the only two commercial herbicides in this group with paraquat being the most commonly used. Paraquat’s herbicidal property was discovered by the company Imperial Chemical Industries (ICI) in 1955 and it was first made commercially available in 1962. The bipyridyls are nonselective, postemergent, very fast-acting contact herbicides and crop desiccants. There are no homeowner or residential uses for paraquat. In the United States, it is sold as a Restricted

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Use Pesticide (RUP) and thus may be purchased and used only by certified applicators. Diquat is not an RUP and there are homeowner and residential uses in the United States. Both paraquat and diquat may be toxic to animals. The rat acute oral LD50 of paraquat is 150 mg/kg, while that of diquat is 231 mg/kg. The dermal LD50 of paraquat in rabbits is 236–325 mg/kg. TOXICOLOGY, CLINICAL SIGNS, AND PATHOLOGY The MOA of the bipyridyls involves cyclic

reduction–oxidation reactions that deplete nicotinamide adenine dinucleotide phosphate (NADPH) and generate reactive oxygen species (ROS), which cause cell death by polymerizing unsaturated lipids in cell membranes. The lungs accumulate paraquat at about a 10-fold greater rate than other tissues, mostly in Type I and Type II alveolar epithelial cells (see Respiratory System, Vol 5, Chap 4). Paraquat’s effects in the lung are similar to those of other oxidizing agents, including oxygen, ozone, and nitrogen dioxide. Interestingly, rabbit lung microsomes incubated with paraquat do not generate superoxide radicals, which correlates with the insensitivity of this species to the toxic pulmonary effects. Mammalian toxicities of paraquat and diquat reflect their different tissue distribution patterns. Both herbicides affect the kidneys, while paraquat alone causes significant lung damage. Diquat is more likely to cause indirect clinical central nervous system (CNS) effects. Bipyridyl herbicides are absorbed relatively slowly from the gastrointestinal tract, but are widely distributed to tissues within 6–18 h. They are absorbed less efficiently through intact skin. Dogs are more sensitive to the oral toxicity of bipyridyl herbicides than rodents. The great majority of orally administered paraquat and diquat is excreted in feces. Dogs may develop clinical signs within a few hours of acute, high-dose exposure, including pulmonary edema, vomiting, depression, dyspnea, and cyanosis. Death usually occurs within 8 days. Clinical signs in acutely poisoned cattle are generally delayed for 1–3 days after exposure, and include anorexia, accelerated and labored breathing, muscular twitching, and laminitis. The exceptional surviving animal may require months of convalescence. Diquat poisoning causes prominent diarrhea but lacks the pulmonary effects of paraquat. Rats and

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mice given doses of paraquat above the median lethal dose show signs of hyperexcitability, ataxia, and convulsions and usually die within a few hours; however, deaths can also occur after 10–12 days due to lung damage, pulmonary edema, and respiratory failure (Lock and Wilks, 2010). At high doses, the bipyridyl herbicides can cause renal proximal convoluted tubular necrosis, which limits subsequent active excretion of the material thus exacerbating toxicity. Both materials are extremely corrosive to all alimentary mucosal surfaces and may cause esophageal perforation. With paraquat, severe acute exposures may cause widespread pulmonary epithelial necrosis, alveolar basement membrane denudation, and pulmonary edema within several hours to days. Alveolar Type II pneumocyte hyperplasia follows. At acutely survivable doses, intra-alveolar pulmonary fibrosis may be lethal one to several weeks later. With diquat, only mild lesions are observed in lungs and no progressive pulmonary fibrosis is noted unlike with paraquat poisoning. There may be centrilobular hepatocyte necrosis with attendant clinical chemistry changes. There may also be focal necrosis of skeletal and heart muscle. Oral exposures can also result in cerebral edema. At necropsy, lesions include moderate neuronal depletion and demyelination of the central white matter around the third and lateral ventricles. Electron microscopy reveals edema and destruction of myelin with myelin breakdown products and astrocytic fibrous gliosis. The primary cause of human poisoning with paraquat is from deliberate suicide attempts and less often by accidental ingestion from storage in bottles previously used for beverages, or from exposure during occupational mixing or loading of the undiluted material. When used according to label instructions, there is no concern for human health risk. New packaging requirements and other risk mitigation measures required by the US EPA will eliminate the illegal transfer of the containers. When ingested, paraquat has lifethreatening effects on the gastrointestinal tract, kidney, liver, heart, lung, and other organs. The potential minimum human lethal dose of paraquat (ion) when orally consumed as a formulation is 2 g paraquat ion with an approximate human median lethal dose (MLD) of 3 g paraquat ion. The gastrointestinal tract is the site of initial

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contact leading to swelling, erythema, and ulceration of the mouth, pharynx, esophagus, stomach, and intestine. Early symptoms in humans include burning pain in the mouth, throat, chest, and upper abdomen. Higher dose levels can cause liver injury with increased enzyme activities such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP). Damage to renal proximal tubules occurs and is often reversible, but impaired renal function plays a role on the outcome of paraquat poisoning as it results in a build-up of tissue paraquat concentrations predominantly in the lungs. Lung damage in rats includes initial acute destructive damage to Type I and II alveolar epithelial cells within a day of dosing, development of alveolar edema, hemorrhage into the alveoli, and inflammatory cell infiltration (Lock and Wilks, 2010). Rats that survive for up to 10–12 days after dosing develop extensive hypercellular lesions in the lung with the predominance of fibroblast proliferation (Lock and Wilks, 2010). The later, proliferative phase includes severe intra- and inter-alveolar fibrosis and associated edema which cause anoxia and death (Lock and Wilks, 2010). Significant paraquat absorption may occur through damaged skin and chronic dermal exposure may damage intact skin. The risk of toxicity is much lower for those exposed to diluted spray mists. Although toxicity from inhalation is low, the lung is the primary target organ manifesting as the most lethal and least treatable effects. In comparison with paraquat, diquat does not selectively concentrate in lung tissues. Dermal signs are common in agricultural workers and minor irritation of the eyes and nose is also observed, while prolonged skin exposure and compromise to skin’s barrier integrity has resulted in discolored, deformed, or lost fingernails. Diquat poisoning is expected to cause greater renal damage and toxicity to CNS that is not typical of paraquat poisoning. The diquat CNS findings are caused by fluid and electrolyte imbalances. Phosphonic Acids GLUFOSINATE

Glufosinate is a phosphonic acid that works by inhibiting glutamine synthetase in both plants and animals (USEPA, 2012). Glufosinate is

eliminated via feces primarily as the parent compound and is poorly absorbed from the gastrointestinal tract. In animals, glutamine synthetase has a minor role in ammonia homeostasis by the liver, which is easily compensated for by other pathways. The inhibited enzyme is also important in the CNS glutamine–glutamate shunt between g-aminobutyric (GABA) and glutamate and thus results in neurotoxicity. TOXICOLOGY, CLINICAL SIGNS, AND PATHOLOGY No evidence of acute toxicity has been

observed in doses up to 500 mg/kg/day, but signs of neurotoxicity were observed in repeatdose studies in rodents. Effects related to inhibition of glutamine synthase were observed in mouse liver and kidney (increased weights with increases in serum AST and ALP activities). An increase in fetal mortality was observed in both rat and rabbit developmental toxicity studies at doses not associated with maternal toxicity (USEPA, 2012). Alterations in brain morphometrics were also observed in the adult offspring exposed in utero or during lactation, at dose levels not associated with maternal toxicity, in the rat developmental neurotoxicity study. Increased mortality was observed in chronic rat, mouse, and dog studies. Increased occurrence of retinal atrophy in chronic rat studies and electrocardiogram changes in dogs were also observed. There is no concern for mutagenicity or carcinogenicity potential. The oral LD50s for glufosinate ammonium in rats is 4010 mg/kg and dermal LD50 is > 2000 mg/ kg. It is not a dermal irritant or sensitizer, but has been shown to be an eye irritant with effects reversible within 72 h. In rats fed 1000 ppm (64– 90 mg/kg/day) glufosinate ammonium for 6, 13, or 90 days, glutamine synthetase levels were significantly decreased in liver, kidney, and brain (USEPA, 2012). Dosage of beagle dogs fed glufosinate ammonium during a 6- or 12month oral toxicity study had to be decreased from a high dose of 10.8 mg/kg/day to 8.4 mg/kg/day after 11 days of dosing, due primarily to neurological signs, including trismus (spasm of the jaw muscles), salivation, and hyperactivity followed by somnolence and hypoactivity. They also showed stereotypic gait, tremor, ataxia, and tonic-clonic spasms. Two of these high-dose dogs died in the second

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week of the study due to myocardial necrosis. Acute and chronic dietary exposure estimates, when calculated, were found to be below the level of US EPA’s concern. HUMAN RISK All occupational exposure scenarios, except for certain mixer/loader/applicator scenarios with a pressurized handgun, indicate that the risks are minimal. Other exposures in humans include suicidal cases (mostly reported in Japan) and a few cases of misuse with initial effects observed related to the gastrointestinal tract such as nausea, vomiting, and diarrhea and later neurological effects including trembling, convulsions, coma, and respiratory failure (Watanabe and Sano, 1998; EFSA, 2005).

Ureas and Thioureas Herbicides of the urea and thiourea group are used for selective pre- and postemergent weed control. They are absorbed through the plant’s roots and inhibit photosynthesis in susceptible species. The first urea herbicide, DuPont’s monuron (N,N-dimethyl-N1-[4-chlorophenyl]urea), was introduced in 1952. Since then, many urea and thiourea herbicides have become available including diuron, flumeturon, isoproturon, linuron, and numerous others with the “-uron” suffix. They are often used in combination with other herbicides. They may remain phytotoxic in the soil for extended periods. The information contained in this section is based on USEPA, 1995, and Liu (2001). TOXICOLOGY, CLINICAL SIGNS, AND PATHOLOGY

The urea and thiourea herbicides are of low mammalian toxicity. As urea herbicides are metabolized to aniline derivatives, which are potent oxidants of hemoglobin, methemoglobinemia (18%–80%) has been reported as well as hemolysis in rodents and dogs. The acute rat oral LD50 of diuron is 2900 mg/kg and that of isoproturon is similar, while the LD50 for linuron is 1500 mg/kg. These materials may cause eye and skin irritation. They are weakly mutagenic in some test systems and have been shown to induce tumors in several organs in lifetime rodent bioassays (see below). Herbicides of this group are readily absorbed from the gastrointestinal tract in rats and dogs and are mainly metabolized by dealkalization of the urea methyl groups. Diuron is excreted

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partially unchanged in feces and urine. The predominant urinary metabolite is N-(3,4dichlorophenyl)-urea. Diuron and its metabolites do not accumulate in tissues. Diuron and monuron are potent inducers of hepatic metabolizing enzymes. Male rats are more sensitive than females to the enzyme-inducing activity of diuron and recovery from diuron intoxication occurs within 72 h. In general, at moderate doses, chemicals of this class induce methemoglobinemia, leukocytosis, slight anemia, and increased splenic weights. Diuron at moderate to high doses causes kidney, urinary bladder, uterine, and mammary cancers in mice. Linuron caused a dose-related increase in testicular interstitial cell tumors in rats and hepatocellular adenomas in mice. Linuron has been shown to be antiandrogenic, reducing testosterone production in fetal rat testes during in utero exposures (Cook et al., 1999). Buturon, linuron, and monolinuron are weak teratogens in experimental animals. Isoproturon administered orally at high dosages for 60 days in rats caused decreased sperm numbers and motility. HUMAN RISK

Humans could be exposed from ingestion of food and water with residues; however, these herbicides do not pose serious risks to humans with normal use. Triazines There are numerous triazines, including atrazine, simazine, propazine, and prometon. They are popular in agriculture and forestry applications because they are selective pre- and postemergent herbicides. The triazines work by inhibiting photosynthesis in plants, specifically by competing with plastoquinone for the binding site on the D1 protein subunit of photosystem II, an irrelevant biochemical pathway for animals. They can be used singly or in combinations (e.g., atrazine and simazine together are called simazat). Atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine) is one of the most widely used herbicides in the United States for control of broadleaf weeds and grasses in crops of corn, sugar cane, and sorghum. Atrazine, propazine, and simazine are collectively referred to as chlorotriazines. These chlorotriazines, along with their three common chlorinated

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metabolites, share a common neuroendocrine mechanism of action and are considered a Common Mechanism Group (CMG) by US EPA for cumulative risk assessment. All other triazines are excluded from this CMG as they do not share this common toxicity profile. TOXICOLOGY, CLINICAL SIGNS, AND PATHOLOGY

In general, the triazines are of low toxicity for most mammalian species. Triazine herbicides induce liver microsomal enzymes and are metabolized to N-dealkylated derivatives with the main route of excretion being urinary. Atrazine and other triazine herbicides are not mutagenic or teratogenic and are not considered human carcinogens. The most common toxic effects in repeatdose experimental toxicology studies across species were reduced food consumption, body weight, body weight gain, and liver enlargement. Other effects in repeat toxicology studies included mild anemia and altered hematological parameters at high doses. Cardiotoxicity has been observed in the dog after chronic exposure to high doses of atrazine (atrial fibrillation, ECG changes, and gross and microscopic cardiac lesions). Atrazine’s toxicological database is exceptionally large as it was researched extensively to understand the mode of action underlying the development of mammary gland tumors in the female Sprague–Dawley rat. At high doses in the rat, the mode of action of the CMG triazines involves perturbation of the hypothalamic–pituitary–gonadal (HPG) axis resulting in attenuation of the luteinizing hormone (LH) surge. As a result of an attenuated LH surge, the female Sprague–Dawley rat fails to ovulate which ensures continued secretion of estrogen from the ovarian follicles and associated elevations in prolactin secretion. This leads to a prolonged stimulation of mammary gland tissue that promotes the development of mammary tumors. This is similar to the spontaneous development of mammary tumors in the female Sprague– Dawley rat during normal reproductive senescence, which is characterized by neuroendocrine failure and persistent estrus. This carcinogenic mode of action is not relevant for human health risk assessment as reproductive senescence in the female human (menopause) is caused by the depletion of ovarian follicles and a concomitant decrease in estrogen (while the LH surge remains normal). As this neuroendocrine mode

of action is relevant to noncarcinogenic health effects (e.g., reproduction and development), the endpoint identified for human health risk assessments is reduction of the LH surge after 4 days of exposure, an effect which also protects from the downstream adverse endocrine-related effects as LH surge attenuation is the most sensitive effect of atrazine exposure in this comprehensive toxicological database. Despite atrazine’s relatively benign toxicological profile in most mammalian species, there is concern among some environmental groups about its endocrine-disrupting potential and concerns about aquatic contamination and effects on fish and amphibians. Due to its moderate water solubility and intermediate KOC (organic carbon-water partition coefficient) value, atrazine is susceptible to storm-induced runoff from treated fields in sheet water flow and/or transportation with soil particles in a runoff event. Consequently, atrazine is frequently detected in surface waters, particularly in vulnerable watersheds experiencing high use. Early research suggested that atrazine could act as a potential endocrine disruptor in fish, amphibians, and reptiles primarily via induction of aromatase; however, more rigorous studies conducted subsequently have refuted these initial findings (Hanson et al., 2019). Dozens of studies have now been conducted on a variety of species representing these taxa and the quantitative weight of evidence indicates that atrazine does not adversely affect fish, amphibians, and reptiles, at environmentally relevant concentrations ( 2000 mg/kg body weight (bw)) but with inhalation (LC50 > 5.093 mg/L) toxicity in the rat. It is neither a skin nor an eye irritant and is negative in skin sensitization tests (EFSA, 2008). Besides the expected effects on the liver, the adrenal glands are a target organ, as the presence of hypertrophic and vacuolated zona fasciculata cells was observed in the rat 90-day oral study (EFSA, 2008) (see also Endocrine System, Vol 4, Chap 7). Tebuconazole is not genotoxic. In a mouse carcinogenicity study over 21 months, the Naval Medical Research Institute (NMRI) mice showed decreased body weight gain with concurrent increased food consumption, and increased plasma liver enzymes starting at 500 ppm. Histologically, microvesicular fatty vacuolation corresponding to increased relative liver weights was a feature. A statistically significant increase in the number of hepatocellular adenomas (males) and carcinomas (males and females) after 1500 ppm were considered not relevant for humans, since this is a common background lesion for this strain and the tumors occurred only at a dose exceeding the maximum tolerated dose (EFSA 2008, US GPO 2013). No tumors were observed in the 2-year study performed in Han Wistar rats. Treated female mice from the carcinogenicity study showed a higher incidence of ovarian atrophy; however, tebuconazole did not cause effects on reproduction in a two-generation study in rats. In 2007, Taxvig and coworkers (2007) described endocrine disrupting properties of

different azole fungicides (including tebuconazole); however, the in vivo effects seen were for dose levels far above NOAEL. In the Hershberger assay, a screening assay intended to identify test articles with androgenic and antiandrogenic activity, propiconazole and tebuconazole (50, 100, and 150 mg⁄kg bw⁄day (d) each) were investigated for antiandrogenic effects in castrated testosterone-treated male rats (Taxvig et al., 2008) and were negative. The developmental toxicity of tebuconazole was assessed in a series of tests in rats, mice, and rabbits. Based on the effects observed, including malformations, postimplantation loss, and resorptions (see Embryo, Fetus and Placenta, Vol 5, Chap 11), in the absence of overt maternal toxicity, a classification of Reproductive Category 3 was proposed (EFSA, 2008). The developmental and reproductive effects of propiconazole, tebuconazole, and epoxiconazole were also investigated by Taxvig et al. (2008). Tebuconazole and epoxiconazole induced a high frequency of postimplantation losses and epoxiconazole also caused a marked increase in late and very late resorptions. No significant effects of propiconazole, tebuconazole, or epoxiconazole on the anogenital distance were observed. In 2018, EFSA modified the maximum residue levels for tebuconazole in olives, rice, and herbs. However, since the toxicological profile of tebuconazole was not changed and considered sufficient, the acceptable daily intake (ADI), the acceptable operator exposure level (AOEL), and the acute reference dose (ARfD) were confirmed at 0.03 mg/kg bw/d (EFSA et al., 2018a). Recently it has been postulated that tebuconazole may have cardiotoxic potential causing increased myocardial fibrosis in male Wistar rats at doses of approximately 30 mg/kg (Othme`ne et al., 2020); however, no indication of cardiotoxicity has been observed in any of the regulatory studies, even with higher exposure. Prothioconazole Prothioconazole was introduced to the market in 2004 and rapidly gained market dominance due to its broad spectrum of activity, covering all important cereal fungal diseases. The toxicology database is extremely large, particularly since it contains a number of complex toxicity studies. Residues of prothioconazole-desthio, the major

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metabolite in plants, together with residues of its 4hydroxy metabolites, may also be found in edible ruminant tissues and milk. Prothioconazole is rapidly and almost completely absorbed (>90%) following oral dosing. Excretion is extensive and relatively rapid, mainly via feces. Prothioconazole is of low acute toxicity via oral (LD50 > 6200 mg/ kg bw), dermal LD50 > 2000 mg/kg bw), or inhalation (LC50 > 4990 mg/m3) routes and does not show any eye and skin irritation or sensitizing potential. Short-term studies up to 13 weeks revealed liver and kidney as target organs in rodents and dogs. Prothioconazole is not genotoxic in vivo. Although prothioconazole gave inconsistent or equivocal results in an in vitro rat liver unscheduled DNA synthesis (UDS) assay and induced chromosome aberrations in cultured Chinese hamster lung (CHL) cells, in vivo testing (a rat liver UDS assay and two mouse bone marrow micronucleus assays) revealed negative results. No carcinogenic potential was observed in either a 2-year carcinogenicity study performed in Wistar rats or an 18-month mouse carcinogenicity study using CD1 mice. Nonproliferative changes consisted of hypertrophy and cytoplasmic changes in the liver. In the kidneys, increased weight and increased severity grades of chronic progressive nephropathy were observed in rats while mice showed decreased kidney weights and (subcapsular) degeneration and interstitial fibrosis (see Kidney, Vol 5, Chap 2). Chronic renal interstitial fibrosis was also recorded in the 1year study in beagle dogs, leading to an overall NOAEL of 5 mg/kg bw/d (EFSA Scientific Report 2007 106, 1-98, Conclusion on the peer review of prothioconazole). Reproductive effects with prothioconazole occurred only in parental females at high doses with concomitant systemic toxicity. Two developmental studies were performed with prothioconazole using different Wistar rat substrains, both showing increased numbers of fetal supernumerary rudimentary ribs. In the first oral study in Wistar rats (substrain Hsd Cpb:WU), a treatment-related increase in the incidence of fetuses and litters with microphthalmia was reported at the very high dose of 1000 mg/ kg bw/d with evidence of overt maternal toxicity. This substrain of rat is known to have a larger background incidence of this malformation (van Eden and Mullink, 1986). In

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a supplementary oral study in a rat strain with a virtually zero background incidence of ocular malformations (Crl:WI(HAN)), no cases of microphthalmia or other ocular malformations were recorded, even at doses up to 750 mg/ kg bw/d, which were severely maternally toxic (ECHA, 2019). In a recent review of the study data, the presence of polyovular follicles in the chronic dog study raised concern by an auditing authority since it was noted in a few treated females. However, the presence of follicles containing multiple oocytes is a finding which is usually not recorded by pathologists, since it represents a frequent background observation in dogs (Payan-Carreira and Pires, 2008; Elmore et al., 2019) and thus should not be confused with a treatment related effect in this species. Mefentrifluconazole As one of the latest sterol biosynthesis inhibitors, mefentrifluconazole [(2RS)-2-[4-(4-chloropheno xy)-a,a,a-trifluoro-otolyl]-1-(1H-1,2,4-triazol-1yl)propan-2-ol] was brought to the market recently. Both CYP51 and aromatase (CYP19) are involved in the 14-a-sterol demethylase process and thus CYP inhibition was identified as a potentially useful surrogate endpoint to predict the reproductive toxicity of azoles (Tesh et al., 2019). Mefentrifluconazole absorption is extensive. The active substance is widely distributed and metabolized, and a preferential metabolism and elimination is observed for the S-enantiomer in rats. The active substance is rapidly excreted, largely via the biliary pathway. In mammalian toxicity studies, mefentrifluconazole showed low acute toxicity by the oral, dermal, or inhalation routes. No skin or eye irritation or phototoxic potential were attributed to the active substance; however, a potential classification as skin sensitizer was suspected (EFSA, 2018a,b,c). The liver is the main target organ of mefentrifluconazole in mice, rats, and dogs. The main findings at higher dose levels include reduced body weight gain and food consumption, altered liver enzyme activities, and increased liver weight, which at higher dose levels is accompanied by hepatocyte hypertrophy and/or liver cell necrosis (Tesh et al., 2019). All genotoxic tests were negative and mefentrifluconazole revealed no carcinogenic

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effects up to 36 mg/kg/d in an 18-month mouse study and up to 163 mg/kg bw/d in a 24month, repeat-dose carcinogenicity study performed in Wistar rats. The results in the two-generation study with mefentrifluconazole did not suggest any adverse effects related to an endocrine-mediated MOA at dose levels up to 200 mg/kg bw/d. There was no consistent effect on gestation length between the F0 and F1 generations, no increase in postimplantation loss, and no evidence of feminization or masculinization of pups that had been exposed in utero or during postnatal life. Thus, mefentrifluconazole did not exert adverse effects on reproduction or fertility (Tesh et al., 2019). Both Taxvig et al. (2007, 2008) and Tiboni et al. (2009) observed a high incidence of both early and late fetal resorptions with older aromatase inhibiting azoles when treatment was administered from gestation day 7–21, especially in the absence of overt maternal toxicity. Investigations by Stinchcombe et al. (2013) revealed that the depletion of maternal estradiol blood levels was identified as the key event for the observed increase in late fetal resorptions. Corresponding morphological differences between treated, coadministered, and control rats led to the suggestion to regularly include histopathological examination of the placenta, since it is the materno-fetal interface and is crucial for fetal maintenance (Rey Moreno et al., 2013; see Embryo, Fetus and Placenta, Vol 5, Chap 11). The relevant NOAEL for mefentrifluconazole is 3.5 mg/kg bw/d derived from the 90-day and 18-month studies in mice. The ADI for mefentrifluconazole has been set at 0.035 mg/ kg bw per day based on the NOAEL of 3.5 mg/kg bw per day for hepatotoxicity from the 18-month mouse study and applying an uncertainty factor (UF) of 100.

3.3. Succinate Dehydrogenase Inhibitor Fungicides Succinate dehydrogenase inhibitor (SDHI) fungicides include benodanil, enzovindiflupyr, bixafen, boscalid, carboxin, fenfuram, fluindapyr, fluopyram, flutolanil, fluxapyroxad, furametpyr, inpyrfluxam, isofetamid, isoflucypram, isopyrazam, mepronil, oxycarboxin, penflufen,

penthiopyrad, pydiflumetofen, sedaxane, and thifluzamide. SDHI fungicides act by inhibiting the enzyme succinate dehydrogenase (SDH, the so-called complex II in the mitochondrial respiratory chain), which is a functional part of the tricarboxylic acid cycle, linked to mitochondrial electron transport. Energy production is curtailed, which results in inhibition of spore germination, germ tubes, and mycelial growth within the fungus target species. These fungicides can be used preventively or at the early stages of disease development. Many candidates show excellent biological efficacy and selection of recent SDHIs has been driven by their safety profile. Fluopyram, a typical phenylamide fungicide, is widely applied to protect fruit and vegetables from fungal pathogens responsible for yield loss (e.g., white dot, black mold, botrytis) (Tinwell et al., 2014). SDH consists of four subunits (A, B, C, and D), and fluopyram, similar to a number of SDHIs, acts by blocking the enzyme binding site for ubiquinone, which is formed by the subunits B, C, and D (JMPR, 2005). Fluxapyroxad belongs to the carboxamide class of chemicals and its MOA is also the inhibition of SDH in complex II of the mitochondrial respiratory chain. Isoflucypram is a more recent molecule in the cereal fungicide market. The agreed ADI for fluopyram is 0.012 mg/kg bw per day (EFSA, 2013). The liver and thyroid gland have been identified as target organs in the rat, mouse, and dog for most SDHIs (see Liver and Gall Bladder, Vol 4, Chap 2 and Endocrine System, Vol 4, Chap 7). The hepatic effects consist of increased liver weight and hepatocellular hypertrophy. Hepatocellular adenomas or carcinomas are observed at the end of the rodent cancer bioassays with some SDHI compounds. In the repeat-dose studies, slight changes are observed in thyroid (i.e., follicular cell hypertrophy) though not necessarily accompanied by thyroid hormone changes. No follicular cell adenomas or carcinomas were observed at the end of the cancer bioassay with isoflucypram, but these changes were observed with other SDHIs such as fluopyram (only in mice). All of the above compounds were selected for absence of genotoxicity. The toxicological MOA consists of an initial effect on the liver by activating the

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constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors (Tinwell et al., 2014; Peffer et al., 2018) which are not human relevant. Significantly increased activity of hepatic Phase II enzymes was consistently observed in several in vivo and in vitro studies causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH) (Rouquie et al., 2014), providing evidence that the thyroid effects are liver mediated. Differences between the in vitro response of human hepatocytes and hepatocytes of several animal models have also been demonstrated. Additional in vitro assays have been performed to exclude alternative MOAs for the observed thyroid effects such as inhibition of thyroperoxidase (TPO) or sodium/iodide symporters (NIS). The nonrelevance of this MOA in humans for the liver and thyroid tumors has been established. Recently potential adverse effects via the disruption of the mitochondrial respiratory chain by SDHIs have been postulated based on published in vitro cell viability data, particularly as an SDH complex deficiency syndrome in humans has recently been characterized by the occurrence of multiple tumors (Rasheed and Tarjan, 2018). However, these effects have not been demonstrated either in animal studies or in human epidemiological studies (Kamp et al., 2021).

3.4. Strobilurins or Quinol Oxidation Site of Complex III Inhibitor Fungicides Strobilurin fungicides (now more properly referred to as QoI (quinol oxidation site of Complex III inhibitor) fungicides) are a group of compounds derived from the natural substance strobilurin A, isolated from wood-rotting mushrooms, including Strobilurus tenacellus. Azoxystrobin was the first marketed strobilurin. Today, strobilurins include kresoxim-methyl, fluoxastrobin, picoxystrobin, pyraclostrobin, pyraoxystrobin, trifloxystrobin, and others (Wang et al., 2021). The strobilurins constitute the most widely used branch of fungicides worldwide and have been registered for use in soybeans, rice, cereals, vegetables and fruit trees, as well as other plants. Metyltetraprole is considered to be a novel QoI and is used for controlling diseases affecting cereal crops and overcoming

pathogen resistance to existing fungicides (Matsuzaki et al., 2020). The molecular target of strobilurin fungicides is the mitochondrial respiratory complex III, which is an integral membrane protein complex that couples electron transfer. Strobilurins act by binding to the Qo site of the complex III and blocking electron transfer between cytochrome b and cytochrome c1 across the membrane. This results in loss of adenosine triphosphate (ATP) synthesis which inhibits cellular respiration in eukaryotes (OECD Series on Testing and Assessment No. 327). Strobilurins are considered relatively nontoxic to humans, mammals, and birds; however, data suggest that these compounds are highly toxic to aquatic species (Wang et al., 2021). In the 90day rat study with trifloxystrobin, the NOAEL was 30.6 mg/kg bw per day based on reduced body weight gain and food consumption and increased liver and kidney weights. In the 1year dog study, the NOAEL was 5 mg/kg bw per day based on reduced body weight gain and food consumption and hepatotoxicity (increased weight, liver clinical chemistry, and hepatocellular hypertrophy). In the 2-year rat study, the systemic NOAEL was 10 mg/kg/bw per day based on decreased body weight gain. In the 18-month mouse study, the systemic NOAEL was 35.7 mg/kg bw per day based on reduced body weight gain, increased liver weight, and microscopic changes in liver (single cell necrosis). There was no evidence of a carcinogenic effect in mice. On the basis of the available information, the evidence was considered not sufficient to propose a classification for carcinogenicity. Trifloxystrobin is considered unlikely to be an endocrine disruptor in mammals (EFSA, 2017a,b).

4. INSECTICIDES 4.1. Introduction Based on their chemistry, insecticides can be classified into four main groups: organophosphates, carbamates, organochlorines, and pyrethrins and/or pyrethroids. Several new insecticide compounds have been discovered including mectins. Insecticides have differing levels of toxicity to vertebrates, dictated by

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divergent mechanisms of action and toxicokinetic profiles, but all have the capacity for some degree of toxicity. More than 30% of today’s agrochemicals contain at least one sulfur atom, mainly in fungicides, herbicides, and insecticides and leading sulfur-containing pesticidal chemistries. Families include sulfonylureas, sulfonamides, sulfur-containing heterocyclics, thioureas, sulfides, sulfones, sulfoxides, and sulfoximines (Devendar and Yang, 2017). Sulfluramid which was introduced in 1989 degrades to perfluoro-octane sulfonic acid which is listed under the Stockholm convention as a persistent organic pollutant and is therefore under pressure to be phased out. Recent insecticide targets include acetylcholinesterase, GABA-gated chloride channel, sodium channel, nicotinic acetylcholine receptor, glutamate-gated channel, juvenile hormones, chordotonal organ transient receptor potential, vanilloid channel, chitin synthase 1, insect midgut membranes, mitochondrial ATP synthase, oxidative phosphorylation (Li et al., 2020), acetyl CoA carboxylase, and the ryanodine receptor.

4.2. Organophosphates and Carbamates The organophosphorus and carbamate insecticides are represented by a wide variety of chemical structures. Toxicity to insects and mammals is determined by several factors that may affect the insecticides as they are absorbed, translocated to the target site and as they inactivate the target, leading to poisoning (Fukoto, 1990). Organophosphates and carbamates both inhibit acetylcholinesterase (AChE) enzymes, leading to excess acetylcholine accumulation at nerve terminals (King and Aaron, 2015). The clinical syndromes result from excessive nicotinic and muscarinic neurostimulation. The toxic effects from organophosphates and carbamates differ with respect to reversibility, and subacute and chronic effects (King and Aaron, 2015). Organophosphates (malathion, parathion, chlorpyrifos, etc.) comprise over 200 complex esters of phosphoric acid that may be divided into 13 types. There are more than 24 carbamates (e.g., aldicarb, carbofuran, carbaryl, propoxur, methomyl, oxamyl, etc.), which are esters of carbamic acid. Carbamate insecticides belong to four chemical classes, i.e., oxime-N-methyl carbamates, aryl N-methyl carbamates, N-phenyl

carbamates, and methyl esters of substituted carbamic acids which include pirimicarb (Knaak et al., 2008). As with their herbicidal counterparts, many compounds of both classes are extremely toxic and lack species selectivity. The inadvertent or accidental use of organophosphates and carbamates continues to pose a serious threat to human and animal health, particularly terrestrial wildlife and aquatic organisms following environmental exposures. Pirimiphos, a broad-spectrum organophosphorus insecticide, has emerged as an alternative to malathion as it is relatively more effective against target species and equally safe to nontarget species (Deo et al., 1995). Profenofos [O-(4-bromo-2chlorophenyl) O–Et S–Pr phosphorothioate] is one of the most widely used organophosphate insecticides on field crops, vegetables, and fruit crops and is classified by the World Health Organization as moderately hazardous (Toxicity Class II) (Kushwaha et al., 2016). Toxicology The route of exposure, lipophilicity, volume of distribution, serum paraoxonase activity (an intrinsic enzyme capable of hydrolyzing certain organophosphates), and elimination all play a role in the pharmacokinetics of organophosphates and carbamates (Aaron and King, 2015). The organophosphates may follow either activation or detoxification pathways of metabolism or both. Activation (lethal synthesis) implies that the metabolite is more toxic than the parent compound (e.g., the conversion of malathion to malaoxon). Detoxification implies that the metabolite is less toxic than the parent compound (e.g., the conversion of malathion to its mono- and diacids). Unlike organophosphates, carbamates are metabolized only to less toxic compounds. Depending on the dose and exposure frequency, toxicity with carbamates can be acute or chronic, while the toxicity of organophosphates can be acute, intermediate, or chronic (e.g., organophosphate-induced delayed peripheral neuropathy or OPIDN) (Figure 11.1). Cholinergic signs can be classified as muscarinic, nicotinic, and central. Muscarinic stimulation of parasympathetic ganglia causes smooth muscle contraction and other effects that include diarrhea, diaphoresis (excessive sweating), urination, miosis, bronchorrhea, bronchospasm, bradycardia, emesis, lacrimation, and salivation.

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FIGURE 11.1 Cross-section of sural nerve from an adult rat with organophosphorus-induced delayed neuropathy (OPIDN). The lesion was attributed to administration of tri-ortho-tolyl phosphate (TOTP) given at 300 mg/kg by oral gavage in two courses of seven doses each administered between study days 14 and 28, and 49 and 63; the animal was necropsied on study day 90. Note the axonal degeneration (indicated by axoplasmic vacuolation; red arrow) and fiber breakdown (black arrow). Epoxy resin–embedded section stained with toluidine blue and safranin. Bar ¼ 20 mm. Figure from In Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, 2013, Academic Press, Figure 42.1, p. 1361, with permission.

The nicotinic effects on the autonomic (sympathetic) ganglia and striated muscles may cause muscle twitching and tremors, paresis, mydriasis, hypertension, tachycardia, and flaccid paralysis (Aaron and King, 2015). The acetylcholine (ACh) receptors are dispersed widely in the central nervous system and the central cholinergic effects vary based on species and the specific toxicant. They may include stimulation and seizures or CNS depression as well as confusion, ataxia, respiratory or cardiovascular depression, and coma. The signs of carbamate poisoning are similar to those of organophosphates, except that carbamates inactivate faster, the toxicity is usually less intense, and the recovery is faster (within 4 to 24 h) (Aaron and King, 2015). Organophosphates cause toxicity by the inhibition of acetylcholinesterase, which results in the synaptic accumulation of acetylcholine (ACh), the ultimate toxicant. Death may follow paralysis of the respiratory muscles (diaphragm and intercostal muscles). Evidence suggests that while cholinergic mechanisms are critical in

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acute toxicity, delayed organophosphate neuropathy occurs through noncholinergic mechanisms, particularly inhibition of neurotoxic esterase (NTE). With carbamate insecticides, the mechanism is essentially the same, but inhibition of AChE is reversible (Aaron and King, 2015) and caused by carbamylation instead of phosphorylation. Carbamates exert toxicity through cholinergic and noncholinergic mechanisms and thereby have multisystemic effects. Brain and skeletal muscles are the major target organs, but cardiovascular, respiratory, reproductive, and immune systems are also affected. Clinical Signs and Pathology The acute histopathological effects of acetylcholinesterase inhibitors in rats are typified by neuronal death in several parts of the brain (hippocampus, cerebral cortex, amygdala, and thalamus, see Nervous System, Vol 4, Chap 8). Skeletal muscle necrosis (see Muscle and Tendon, Vol 4, Chap 4) follows severe (85%) acetylcholinesterase inhibition in the rat and can be prevented by efferent nerve transection. Intermediate syndrome may develop 1 to 4 days after acute organophosphate insecticide intoxication (Karalliedde et al., 2006). This condition is characterized by progressive loss of peripheral nerve function and weakness (Aaron and King, 2015), particularly those that supply skeletal muscles, and often ends in death due to respiratory failure. In addition to the muscles of respiration, proximal limb muscles, neck flexors, and muscles innervated by cranial motor nerves are affected. The cardiac histopathology of intermediate syndrome in humans is characterized by interstitial edema and patchy inflammation, often with mural thrombosis (see Cardiovascular System, Vol 5, Chap 1). With chronic exposure, certain organophosphates cause inhibition and aging of NTE, which results in organophosphate-induced delayed neuropathy (OPIDN) (Aaron and King, 2015), although this has been questioned (Ding et al., 2017). This late-onset condition is characterized by axonal swelling and subsequent degeneration in the distal parts of long somatic nerves (distal axonopathy) in the limbs as well as the corresponding white matter tracts in the spinal cord. This neurodegenerative disorder is characterized by ataxia progressing to paralysis. The

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potency of this effect is independent of acetylcholinesterase inhibition, so the neuropathic organophosphates are not necessarily effective as insecticides. The widely used organophosphate insecticide chlorpyrifos, which can cause intermediate syndrome in certain animal models, does not cause OPIDN in humans at sublethal dosages. Albers and coworkers (2007) indicate that there is little support for the hypothesis that chronic chlorpyrifos exposures, at levels in the range associated with appreciable inhibition of B-esterases, produce adverse dose effects on peripheral nerve electrophysiology suggestive of subclinical neuropathy. In susceptible vertebrates, NTE inhibition appears to disrupt membrane phospholipids and endoplasmic reticulum functions, including axonal transport and glial–axonal interaction. In a reproductive developmental study in rats, the organophosphate insecticide diazinon caused inconsistent fetotoxic effects but only at doses that caused marked maternal toxicity. Accordingly, the effects were considered to be secondary to the maternal toxicity and unrelated to cholinesterase inhibition. Human Risk Organophosphates and carbamates can be neurotoxicants, if not used correctly. Residues can be present in food, water, and air, and residues or their metabolites can pass through the milk. Direct ocular and respiratory organophosphate exposure may be an immediate matter of life and death, particularly for pilots performing aerial applications. The largest industrial accident in history occurred in India in 1984 and involved the release of methyl isocyanate during the production of carbamate carbaryl (Aaron and King, 2015). An effective antidote to carbamate toxicity is atropine. Diagnosis of organophosphate toxicity is based on cholinergic clinical signs, determination of acetylcholinesterase activity in whole blood or brain, and the detection of agentspecific residues in body tissues or fluids.

4.3. Organochlorines Development and Use Organochlorines are renowned for their high lipid solubility, bioaccumulation, slow

degradation, high persistence, low polarity, and low aqueous solubility (Jayaraj et al., 2016). Organochlorines are generally highly lipophilic and stable, with environmental half-lives measured in years or decades. They are rarely used today in the developed world because they have negative effects on a variety of nontarget species. In addition, many of the organochlorine compounds are prohibited or severely restricted since 2004 by the Stockholm Convention on Persistent Organic Pollutants. The organochlorines may be divided into three classes: the dichlorodiphenyltrichloroethanes (DDT, dicofol, methoxychlor, and perthane); the chlorinated cyclodienes (aldrin, dieldrin, endrin, chlordane, endosulfan, and heptachlor); and the hexachlorocyclohexanes (benzene hexachloride, toxaphene, lindane, mirex, and chlordecone). Though banned in the United States since the early 1970s, DDT is still used in many parts of the world to control the insect vectors responsible for many parasitic and viral diseases, and it played an essential role in the eradication of malaria around the globe. In the United States, the organochlorine lindane is still used as a second line agent for head lice and scabies, although lindane has been associated with numerous severe and fatal adverse reactions due to its neurotoxic effects (Nolan et al., 2012). The most serious events and fatalities involving lindane occur in pediatric and geriatric populations. The DDT pesticides act as teratogens, are neuroendocrine disruptors, suppress the immune and reproductive systems, and dysregulate lipids and metabolism (Martyniuk et al., 2020). Toxicology Generally, the organochlorines are rapidly absorbed by oral or inhalation routes, while dermal absorption is variable. Signs of intoxication appear early and may be prolonged due to persistence in fatty tissues, including the brain. The DDT class of organochlorines affects axonal transmission by slowing sodium influx and inhibiting potassium outflow. In contrast, the cyclodienes and hexachlorocyclohexanes inhibit GABA-mediated chloride influx in the CNS, leading to neuronal hyperexcitability and repetitive firing after a single stimulus.

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Clinical Signs and Pathology The acute signs of intoxication by DDT (or its analogs) and the chlorinated benzenes include oral and facial paresthesia, apprehension, tremors, and seizures and the latter may cause hyperthermia. The signs of acute cyclodiene or hexachlorocylohexane intoxication include dizziness, nausea, agitation, confusion, and seizures. Death may result within 24–72 h from respiratory paralysis. Inhalation or aspiration of organochochlorines can cause chemical pneumonitis and, in severe cases, hemorrhage and necrosis of lung tissue. Cardiotoxicity is also reported with organochlorines (Georgiadis et al., 2018). Environmental DDT is well known for causing dose-related eggshell thinning after bioaccumulation by apex predators, which led to the decline or endangerment of many species of raptors, waterfowl, and songbirds. In rodent carcinogenicity studies, DDT increases the incidence of hepatocellular tumors in rats, and serves as a promoter in several initiation– promotion studies for hepatic neoplasia (Diwan at 1994). Environmental DDT is also considered an endocrine disruptor, primarily for o,pDDT’s weak estrogenic activity, although DDE may act as an antiandrogen (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). Human Risk Due to their lipophilicity, organochlorines may be passed through the milk. Data suggest that childhood exposure to DDT increases the chance of breast cancer development in women (Cohn et al., 2007, see Mammary Gland, Vol 5, Chap 8). Diagnosis is based on clinical signs and measurements of insecticide residue in body tissues and fluids.

4.4. Pyrethrins and Pyrethroids Development and Use Pyrethrins are insecticides extracted from the flowers of Chrysanthemum cinerariaefolium. Pyrethroids are their newer, more stable, and highly effective synthetic analogs. These agents break down quickly in air or sunlight and thus have gained global acceptance as insecticides of choice. Many commercial products contain pyrethroids due to their enhanced stability and diminished toxicity to mammals. This is because

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the amount of pyrethroid is lower in the formulation compared to the mixture active ingredients and the pyrethroid application rate per hectare is quite low compared to other insecticides. Pyrethrins are readily absorbed from the gastrointestinal tract and respiratory tract, but poorly absorbed through skin. The active components are rapidly and extensively metabolized in the liver. Pyrethrins act on sodium channels resulting in nervous system overactivity (Proudfoot, 2005). There are two types of pyrethroids. Type I compounds lack an a-cyano substituent and include pyrethrin 1, allethrin, tetramethrin, kadethrin, resmethrin, phenothrin, and permethrin. Type II compounds contain a stabilizing a-cyano-3-phenoxybenzyl component and include cyfluthrin, cypermethrin, fenpropanthrin, deltamethrin, cyphenothrin, fenvalerate, cyhalothrin, tefluthrin, and fluvalinate (Bradberry et al., 2005). Stabilization delays breakdown of the pyrethroids and apparently increases their toxicity, though to a variable degree. Products that incorporate pyrethrins are sometimes combined with an inhibitor of insect microsomal oxidases, piperonyl butoxide, which prevents pyrethrin clearance and maximizes insect lethality. This inhibitor has low mammalian toxicity and no substantive impact on their metabolic pathways. Toxicology Pyrethroids cause neuronal hyperexcitation resulting in repetitive synaptic firing and persistent depolarization. The molecular targets of the pyrethrins and pyrethroids are similar in mammals and insects and include voltagegated sodium, chloride, and calcium channels, GABA-gated chloride channels, nicotinic ACh receptors, and intercellular gap junctions. Type I pyrethroids produce a neurological syndrome through their CNS and peripheral nervous system effects, with signs including tremors, incoordination, prostration, seizures, and death (Ray and Forshaw, 2000). Type II pyrethroids work primarily by CNS mechanisms to elicit the choreoathetosis (involuntary twitching)/salivation syndrome, which is characterized by hyperactivity, hunched back, salivation, tremors, and incoordination progressing to sinuous writhing movements (Ray and Forshaw, 2000). Pyrethroid insecticides affect the nerve

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fiber by binding to a protein that regulates the voltage-gated sodium channel and may be toxic to fish and amphipods (He et al., 2008). Clinical Signs and Pathology There are few morphologic lesions attributable to the pyrethroids except at high toxic doses. Human Risk The number of reports of toxicity caused by pyrethrins has greatly decreased over recent years (Proudfoot, 2005). The pyrethrins are generally of low acute toxicity but convulsions may occur if substantial amounts are ingested (Proudfoot, 2005). Mild, acute occupational exposure may result in transient dizziness, headache, nausea, anorexia, and fatigue. At high doses, such as dermal soaking with concentrated pyrethroids or intentional ingestion, skeletal muscle fasciculations, convulsions, pulmonary edema, and coma have been reported (Proudfoot, 2005). Dermal exposure to Type II pyrethroids may cause temporary skin irritation and paresthesia localized to the exposure site (Proudfoot, 2005). Allergic reactions of various types have been reported; however, the chief allergen, a lactone termed pyrethrosin, has been removed from current formulations. Ocular exposure has resulted in corneal erosions (Proudfoot, 2005). Diagnosis is based on clinical signs, a history of exposure, and determination of insecticide residue in body tissues and fluids.

4.5. New Insecticides Neonicotinoids Neonicotinoid insecticides such as thiamethoxam have favorable safety profiles, due to their preferential affinity for nicotinic receptor (nAChR) subtypes in the CNS of insects, poor penetration of the mammalian blood–brain barrier, and low application rates (Sheets et al., 2016). Examples include imidacloprid, acetamiprid, thiacloprid, clothianidin, thiamethoxam, and dinotefuran (Sheets et al., 2016). At present, the truly commercial and valuable insecticides mainly target nicotinic acetylcholine. Neonicotinoids have a low risk in nontarget animals and the environment (Cresswell, 2011). Neonicotinoid compounds have been selected to be

specific for subtypes of nicotinic receptors that occur in insects to reduce toxicity and in addition, neonicotinoids do not cross the blood–brain barrier (Ensley and Gupta, 2018). Excretion of neonicotinoids is predominantly via the urine (Ensley and Gupta, 2018). Hypertrophy of hepatocytes and sporadic cell necrosis has been noted with imidacloprid in the livers of male rats treated at a dose of 300 mg/kg over 13 weeks (Ensley and Gupta, 2018). Imidacloprid is considered to be nonmutagenic, nonembryotoxic, and nonteratogenic (Ensley and Gupta, 2018). The principal effects in reproductive studies in rats are associated with decreased body weight (delayed sexual maturation, decreased brain weight, and morphometric measurements) and acute toxicity (decreased activity during exposure) at high doses, without neuropathology or impaired cognition (Sheets et al., 2016). Phenylpyrazoles Fipronil is a member of a group of insecticides called the phenylpyrazoles which is widely used for pest management on dogs and cats (with no effects in human pet handlers) as well as residential insect control, pest control in rice, and cotton production and turf grass production (Gupta and Anadon, 2018). Fipronil inhibits GABAAinduced ion influx by targeting GABAA-regulated chloride channels (Gupta and Anadon, 2018). Fipronil may exert toxicity with the same mode of action, causing hyperexcitation (Gupta and Anadon, 2018). Occasional poisonings occur in dogs and cats due to accidental ingestion, licking of the product, or accidental dosing of the dog product in cats (Gupta and Anadon, 2018). In rats, fipronil is largely excreted in the feces, although significant amounts of metabolites can persist in the adipose tissues for 1 week posttreatment, possibly reflecting the long half-life of fipronil in the blood (Gupta and Anadon, 2018). Fipronil is a CYP450 isoform inducer and may be a hepatotoxicant at higher than induction doses (Gupta and Anadon, 2018). Macrocyclic Lactone Endectocides (Mectins) Ivermectin, abamectin, and eprinomectin belong to the group of avermectin compounds called macrocyclic lactones, which are used as insecticides, acaricides, and nematicides on

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a variety of agricultural and horticultural crops. Ivermectin/abamectin is a leading acaricide/ insecticide/nematicide produced from a natural fermentation process and is derived from Streptomyces spp. It is extensively used worldwide to control a diverse range of pests and is considered critical for insect resistance management. The compounds act by increasing the membrane permeability to chloride ions (chloride channel activator), mainly stimulating the release of GABA, an inhibitory neurotransmitter. There is a correlation between activation of glutamategated chloride channel current, membrane binding, and nematocidal activity. Abamectin is used as an anthelmintic drug in veterinary medicine and ivermectin has proved to be safe and effective for the treatment of a number of parasitic diseases in domestic and food animals and humans (Shoop and Soll, 2002). Despite over 25 years of product use against agricultural and horticultural pests, there are only a small number of recorded cases of resistance to the chloride channel activator pesticides. In target organisms, the MOA is receptor mediated and ligand-gated chloride channels are the target proteins for this class of compounds. Avermectins potentiate and/or directly activate arthropod and nematode glutamate-gated chloride channels. Modulation of other ligand-gated chloride channels, such as those gated by the neurotransmitter GABA, may also be involved. Mammals do not have glutamate-gated chloride channels; thus the macrocyclic lactones have a low affinity for other mammalian ligand-gated chloride channels and they do not readily cross the blood–brain barrier (Shoop and Soll, 2002). Ivermectin is not approved for use in lactating cows, sheep, and goats (Gwaltney-Brant et al., 2018). Clinical signs of toxicity include CNS depression (GwaltneyBrant et al., 2018). The observed LD50 values in experimental animals, measured in units of milligrams per kilogram of body weight, are well above the microgram per kilogram dosages used in humans and against target species for antiparasitic activity (Burkhart, 2000). This, together with the low affinity for mammalian ligand-gated chloride channels and the minimal accumulation of ivermectin in the CNS of mammalian species, confers a wide margin of safety to the avermectins (Shoop and Soll, 2002).

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Diamide Insecticides Diamide insecticides have emerged as one of the most promising new classes of insecticide chemistry owing to their excellent insecticidal efficacy and high margins of mammalian safety (Lahm et al., 2009). Currently available diamide insecticides include chlorantraniliprole, cyantraniliprole, tetraniliprole, and flubendiamide (Texiera and Andoloro, 2013). Breakthroughs have been made in the development of the insecticides benzamide and chlorantraniliprole, which target the ryanodine receptor as well as cyclaniliprole. These were discovered based on the allosteric ryanodine receptor structure (Lahm et al., 2009). Chlorantraniliprole and flubendiamide, the first two insecticides from this class, demonstrate exceptional activity across a broad range of pests in the order Lepidoptera which includes butterflies (Lahm et al., 2009). This chemistry has been confirmed to control insects via activation of ryanodine receptors which leads to uncontrolled calcium release in muscle (Lahm et al., 2009). The high levels of mammalian safety are attributed to a strong selectivity for insect over mammalian receptors (Lahm et al., 2009). Cyromazine Cyromazine acts as a growth regulator by blocking the development of dipterous larvae (Loosli, 1994). Diafenthiuron Diafenthiuron is one of the newer products on the market performing as an insecticide and acaricide. It is active against a wide range of sucking insects and some lepidopteran pests (Kayser and Eilinger, 2001). Biological and biochemical studies finally revealed that the thiourea diafenthiuron is not itself active, but has to be converted into the respective carbodiimide, Diaf-CD. This carbodiimide product inhibits respiration of coupled mitochondrial ATPase. In addition to ATP synthase, located in the inner mitochondrial membrane, both of these carbodiimides also target porin, a 30-kDa voltage-dependent anion channel in the outer mitochondrial membrane (Kayser and Eilinger, 2001). Fenoxycarb A class of insecticides called insect growth regulators (IGRs) include synthetic compounds

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that mimic the action of juvenile hormones (Jindra and Bittova, 2020). The juvenile hormones block metamorphosis of insect larvae to reproductive adults (Jindra and Bittova, 2020). Conclusions after the peer review of the pesticide risk assessment of the active substance fenoxycarb indicate a low risk for toxicity for birds and mammals (EFSA, 2010). Lufenuron Lufenuron belongs to the group of benzoylurea compounds which are used as insecticides, belonging to the class of chitin synthesis inhibitors (EFSA, 2017a,b). Lufenuron is an IGR used to control biting and sucking insects including Lepidoptera and Coleoptera larvae (Aizawa, 2014). Pymetrozine The heteromeric channel composed of Nanchung and Inactive vanilloid TRP (TRPV) channel subunits is the target of the selective feeding blockers pymetrozine and pyrifluquinazon (Salgado 2017). The TRP channel causes feeding inhibition (Salgado 2017). The TRPA1 channel has been investigated as a suitable target for repelling honeybee parasites such as varroa mites (Salgado, 2017). Spiropidion Following release inside the plant, the insecticidally active dione metabolite 2 of tetronic and tetramic acid derivative is a translaminar and two-way systemic (both xylem and phloem mobile) for full plant protection against arthropod pests (Muehlebach et al., 2020).

5. RODENTICIDES 5.1. Introduction The control of rodent populations and the use of rodenticides is required for the protection of public health, including prevention of transmission of disease, prevention of contamination of food and feed, protection of buildings including pipes and cables, protection of livestock, social abhorrence and stigma, and as a legal requirement (Regulation EU No 528/2012 Assessment report 87596_Brodifacoum 2016 Netherlands and Italy) and (US EPA Risk Mitigation Decision for Ten Rodenticides 2008); (ECHA-18-H-23-EN

2018) [revised Emission scenario document]. For more information about rodent-borne diseases please refer to Meerburg et al., (2009). The use of rodenticides requires careful risk mitigation as most are nonspecific toxins for vertebrates which also endanger nontarget species. Both the United States and European legislation are currently trying to balance the need for rodent control with risk mitigation measures such as inclusion of dyes and bittering agents into rodenticide formulations, a requirement to use tamperresistant bait stations, restrictions on the use of certain rodenticide classes by nonprofessional users, limitations on package sizes, and constraints on the use areas of a rodenticide (EUBEES Emission Scenario Document Use Areas 1. ‘In and around buildings’ 2. ‘Sewers’ 3. ‘Open areas’ 4. ‘Waste dumps’). A detailed account can be found in the respective documents issued by the US Environmental Protection Agency (EPA) and the European Chemical Agency (ECHA): (US EPA Risk Mitigation Decision for Ten Rodenticides 2008); (ECHA-18-H-23-EN 2018) [revised Emission scenario document]. The characteristics of an ideal rodenticide (Prescott et al., 2015) are: 1. Onset of clinical signs should be slow to avoid bait shyness 2. Lethal in a normal amount of food 3. Palatable to rodents 4. Inexpensive 5. Easily formulated 6. Easily degraded in the environment 7. No difference in susceptibility due to variations in age, sex, or strain 8. Resistance should not develop 9. No secondary poisoning hazard 10. No danger to man or domestic animals 11. Specific to the target species So far, rodenticides have fallen short of these ideal characteristics in several ways and therefore the need for new developments in this field remains, especially since no new substance has been developed in the last 30 years. Currently, the anticoagulant rodenticides are the preferred class of rodenticide because of their advantages of overcoming bait shyness, excellent efficacy (if not hampered by resistance of the target species), and available antidote. Disadvantages include persistence in the food chain, resistance in the target species, and high toxicity for nontarget animals (Prescott et al., 2015).

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Various classes of chemicals have been used in rodent control. For a list of currently approved substances in both the United States and Europe see Table 11.2. Historically, other chemicals have also been used but have been discontinued due to poor effectivity and/or unacceptable toxicity (Table 11.3). However, there is a need for effective rodenticides which are less persistent than second-generation anticoagulants (Table 11.2). Ideally, alternatives to existing anticoagulants would combine limited persistence, availability of an antidote, humaneness, and high efficacy; however, this is a significant challenge. Research includes the retrieval and retention of older alternatives and the development of novel rodenticides. A possibility pursued by researchers from New Zealand is to improve the performance of older, nonanticoagulant rodenticides, the optimization of existing compounds belonging to the first generation of anticoagulants and seeking alternatives to TABLE 11.2

anticoagulants, e.g., methemoglobinemia inducers (Eason et al., 2011). For a critical review of alternatives to chemical control with anticoagulants, including repellents and attractants (e.g., pheromones), chemosterilants, and trapping, as well as integrative rodent population management strategies with resistance management and nonchemical measures as rodent-proofing of buildings, see Berny (2013).

5.2. Anticoagulant Rodenticides Development and Use Anticoagulant rodenticides (ARs) are used in the vast majority of rodent control operations in the EU and United States and this will continue for the foreseeable future (Berny 2013). The reasons for this include an excellent safety record, the existence of an antidote (vitamin K1), and the avoidance of bait shyness due to

Currently Used Rodenticides and Their Chemical Classes (as of 2021).

Anticoagulants

European Union

United States

First generation

Warfarin, coumatetralyl

Warfarin

Second generation

Brodifacoum Bromadiolone Difenacoum Flocoumafen

Brodifacoum Bromadiolone Difenacoum

1,3-Indandiones (first generation)

Chlorophacinone

Chlorophacinone Diphacinone

Thiocoumarine (second generation)

Difethialon

Difethialon

Calciferole

Cholecalciferol

COUMARINS

Cholecalciferol a

Inorganic compounds (baits)

Zinc phosphide

Inorganic compounds (fumigants)

Aluminum phosphide, Hydrogen cyanide, CO2

Organochlorine

Alphachloralose

Respiratory toxins Other Convulsants a

Zinc phosphide

Alphachloralose Bromethalin

Starch (corn cob)

Starch (corn cob) Strychnine (gophers)b

Zinc phosphide is the only remaining plant protection rodenticide available for field use in the EU. It is generally regarded as effective for controlling small field rodents such as common voles (Microtus arvalis) but palatability is low (Jacob et al., 2010). It is not regulated as a biocide but as a pesticide per Plant Protection Products regulations (CE1107/2009). b Strychnine is currently under reevaluation by EPA and may only be used underground against gophers. Printed in bold: Example compounds for each class that are described in more detail below.

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TABLE 11.3 Historically Used Rodenticides and Their Chemical Classes, now Banned in the United States and European Union. Compound Class

Compound Examples

Organophosphorus

Phosacetim

Respiratory toxin (blocker citric acid circle)

Fluoroacetamide 1,3-Difluoro-2propanol (Gliftor) Sodium fluoroacetatea

Organosulfur (free radicals damage lung tissue)

a-Naphthylthiourea

Ca-channel blocker

Norbormide

Nicotinamide

Pyrinuron

Red squill (Glycoside)

Scilliroside

Piperidines

Flupropadine

Inorganic

Arsenic Barium carbonate Calcium phosphide Cyanide Thallium sulfate

a

Sodium fluoroacetate (“1080”) is still used in some countries such as Australia and New Zealand. Main indications are the control of foxes, rabbits, wild dogs, and feral pigs. Native species are often resistant to this toxin as many plants in Western Australia contain this toxin naturally (Government of Western Australia, 2018: https://www.agric.wa.gov.au/1080/1080characteristics-and-use). In the United States, the sole indication is “sheep coyote collars.”

longer time between poisoning and symptoms (Berny et al., 2014). First-generation anticoagulant rodenticides (FGARs) have been developed in the 1950 and 1960s, the first being warfarin, after 4hydroxycoumarin was discovered as the toxic principle in sweet clover disease in 1940 (Stahman et al., 1941). Unfortunately, resistance developed in target species (Rattus norvegicus, Rattus rattus, Musculus domesticus). In Norway rats, mutations of the target enzyme VKOR have been described (Li et al., 2004; Rost et al., 2004) as well as increased metabolism (Ishizuka et al., 2008). In black rats, increased metabolism of warfarin by CYP3A2 is proposed as a mechanism. CYP3A2

was found to be increased in resistant rats (Ishizuka et al., 2007). House mice seem to be more frequently resistant to FGAR than rats (Buckle et al., 1994; Buckle and Prescott, 2012). This may be due to mutations in VKORC1 (Pelz et al., 2012). The hybridization of the Algerian mouse (Mus spretus) with the house mouse, as demonstrated in Germany and some parts of Spain, has also caused resistance (Song et al., 2011). Therefore, FGARs are currently not recommended for use against mice (Prescott et al., 2015). A second generation of anticoagulants (SGARs) was developed in the 1970 and 1980s (Rodenticide Resistance Action Committee, Website (RRAC, 2021)). These rodenticides share the same mechanism of inhibition of coagulation by impaired vitamin K recycling but have a much higher halflife than FGARs. They are often more active but are also more toxic for nontarget species than FGARs. Some SGARs (bromadiolone, difenacoum) have lower potency and require multiple feeding and resistance has been observed. Others (brodifacoum, difethialone, flocoumafen) are more potent, require a single dose to be effective, and resistance has not been observed so far. Some genetic alterations may induce resistance to low potency SGARs, as observed in the United Kingdom and Germany (Buckle and Prescott, 2012; Pelz, 2007) and this resistance is seen quite commonly in mice (Pelz et al., 2012). If used inappropriately, low potency SGARs will not control rodent populations effectively and may increase the selection of resistance in target animals, leading to longer use and increased risk to nontarget species (Buckle and Prescott, 2012; Prescott et al., 2015). In Table 11.4, two FGARs, a 4-hydroxycoumarin (warfarin) and a 1,3 indanedione (chlorophacinone), and two SGARs, a second-generation coumarin (brodifacoum) and a thiocoumarin (difethialone), are discussed as an example of the properties of the different chemical classes used as anticoagulants. Data are taken from the respective Assessment reports (Regulation EU No 528/2012 Assessment report, (Regulation EU No 528/2012 Assessment report 80815_Warfarin 2016 Ireland) (Regulation EU No 528/2012 Assessment report 77126_Difethialon 2016 Norway), (Regulation EU No 528/2012 Assessment report 80186_Chlorophacinone 2016 Spain), (Regulation EU No 528/ 2012 Assessment report 87596_Brodifacoum 2016 Netherlands and Italy)).

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TABLE 11.4

The Use Areas of Warfarin, Chlorophacione, Brodifacoum, and Difethialone. Use Area

FGAR

SGAR

In and Around Buildings

Open Areas

Waste Dumps

Sewers

Warfarin

X

X

X

X

Chlorophacinone

X

X

X

X

Brodifacoum

X

X

X

X

Difethialon

X

X

Toxicology The mechanism of both first- and secondgeneration anticoagulants is the inhibition of vitamin K epoxide reductase, which causes the inhibition of synthesis of clotting factors II, VII, IX, X, and protein C and S. The antagonist is vitamin K1. Metabolism Oral absorption is up to 100% for warfarin and brodifacoum. The highest tissue levels of all four compounds have been found in the liver. Both FGARs are metabolized and then excreted via feces. Warfarin metabolites (warfarin alcohols) have anticoagulant activity in humans. Chlorophacinone enters the enterohepatic circulation and then is excreted through the feces. In studies dosing 1–1.4 mg/kg, there was rapid absorption and relatively rapid metabolism in the liver and 100% elimination within 4 days. Maximum blood concentrations are reached after 4 h. The parent compound accounts for only approximately 20% of excreted substance, while approximately 45% are hydroxylated metabolites, one in the indandione group and the other in the biphenyl portion of the molecule, both are of unknown toxicity. Both SGARs accumulate in the liver and are excreted almost unmetabolized. Brodifacoum has a half-life in the liver calculated in the range of 282–350 days and brodifacoum is excreted in mostly (50%–80%) unmetabolized form in the feces. During the first 3- to 4-day long phases, clotting factor synthesis is impaired and this returns to normal function in later phases. Difethialone accumulates with a half-life of 18 weeks in rat liver. Elimination is exclusively in the feces as unchanged parent material. Due to its

accumulative properties, difethialone poses a high risk of primary and secondary poisoning to nontarget mammals and birds. The toxicity data from appendix 1 of the respective assessment reports for the four compounds are shown in Table 11.5. Note the similarities in the toxicity profile regarding acute and repeateddose toxicity, note the differences in aquatic and bird toxicity and the potential to bioaccumulate especially between Warfarin and the later compounds and also the differences in reproductive toxicology explained later in the text. Clinical Signs and Pathology Clinically, pallor, ataxia, or weakness/limb paralysis and breathing difficulty are seen. There is prolongation of the prothrombin time. At necropsy, multifocal hemorrhages and serosanguineous exudate in the pleural and abdominal cavity are observed. Centrilobular liver necrosis has also been observed. Chronic administration is not possible in rodents. Reproductive toxicity classification of warfarin (and brodifacoum) is Cat1A based on observation of Fetal Warfarin Syndrome. This is a hemorrhagic syndrome observed after use in humans in the first trimester of pregnancy as an anticlotting therapy. Rat fetuses show structural malformations of the hind limbs, internal hydrocephalus, metabolic damage of fetus livers (rat, repeated dose of 0.04–8 mg/kg bw), maxillonasal hypoplasia, and calcium deposits in cartilage of the nasal septum and epiphyseal cartilage of vertebrae and long bones (rat, 100 mg/kg bw subcutaneous injection). Exposure throughout pregnancy or during the second and third trimester is associated with adverse effects on CNS development (human, 2.5–20 mg/day).

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Toxicity Data From Assessment Reports for Warfarin, Chlorophacione, Brodifacoum, and Difethialone (Regulation EU No 528/2012 Assessment Report 80815_Warfarin 2016 Ireland, 77126_Difethialon 2016 Norway, 80186_Chlorophacinone 2016 Spain, 87596_Brodifacoum 2016 Netherlands and Ireland).

Substance

Warfarin

Chlorophacinone

Brodifacoum

Difethialone

Class

Hydroxycoumarin FGAR

1,3-Indandiones FGAR

Hydroxycoumarin SGAR

Thiocoumarin SGAR

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TABLE 11.5

SAFETY CLASSIFICATION

Reproductive toxicity

Repr. 1A, H360D (Proven Repr. 1B; H360D (May damage to the unborn child) damage the unborn child)

Repr. 1A; H360D (Proven Repr. 1B; H360D (May damage to the unborn child) damage the unborn child)

Acute toxicity

Acute tox. 1; H300 (fatal if swallowed), acute tox. 1; H310 (fatal in contact with skin), acute tox. 1; H330 (fatal if inhaled)

Global harmonized system codes

GHS06 (acute toxicity), GHS08 (health hazard), GHS09 (hazardous to the aquatic environment)

Eye toxicity

Not classified

Environmental toxicity

Aquatic chronic 2, H411 (toxic to aquatic life with long-lasting effects)

Aquatic acute 1, H400 (very toxic to aquatic life) Aquatic Chronic 1; H410 (very toxic to aquatic life with long-lasting effects)

LD50 rat oral mg/kg bw

5.62

Combined sexes: 6.26

LD50 (mg/kg bw) other species

Birds acute toxicity

EUH070 (toxic by eye contact)

0.49

Combined sexes 0.4e0.8.

Mouse: 374 Rabbit: 800 Pig: 1e5 Dog: 20e50

Mouse: 0.4

Dog: 11.81

LD50 > 2000 mg/kg bw

LD50 ¼ 0.31 mg/kg bw (mallard duck)

LD50 ¼ 0.264 mg a.i./kg bw (bobwhite quail) Oncorhynchus mykiss 51 mg/L

LC50, 96h mortality Salmo gairdneri (most sensitive fish 65 mg/L tested)

Oncorhynchus mykiss 0.45 mg/L

Oncorhynchus mykiss 0.04 mg/L

Bioaccumulation (L/kg)

BCFfish 22.75

Considered bioaccumulative BCFfish 40000 BCFearthworm 23943

Not persistent

11. AGROCHEMICALS

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Specific target organ STOT RE1; H372 (causes damage to organs through prolonged or repeated exposure) (blood) toxicant, repeated exposure (STOT RE)

5. RODENTICIDES

In rodents and rabbits, no reproductive toxicity is seen after treatment with chlorophacinone and difethialone and they are classified as Cat1B because their mechanism of action is the same as warfarin. ((Regulation EU No 528/ 2012 Assessment report 80815_Warfarin 2016 Ireland), 77126_Difethialon 2016 Norway (Regulation EU No 528/2012 Assessment report 77126_Difethialon 2016 Norway), 80186_Chlorophacinone 2016 Spain (Regulation EU No 528/ 2012 Assessment report 80186_Chlorophacinone 2016 Spain).) HUMAN RISK

Warfarin is used as a human therapeutic agent. A few cases of skin necrosis and hepatotoxicity have been observed after errors of dosing during medical therapy. A risk of human carcinogenicity was not observed in retrospective studies. There is a risk of reproductive toxicity in humans as described above. Signs include miscarriage, CNS malformations (microcephaly, hydrocephaly), nasal hypoplasia, bone anomalies, and growth retardation. Cases of poisoning in workers exposed to warfarin are extremely rare. A few cases of accidental poisoning of workers exposed to brodifacoum or difethialone have been reported in the assessment reports but none for chlorophacinone. A comparison between human and animal exposure recently demonstrated that anticoagulant exposure was rare and usually involved young children. Most cases resulted in no harmful exposure and resulted in very limited (if any) clinical signs. Intentional exposure (suicidal attempts for instance), although uncommon, resulted in more severe cases (Berny et al., 2010). Human exposure is fairly limited, mostly documented in young children. Thanks to the use of bittering agents, the vast majority of anticoagulant exposure in humans does not result in clinical signs. Only suicidal attempts may result in severe poisoning cases, but, generally speaking, anticoagulant exposure in humans does not result in prolonged monitoring of patients and does not necessitate hospitalization (Caravatti et al., 2007).

5.3. Cholecalciferol Development and Use Cholecalciferol (vitamin D3) was developed as a rodenticide in the 1970s. It has a relatively low risk of secondary poisoning and low toxicity to

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birds (RRAC, 2021). The intended use is the control of mice and rats (brown rats) in and around buildings by professional and nonprofessional users. Low doses of cholecalciferol have been added to anticoagulant containing baits to increase their effectiveness. There is some proof that low doses of cholecalciferol added to anticoagulants, like coumatetralyl, can significantly increase the efficacy of the FGAR in resistant Norway rats (RRAC, 2021). In laboratory studies calciferol (vitamin D2) was found to cause a stopfeed effect in Norway rats, with female animals reducing food consumption by 80% and male animals halting food consumption completely within 24 h. The calciferol also causes conditioned bait aversion or bait shyness in the rats (Prescott, 1992; Quy et al., 1995). Thus, prebaiting is important to obtain reasonable control in a field treatment (Quy et al., 1995). Toxicology To become biologically and toxicologically active, cholecalciferol must undergo metabolic conversion to 25-hydroxycholecalciferol. The latter metabolite is the most biologically active form of vitamin D3 (RRAC, 2021). Absorption reaches up to 50% after oral administration in humans. It is distributed in chylomicrons and lymph and bound to a specific binding protein, vitamin D–binding protein (DBP) in plasma. Adipose tissue shows the greatest accumulation. Excretion of cholecalciferol and its metabolites is via bile (feces). A minor part is eliminated as metabolites in urine. No specific antidote is available (Morrow, 2001) and current toxicity classification is H300 (fatal if swallowed), H310 (fatal in contact with skin), H330 (fatal if inhaled), and H372 (causes damage to organs through prolonged or repeated exposure). The Rat LD50 oral dose is 41 mg/kg. Clinical Signs and Pathology Cholecalciferol causes a lethal hypervitaminosis D. The onset of mortality in the rodent is delayed (occurs generally 3 to 10 days after baiting with cholecalciferol). Clinical signs of cholecalciferol toxicity prior to death include reduced eating, loss of body weight, hunched posture, piloerection, oligemia, anergia, and hypothermia. The cause of death is calcification of blood vessels and heart failure (RRAC, 2021). Carcinogenicity and reproductive toxicity studies were waived for the registration process.

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In rodents, acute toxicity studies showed calcification of heart, spleen, kidney, and blood vessels at a dose of 25 mg/kg bw and above (see Figure 11.2; (Regulation EU No 528/2012 Assessment report 80053_Cholecalciferol 2018 Sweden)). Human Risk

effect in a study where personal protection measures were used. There are no known cases of cholecalciferol poisoning in humans from rodenticide use. Symptoms and pathologic lesions of vitamin D poisoning are those of hypercalcemia which are reviewed in EFSA 2012.

MEDICAL DATA

OTHER SPECIES

Medical surveillance data of cholecalciferol in manufacturing workers showed no adverse

The assessment report mentions fatal intoxication with cholecalciferol in a cat and dog after ingestion of cholecalciferol bait. In case reports of cholecalciferol poisoning of dogs, the most frequently affected sites of mineralization are heart (with necrosis), vessels, gastrointestinal tract (with hemorrhage), kidneys, central nervous system, and lungs (Figure 11.2) (e.g., Peterson, 2013; Gunther, 1988; Talcott et al., 1991).

5.4. Inorganic Compounds: Metal Phosphides Aluminum Phosphide/Zinc Phosphide DEVELOPMENT AND USE

FIGURE 11.2 Lung, vitamin D3 toxicity, rodenticide (Quintox) ingestion, dog. (A) Alveolar edema, hemorrhage, and minimal cellular infiltrate. Mineralization is suspected due to the linear appearance of basophilic material (arrows) within the alveolar walls. H&E stain. (B) Pulmonary mineralization. The black staining indicates calcium deposition within the alveolar walls. Von Kossa stain. Figure from In Haschek WM, Rousseaux CG, Wallig MA, editors: Fundamentals of toxicologic pathology, ed 2, 2010, Academic Press, Figure 6.20, p. 123, with permission.

Aluminum phosphide products are laid out in burrow systems in form of fumigant pellets or tablets releasing phosphine gas. They are intended to be used only by trained professional users to control voles (Arvicola terrestris) and Norway rats (Rattus norvegicus) in all types of nonagricultural purposes, including embankments and dikes. Zinc phosphide was first used as a rodenticide in 1911 in Italy. It is an effective acute rodenticide and was the most widely used rodenticide worldwide until the introduction of anticoagulant compounds in the 1940 and 1950s. It is still used as a rodenticide in the United States, Australia, the Asia-Pacific region, Europe, and China (RRAC, 2021). Zinc phosphide can be used against mice in bait form in the United States. In the EU, zinc phosphide is regulated as a pesticide for field use against voles. TOXICOLOGY

The active ingredient, aluminum phosphide, reacts with moisture in the soil and air and releases the toxic gas, phosphine. Aluminum phosphide is unstable in water/moisture and hydrolyzes at pH 3 to aluminum hydroxide and water (Al(OH)3  3 H2O). The Al(OH)3

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5. RODENTICIDES

part is considered to be of no toxicological significance as it is ubiquitous in the environment and aluminum is the most commonly occurring metallic element in the earth crust. Zinc phosphide releases phosphine gas in the acid environment of the stomach, and the gas enters the bloodstream causing heart failure and damage to internal organs (Staples, 2003). There is no specific antidote and the compound is toxic to other vertebrates (Berny, 2013). Phosphine induces oxidative stress in mammalian cells, and administration of high doses causes methemoglobinemia in the rat. Resistance against aluminum phosphide did not occur in relevant susceptible pests. ABSORPTION, DISTRIBUTION, EXCRETION, AND METABOLISM

Studies with other phosphides are regarded as adequate model compounds for aluminum phosphide. Studies with zinc phosphide and phosphine are available. Once formed from the metal phosphide, phosphine is rapidly and completely excreted by exhalation or via urine, after oxidation to hypophosphite or phosphite. The phosphine metabolites, hypophosphite or phosphite, are regarded as less toxic than phosphine itself. There is ready absorption of phosphine through the lungs and after oral exposure and it is widely distributed in tissues. There is no potential for accumulation as excretion is rapid via urine (hypophosphite and phosphite) and via lungs (phosphine). The toxicologically significant metabolite is phosphine. Following oral administration of zinc phosphide, 32P was rapidly absorbed from the gastrointestinal tract. Inhaled phosphine gas is considered to be rapidly and quantitatively absorbed through the lungs. Inhaled 32P was detectable in all organs and tissues, with temporary higher levels in the liver and medulla oblongata. Inhaled phosphine gas is excreted as such with the expired air or, after metabolic oxidation, with the urine in the form of hypophosphite and phosphite (EU assessment report 7237 Aluminum phosphide 2008 Germany). CLINICAL SIGNS

Zinc phosphide is a fast-acting compound, with clinical signs first appearing from 15 min to 4 hours after intake and, following a lethal dose, death generally occurring in 3 to 12 h.

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The emetic action of the zinc portion reduces the toxicity of zinc phosphide to some nontarget species; however, rats lack a vomiting reflex. Death is mediated by a combination of cardiac and respiratory failure (RRAC, 2021). OTHER SPECIES (CLINICAL SIGNS AND PATHOLOGY)

Nontarget vertebrates which use the tunnels of the target organisms as a part of their habitat (least weasel: Mustela nivalis) or living in similar holes in the same habitat (mole: Talpa europaea, ground squirrels: Spermophilus, hamster: Cricetus cricetus) are highly endangered by the arising phosphine gas. Aluminum phosphide and its reaction product, phosphine, may theoretically pose a risk for carnivorous and scavenging terrestrial vertebrates that feed on intoxicated target animals. However, according to the intended use of the substance in underground tunnel systems, the presence of intoxicated animals on the soil surface should be negligible. In addition, in the target organisms, phosphine is metabolized to nontoxic phosphates. Thus, a relevant exposure of these nontarget organisms via the food chain can be excluded and there seems to be no risk of secondary poisoning. Poisoning of horses (Fox et al., 2018) and dogs (Gray et al., 2011) has been observed. Lesions described for horses are edema, congestion, and hemorrhage in multiple organs and also macro- and microvesicular steatosis in the liver (Fox et al., 2018). Clinical signs in dogs collected in an animal poison control center referred most frequently to the gastrointestinal tract while other organ systems affected were the CNS, the respiratory and cardiovascular systems. Notably, over half of the dogs did not show clinical signs after zinc phosphide ingestion (Gray et al., 2011). HUMAN RISK

The case reports submitted in the application process were considered to be representative of the numerous records of poisoning cases, mainly in connection with suicide, which are available from the literature. In developing countries, where aluminum phosphide has been frequently used as a household rodenticide, accidental poisonings are reported in the literature, especially in children, causing numerous fatalities. Diagnosis is mainly based on the history of

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11. AGROCHEMICALS

intake, gastrointestinal symptoms, shock symptoms, and silver nitrate impregnated paper test. The main symptoms are severe circulatory, cardiac, and renal failure, uremia, hepatic damage, changes in ECG, and respiratory distress connected with a high mortality rate. Histopathological changes have been observed in lungs, liver, heart, and kidney. Since an antidote is not available, therapy relies on treatment of the clinical symptoms and administration of high doses of corticosteroids (EU assessment report 7237 Aluminum phosphide 2008 Germany).

5.5. Alphachloralose Development and Use Chloralose is a condensation product of glucose and chloral hydrate (Vetpharm, 2021) and is an acute CNS depressant. It is intended for indoor use in bait form for control of the house mouse (Mus musculus) by both professional and amateur users. It has been introduced as a rodenticide relatively recently (2019 in the United States). Alphachloralose and its metabolite chloral hydrate have been used as sedative and hypnotic drugs in human and veterinary medicine. Toxicology Alphachloralose is metabolized to trichloroethanol, both of which have a depressant action on the reticular formation and an excitatory action on spinal reflexes, which causes convulsions with very small stimuli (Vetpharm, 2021). High acute doses lead to a reduction in body temperature, marked respiratory inhibition, and death. Repeat-dose toxicity at doses above subacute NOAEL (20 mg/kg bw/day) and subchronic NOAEL (15 mg/kg bw/day) showed evident CNS depression effects, within 10– 20 min. Death is caused by hypothermia. Several metabolites of alphachloralose can be found in urine including trichloroacetic acid, chloral hydrate, and conjugate derivatives. Urine metabolite examinations showed that chloral hydrate is the main metabolite of alphachloralose accounting for about 40%. The LD50 after oral exposure in rats is 341 mg/ kg and LD50 values are 100 mg/kg for cats and dogs. After oral administration of chloralose to rats at least 80% of the substance is rapidly

absorbed, widely distributed, metabolized, and excreted. The plasma half-life in rats is between 8.8 and 12.6 h. The greatest concentrations were measured in the gastrointestinal tract and liver. Alphachloralose was detected after 168 h in the large intestine contents. Toxicokinetic evaluation indicates that chloralose and its metabolites are unlikely to bioaccumulate in mammals. Clinical Signs and Pathology Chloralose causes transient and reversible CNS depression (prostration, spastic locomotion, drowsiness, and piloerection) with no apparent neuropathologic changes. Reduced body weight has also been observed. Human Risk Adverse effects in humans after medical use include vomiting, tremors, ataxia, mental confusion, and very seldom leukopenia and eosinophilia. Fatal cases are rare. There is no evidence of carcinogenicity or reproductive toxicity in humans despite therapeutic use and long follow-up time. As chloral hydrate can be formed as a by-product of the chlorination of water, public exposure to chronic low levels of chloral hydrate and its breakdown products can be assumed. There are no data indicating adverse health effects from these chronic exposures. Risk to Environment/Other Species The primary poisoning hazard may be related to grain-eating birds because birds are more susceptible to this active substance than rodents and other mammals that are bigger than mice (EU Assessment report Alphachloralose 2008). Chloralose is to be used indoors and the opportunity for primary poisoning to nontargets is considered negligible. Chloralose is for indoor use only, and immobilization of mice occurs shortly after bait consumption. Although the assessment report states that a secondary poisoning risk for cats and dogs is considered negligible, there are case reports of cats and dogs poisoned with alphachloralose (Segev et al., 2006; Bernhoft et al., 2020). Prognosis was good if animals were kept warm and received supportive therapy. For animals that are not found in time and remain in a poisoned state outside in cold environments, the prognosis is characterized as poor.

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5. RODENTICIDES

5.6. Bromethalin Development and Use Bromethalin was developed in the 1970s. It is a single-feeding rodenticide that is registered for use in the United States, where its use is restricted to bait stations in and around buildings for the control of commensal rodents. Its use is prohibited in the EU, but its use is increasing in the United States due to the ban of long-acting anticoagulants (second generation) for nonprofessional use by EPA in 2008 (US EPA Risk Mitigation Decision for Ten Rodenticides 2008; RRAC, 2021). Toxicology The bromethalin parent compound is rapidly absorbed from the intestines and metabolized in the liver to the N-demethylated metabolite, which is a more potent inhibitor of mitochondrial respiration, i.e., ATP production. Bromethalin and its N-demethylated metabolite (desmethylbromethalin) are lipid soluble and are distributed to the brain where they inhibit oxidative phosphorylation. Disruption of energy production causes microscopic cerebral edema and an increase in intracranial pressure (Coppock 2013). Clinical Signs Bromethalin targets the CNS and is associated with two types of presentation: a convulsant syndrome characterized by muscle tremors, hyperexcitability, hyperthermia, and seizures; and a progressive paralytic syndrome involving ascending ataxia and/or paresis, proprioceptive deficits, and CNS depression (van Lier and Cherry, 1988). Other Species (Clinical Signs and Pathology) There is growing concern about bromethalin being more commonly involved in poisoning of pets (Berny 2013). In the brain of birds, domestic animals, and humans, a cascade of edema and ion pump disruption-linked neurologic damage occurs resulting in a characteristic diffuse spongiosis of the white matter (Coppock 2013). In a case report (Romano et al., 2018), intoxication of dogs and cats is reported without the characteristic myelin lesions, although the metabolite desmethylbromethalin (DMB) which is used for diagnosis of bromethalin exposure was detected in tissues. In cats, edema vacuoles in extracellular spaces and myelin lamellae, hypertrophied

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astrocytes, and oligodendrocytes were observed in the cerebrum, cerebellum, spinal cord, and optic nerve (Dorman et al., 1992).

5.7. Corn Cob Development and Use Stripped corn cobs are ground into powder to produce the active substance corn cob. It is a solid tan colored powder with negligible odor for the eradication of unwanted rats (Rattus norvegicus) and mice (Mus musculus), respectively. It can be used both inside buildings and outdoors. Eradirat and Eradimouse are intended to be used by professional pest control officers and the general public. Toxicology Powdered corn cob is formulated from plant material and contains a percentage of a-cellulose with admixed sweet molasses. The cause of death is dehydration leading to reduced blood volume and blood pressure, tissue ischemia, and circulatory shock. After ingestion, death occurs up to 10 days later (Berny, 2013). The exact mechanism of the development of dehydration is not known but a possibility is absorption of water through the intestinal wall (ECHA Assessment report Powdered Corn Cob 2012 Greece). Some concern exists about the efficacy of corn cob whenever rodents have a choice of other food (Schmolz, 2010; Prescott et al., 2015). Corn cobs decompose mainly to sugars which do not pose a threat to human or animal health or the environment. Clinical Signs and Pathology Animals feeding on powdered corn cob baits do not develop symptoms for several days until the last few hours before dying. The main signs were lethargy, piloerection, and, occasionally, tremors. No gross or histopathologic findings were described in the assessment report. Human Risk Powdered corn cob is of low toxicity to human health. Risk to Other Species Tests on terrestrial animals (included in the dossiers) such as dogs, cats, rabbits, ducks, and chickens all indicated that when ingested, no adverse effects are observed. The material is not palatable to nonrodents.

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5.8. Strychnine Strychnine is currently under reevaluation by the EPA. Its use has been banned by many countries including the EU and Canada. In the United States, it can only be used below ground for pocket gophers and as a “special local need” measure in Nevada against yellow-bellied marmots and ground squirrels (USEPA, 2020a, 2020b). It was derived from seeds of plants in the genus Strychnos; it is a terpene indole alkaloid. It was first registered in 1947. Toxicology It is an antagonist of glycine receptors and causes convulsions as glycine is a transmitter inhibitor of motor neurons. Clinical Signs The interference with glycine leads to hyperexcitability of motor neurons with tremors, convulsions, and eventually death due to asphyxiation. Risk to Other Species Ecotoxicology appears to be a major concern with reported cases of primary and secondary poisoning of birds and mammals and also effects on endangered species (e.g., California redlegged frog (Rana aurora draytonii), California tiger salamander (Ambystoma californiense), and the San Joaquin kit fox (Vulpes macrotis mutica)). Many birds and mammals are highly sensitive to strychnine; affected bird species include corvids passerines, raptors, and waterfowl, among mammals, canids, and mustelids as off-target species are affected anddof coursedrodents. Chronic exposure studies in the mallard showed reduced growth and reduced egg production (USEPA, 2020). None of the rodenticides described in this chapter represents an ideal solution to the tradeoff of target species toxicity and sparing of nontarget species. The development of rodenticides is therefore anticipated to be an area of active research in the future.

6. CONCLUSIONS This chapter has considered the toxicity and toxicologic pathology of a variety of

agrochemicals, including herbicides, fungicides, insecticides, and rodenticides. The challenges of recognizing and adapting the toxicity of various agrochemicals so they can be used safely in the environment and in animals have been considered. Other challenges include pressure from environmental groups and the public to decrease the use of agrochemicals for human and pollinator health. In addition, pesticide legislation policies for risks and hazards continue a trend toward ever greater restrictions.

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C H A P T E R

12 New Frontiers in Endocrine Disruptor Research* Paul S. Cooke1, Cheryl S. Rosenfeld2,3, Nancy D. Denslow1, Christopher J. Martyniuk1, Ana M. Mesa1, John A. Bowden1, Trupti Joshi2, Juexin Wang2, Juan J. Aristizabal-Henao4, Anatoly E. Martynyuk5 1

Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States, 2Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States, 3Thompson Center for Autism and Neurobehavioral Disorders, Columbia, MO, United States, 4Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, United States, 5McKnight Brain Institute, University of Florida, Gainesville, FL, United States

O U T L I N E 1. Introduction 2. Environmental Chemicals Can Disrupt Endocrine Signaling 2.1. History of Endocrine Disruptor Research 2.2. Types of Chemicals With EndocrineDisrupting Activity 2.3. Routes of Exposure to Endocrine Disruptors 2.4. Regulatory Approaches to Endocrine Disruption 3. Mechanisms of Endocrine Disruption

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4. Examples of Disruption of Endocrine Pathways by Some Environmental Contaminants and Emerging Endocrine Disruptors 773 4.1. Phthalates Disrupt Several Endocrine Pathways 774 4.2. Emerging Endocrine Disruptors: Glyphosate 774 4.3. Emerging Endocrine Disruptors: General Anesthetics as Endocrine Disruptors 775

5. Epigenetic Effects of EDCs

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6. From Reactive to Proactive Endocrine Disruptor Analysis

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7. Emerging Models in EDC Research 7.1. Zebrafish Model 7.2. CRISPR Screening

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8. Omics Technologies to Evaluate Endocrine Disruption 8.1. Transcriptomics and Proteomics 8.2. Lipidomics and Metabolomics 8.3. Microbiome 8.4. Exposomics

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9. New Frontiers in Bioinformatics and Integrative and Functional Enrichment Omics Approaches 9.1. Integrative Correlation Analyses 9.2. Integrative MultiOmics Pathway Resolution

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This work was supported in part by NIH grants R03 HD087528 and R21 HD088006, and R01 PR015540 (to P.S. Cooke) and R01 NS091542 and R56 HD102898 (to A. Martynyuk). C.S. Rosenfeld was supported by NIH grant R01 ES025547.

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-443-16153-7.00012-5

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10. Machine Learning and EDCs 10.1. How Machine Learning Works 10.2. Examples of Current Deep Learning Programs for Toxicology

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1. INTRODUCTION Physiological homeostasis is largely maintained by actions of hormones that regulate reproduction, growth, metabolism, and a myriad of other biological processes. The past century has seen rapid progress in our understanding of how hormones are synthesized in endocrine tissues, circulate to target cells and tissues, bind their cognate receptors, and ultimately regulate cellular activity. Starting with data published in the 1950s, it became clear that various aspects of endocrine signaling could be altered by many natural and synthetic chemicals collectively referred to as endocrine-disrupting chemicals (EDCs). EDCs are defined as substances in the environment, food, or various manufactured or pharmaceutical products that are capable of mimicking or inhibiting normal hormone effects and do so through numerous modes of action that can involve hormone biosynthesis, release and transport, and steroid hormone and nonsteroid hormone receptor binding and/or metabolism (Figure 12.1). EDCs can pose significant health risk to humans and animals, including wildlife, but there has been ongoing controversy about the magnitude of this threat. In this chapter, we provide an overview of EDCs and their effects. We will discuss the current state of knowledge with regard to EDCs, their actions, and health implications. We have also focused on recent literature describing new models for assessing endocrine disruption, as well as new endocrine pathways that have been recently shown to be targeted by such chemicals. In addition, we provide an overview of current methods used to test chemicals for endocrine-disrupting activity. Perhaps most critically, we highlight future testing strategies for putative EDCs that would use the powerful

11. Conclusions

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References

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emerging “omics” and computational technologies to effectively assess endocrine-disrupting activity of compounds before they are widely disseminated. These emerging tools are expected to allow society to take a prospective, integrated approach to evaluate current and emerging EDCs, rather than the reactive approach that has typified industrial and governmental regulations over the last few decades.

2. ENVIRONMENTAL CHEMICALS CAN DISRUPT ENDOCRINE SIGNALING 2.1. History of Endocrine Disruptor Research Over seven decades ago, Bennetts and Underwood (1951) reported that consumption of one type of red clover by sheep in Australia produced estrogenic effects that impaired reproduction in these animals. This finding that certain chemicals contained in food could mimic endogenous steroid hormones such as estrogens was groundbreaking. However, as with many novel discoveries, the implications of the findings were initially not fully appreciated and the idea that chemicals in the environment could alter normal endocrine signaling in animals did not attract widespread scientific attention. Further findings over the next decade indicated that some insecticides and other environmental chemicals could also have estrogenic effects in animals, including birds and mammals; this resulted in a growing list of chemicals that had endocrine-disrupting activity. Significant interest was initially focused on the ability of the widely used insecticide DDT to disrupt reproduction in wildlife as documented

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FIGURE 12.1 Various mechanisms have been described by which endocrine-disrupting chemicals (EDCs) can alter hormonal signaling pathways. These include EDC binding to hormone receptors to induce agonistic or antagonistic activities, effects on hormone production through transcriptional effects on hormone production as well as changes in steroidogenic enzymes involved in hormone synthesis, alterations in hormone transport and metabolism, effects on coactivators involved in hormone responses, and changes in hormone receptor synthesis or degradation.

in Rachel Carson’s classic book Silent Spring in 1962 (Carson, 1962). Many people credit this work with launching the environmental movement in the 1960s that resulted in the creation of the US Environmental Protection Agency (EPA) and enactment of various laws designed to regulate potentially harmful chemicals. During the subsequent decades, it became clear that many types of endocrine signals, involving both steroid and nonsteroid hormones, could be disrupted by such compounds. Scientific awareness of environmental endocrine disruptors and their effects were greatly increased by the advocacy and organizational efforts of the late Dr. Theo Colborn. She had initially examined disruption of endocrine and reproductive systems in Great Lakes wildlife by environmental chemicals (Colborn, 1991), and later

organized the Wingspread conferences on EDCs in the environment (Colborn et al., 1993) that catalyzed public and scientific awareness of potential health impacts of these chemicals in man and other animals. The demonstration by Dr. Lou Guillette, Jr., and colleagues that environmental pollutants in the water of Lake Apopka in Florida could alter sexual differentiation of resident alligators further galvanized both the scientific community and the lay public and increased awareness of potential EDC hazards (Guillette et al., 2007). Since work in the EDC field now spans well over half a century, the methodologies used in this area reflect the long historical progression of our technical capabilities. Initial studies typically involved in vivo exposure of animals to EDCs, either as a result of natural exposure in

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the environment or in laboratory settings. As scientific techniques advanced, in vitro studies have complemented in vivo approaches. The rise of molecular techniques over the past few decades has provided new and powerful tools to assess EDCs and their mechanisms of action. More recently, high-throughput analyses (e.g., RNA-seq) have been utilized extensively and proven especially useful when analyzing effects of well-characterized EDCs, namely phthalates and bisphenol A (BPA). Both of these types of chemicals are promiscuous and capable of altering numerous endocrine pathways, and the ability to assess global effects of EDCs that target more than one endocrine pathway has allowed more accurate understandings of the totality of the effects of an EDC, in contrast to older initial studies that typically examined effects on one endpoint or signaling pathway. It is estimated that over 100,000 chemicals are in use in our society, with over 1000 new ones introduced each year. There are over 35,000 registered pesticides in the United States and 3000 approved food additives, and approximately 50 new pharmaceuticals are approved for use each year. All of these chemicals are potential EDCs. Historically, studies initially focused on the ability of one chemical or a small number of related chemicals, for example, a group of pesticides, to disrupt a particular endocrine pathway or endpoint. The extent of our chemical exposure, the large number of chemicals with known EDC activity, and the extensive number of endocrine pathways that can be potentially disrupted by EDCs have driven examining EDC actions with a highthroughput standardized panel of tests to allow EDC effects of any compound to be effectively compared with similar classes of chemicals. The endocrine disruptor screening program, Tox21 programs and CLARITY-BPA Consortium project that brought together FDA scientists and independent investigators, discussed subsequently in this section, are responses to these types of needs that were born out of a need to address critical gaps in our understanding and allow for a more global and integrated assessment of EDC activity.

2.2. Types of Chemicals With EndocrineDisrupting Activity Initial work focused on synthetic chemicals such as pesticides and industrial pollutants (e.g., polychlorinated biphenyls, PCBs) that disrupted endocrine activity. However, a wide variety of chemicals and pharmaceutical products, including those in personal care products, widely used industrial products, and especially food, are capable of altering endocrine signaling. Many of the initial studies in this area examined estrogenic and antiestrogenic effects of EDCs on reproduction, but work from laboratories across the globe has indicated that EDCs in the environment can affect an ever-increasing list of important physiological processes, including metabolism, growth, reproduction, and immune function. Reviews in the area of endocrine disruption have historically attempted to provide comprehensive lists of compounds with this type of activity and a discussion of their effects and mechanisms of action (Cooke et al., 2013). As the EDC literature has expanded, it has become increasingly difficult to list and describe in a concise manner all of the chemicals with ascribed EDC activity. For example, a recent review (Kiyama and Wada-Kiyama, 2015) reported that over 450 chemicals with estrogenic or antiestrogenic effects are known. Also challenging is the increasing awareness that individual chemicals can simultaneously perturb more than one endocrine signaling pathway, making categorization increasingly more difficult. Accordingly, this chapter will not attempt to provide an exhaustive list of EDCs or even those that affect a particular endocrine pathway, but rather point out a number of excellent reviews of EDC activity on specific endocrine signaling pathways that have been published. The interested reader is referred to such articles for a more complete discussion and listing of EDCs that have been shown to have specific types of activity such as the disruption of estrogen and other steroid receptor and nonsteroid receptor signaling pathways (Henley and Korach, 2010).

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2.3. Routes of Exposure to Endocrine Disruptors We are exposed daily to EDCs and other chemicals in our environment through various routes. One of the most important routes of exposure is dietary. Many compounds that can mimic or inhibit the action of various hormones are contained in food. For example, genistein, daidzein, and a number of other isoflavones are present in high quantities in soy and other foods. Consumption of isoflavones by human populations in some countries, such as Japan, is up to 1 mg of isoflavones/kg of body weight/day. Furthermore, human infants given widely used soy-based infant formulas may take in an order of magnitude more isoflavones per day than even adults eating a diet rich in soy because this is initially the infant’s sole source of nutrition. The most abundant isoflavone in soy, genistein, has a marked structural resemblance to E2 (Figure 12.2). This results in significant affinity of genistein for estrogen receptor 2 (ESR2) and to a lesser extent estrogen receptor 1 (ESR1). In addition to EDC compounds that are natural constituents of food, pesticide residues in food and even the plastic packaging used for many food items can be sources of EDCs. The most ubiquitous EDCs in food are often associated with disruption of the estrogen signaling pathway, but human diets can also contain compounds that may affect androgen, thyroid hormone and glucocorticoid signaling, as well as other endocrine pathways. Exposure to EDCs may occur through drinking water, where more than 700 chemical contaminants have been identified. Although we tend to consider man-made compounds being introduced into drinking water and having potential EDC activity, natural arsenic contamination of groundwater used for drinking is a significant problem in certain parts of the United States, in particular New Jersey and West Virginia, and certain other countries (e.g., Bangladesh), and arsenic is a known endocrine disruptor (see Metals, Vol 2, Chap 27, for more information on arsenic). The EDCs in drinking water can also be chemicals or by-products of chemicals used in the water purification process. Industrial and agricultural chemicals and pesticides, many of which have EDC activity, are introduced into water supplies through a variety of routes such as agricultural run-off, direct pollution of bodies of water, and leaching from abandoned industrial sites and chemical depots. However, this list of

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potential EDCs in drinking water also includes pharmaceuticals such as estrogens from birth control pills and Prozac (fluoxetine; a selective serotonin reuptake inhibitor prescribed to treat depression), and other chemicals. These can originate from sewage contamination of some natural water supplies and they are not removed during water purification processes. A classic example is that measurable levels of the estrogen 17a-ethinylestradiol (EE2) derived from birth control pills can be found in municipal water supplies around the world, and this pharmaceutical is suspected of causing endocrine disruption in fish and other aquatic wildlife. This ubiquitous pharmaceutical has been reported to disrupt steroidogenesis, gonadal maturation, and fertility in diverse aquatic vertebrates and can even induce an intersex condition in fish, which is the presence of both male and female gametes in the gonad simultaneously (Williams et al., 2009). Perhaps most surprising is that low ng/L EE2 is sufficient to cause population level declines in fish. This was demonstrated in lake experiments in Canada which were treated with w5 ng/L EE2 over several years (Kidd et al., 2007). The exposure in the lake caused a complete collapse of the local fathead minnow population in only a few years. On a positive note, the fathead minnow population was able to rebound after removal of the EE2. This experiment is a sobering example of how a widely used pharmaceutical can have devastating effects on wildlife populations at concentrations detected in municipal wastewater effluent. In addition to the estrogen component of the birth control pill, there are studies showing the progestin component may also have effects on aquatic species (Fent, 2015). For example, levonorgestrel, a potent progestin used in birth control pills, completely stopped spawning of fathead minnows at an aqueous concentration of 100 ng/ L (Runnalls et al., 2013). Other progestins been shown to have deleterious effects on reproduction and interestingly also have masculinizing effects in female fish in a dose–responsive manner. A 30 min exposure of fathead minnows to 5 ng/L EE2 or 100 ng/L levonorgestrel altered phosphorylation patterns of proteins in the brain involved in neurogenesis and synaptic activity. Each chemical also produced unique alterations, with nervous system development, synaptic transmission, and neuroprotection altered by EE2 and axon cargo transport and calcium ion homeostasis altered by levonorgestrel (Smith et al., 2018).

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FIGURE 12.2 ToxCast bioanalytical plots for bisphenol A (BPA), genistein, methoxychlor, and vinclozolin, all known endocrine disruptors. The Y-axis (scaled top) represents the highest response of each assay divided by the activity cutoff of the assay to be able to represent all assays in the same graph. X-axis is the AC50 (mM) for each assay. The data for these plots can be found on the EPA chemistry dashboard, https://comptox.epa. gov/dashboard. Chemical structures for BPA, genistein, methoxychlor, and vinclozolin were generated with http://molview.org/.

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Some pharmaceuticals may have more subtle effects on endocrine systems that may not manifest until several generations after the initial exposure. Fluoxetine is a highly prescribed pharmaceutical that is sometimes given to pregnant women to combat depression and anxiety. In a study in zebrafish, parents were exposed to fluoxetine and the F3 generation showed an impaired response of the interrenal gland to adrenocorticotropic hormone injections, suggesting that the stress response was epigenetically modified and less robust than in unexposed fish. Such exposures may have important implications for future generations in terms of cortisol and stress responses for descendants, who are exposed to this chemical only through their progenitors (Vera-Chang et al., 2018). Potential translation to humans remains to be seen, but it is clear that increased maternal usage of chemicals such as fluoxetine is associated with pathological changes, such as persistent pulmonary hypertension, in their sons and daughters (Chambers et al., 2006). In addition to ingestion of EDCs in food, water, or items such as personal care products, inhalation and skin (transdermal) contact can expose humans to a wide variety of endocrine disruptors that can be present in the soil, sediment, or dust. These EDCs are constituents of products we use every day, such as the plasticizer BPA that is present in plastic bottles or flame retardants that are used in furniture upholstery, mattresses, carpets, and various household electrical devices.

2.4. Regulatory Approaches to Endocrine Disruption Regulatory approaches to address endocrine disruption started in the mid-1990s in various countries around the world, as it became apparent that there were contaminants in the environment that behaved as estrogens and for which guidelines at the time were unable to address. The efforts in the United States were spearheaded by the EPA with the formation of the EDSTAC (Endocrine Disruptor Screening and Testing Advisory Committee) to help advise on methods to identify EDCs that could cause adverse effects. Japan’s Environment Agency initiated a similar effort, Strategic Programs on Endocrine Disruptors (SPEED), and Europe initiated the European Regulation on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) program. These efforts came together under the auspices of the Organization for Economic Cooperation and Development

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(OECD), an intergovernmental organization charged with devising harmonized protocols across countries to regulate chemicals with EDC activity. In the United States, EDSTAC was formed by the EPA in 1996 in response to Congress’ mandate to develop a testing strategy for chemicals that were likely EDCs. The committee included members from government, academia, and industry. Because of the link between sex steroid hormones and thyroid hormones with cancer, this committee determined they would focus only on three types of EDCs, those that disrupted estrogen, androgen, and thyroid (EAT) hormones. This committee was charged with developing a framework that could lead to regulatory decisions regarding the effects of exposure to chemicals that mimicked these activities or that altered the biosynthesis or metabolism of endogenous hormones. The final report of the committee is available at the EPA website (EDSTAC, 1998). Initial strategies focused on a two-tiered screening and testing system. The first tier included five in vitro assays (to assess mechanistic information such as receptor binding and activity) and six short-term in vivo assays, such as change in organ weight (e.g., uterus, prostate) in rats. The second tier included longer-term assays in mammals, fish, amphibians, and birds and needed to include apical endpoints that showed adverse reactions leading to morbidity or mortality. Since those early years, much has changed in this area. First, in the early 2000s, the US National Research Council came out with a proposal to change how toxicity assessments were done and suggested to predominantly use mechanistic data and cell-based assays. The European Union passed the REACH legislation in 2006 limiting the use of animals for toxicity testing. These two ideas were the impetus needed for the EPA (via Toxcast) and the National Institutes of Health (NIH) (via Tox21) to design a multitude of cell-based assays that could bin chemicals into activity groups. It was clear that over 100,000 chemicals needed to be tested for endocrine disruption, a feat that was impossible with in vivo testing due to cost and time. The high-throughput assays originally developed targeted EDCs with potential effects on the EAT hormones. In the first trial, 310 chemicals, primarily pesticides, were tested with 700 different assays (Kavlock et al., 2012). The selected chemicals had been extensively studied and served as tests of the new approach. Since then, tens of thousands of chemicals have been tested. Data generated for each chemical are publicly

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available. The best resource is the EPA’s CompTox Chemical Dashboard (https://comptox.epa. gov/dashboard) where, to date, information for about 875,000 chemicals can be found. Four chemicals, including BPA, methoxychlor, vinclozolin, and genistein, were randomly selected to illustrate the type of bioanalytical data available on the Dashboard (Figure 12.2). As can be seen in the figure, the structures of these four chemicals are quite disparate, making it hard to predict EDC activity based on chemical structures. In the figure, the Y-axis (scaled TOP) is the highest response of each assay divided by the activity cutoff of the assay to be able to plot all the reactions on the same graph. The X-axis contains the AC50s (concentration at 50% of maximum activity) for each assay. Only assays that relate to hormone receptors and steroid hormones are plotted in these graphs, except for BPA, where Cyp1A1 and Cyp1A2 data were included to illustrate their very low AC50 values. Additional activities for each of these chemicals are presented in the Dashboard. It is clear from this graph that BPA can not only transactivate ESR1 and ESR2 but can also interact with other hormone receptors at higher BPA concentrations, perhaps explaining the various apical endpoints associated with BPA exposure. Methoxychlor, an organochlorine pesticide, is known for having both estrogenic and antiandrogenic activity in vivo. This is amply demonstrated by the activity assays, where interactions with both estrogen and androgen receptors (ARs) can be quantified. Genistein, a phytoestrogen, is known to transactivate ESR1 and ESR2, as shown in this graph. More controversial in the literature is the fungicide vinclozolin. Experiments with rats show it to be antiandrogenic (Monosson et al., 1999), and as demonstrated by the assays it does interact with AR. It should be stressed, however, that the assays are still limited to known receptors and do not test all possible mechanisms of action. Initial efforts with high-throughput assays for EAT have shown excellent results that can be linked directly to observable effects of exposure to a toxic chemical in a test animal, which are termed apical endpoints. Large harmonizing efforts between Japan, the United States (EPA), and Europe (OECD) have streamlined the approach. First efforts focused on estrogens, but now there are large efforts focusing on androgens and thyroid hormones. Clearly other hormonal systems should also be examined soon.

In 2010, Ankley proposed linking molecular initiating events to apical endpoints via the Adverse Outcome Pathway (AOP) paradigm (Ankley et al., 2010). The AOP, first described in fish, but now accepted in human health, posits that chemicals will first interact with a biological organism at the molecular level, initiating a cascade of events through increasingly more complex interactions at the cellular and organ levels that culminate in morbidity or mortality, both adverse apical outcomes. Extensive testing has gone into this paradigm both in human and environmental health. The AOP wiki where researchers can add to the knowledge base can be accessed through https://aopwiki.org. Despite the importance of the EAT hormones, it is increasingly apparent that this focus left many other endocrine-related endpoints unexamined. At present, there are large international efforts to address these shortcomings. For example, one recent global effort to address EDCs that disrupt metabolism and metabolic functions has materialized. The European “GOLIATH” (Generation of Novel, Integrated and Internationally Harmonized Approaches for Testing Metabolism Disrupting Chemicals) project is bringing together scientists from around the world to find suitable testing protocols for metabolic disruption (Legler et al., 2020). The plan is to develop easy-to-use in vitro assays as a screening mechanism and to integrate both in vitro and in vivo assays with endpoints that are clearly adverse for metabolism. It is likely that OMICS technologies will play a prominent role in this endeavor and will allow us to obtain greater understanding about contaminants that influence obesity, nonalcoholic fatty liver disease, and diabetes, all of which have been increasing over the years (see Food and Toxicologic Pathology, Vol 3, Chap 2). Chemicals that are known to be active in this regard include BPA, tributyltin, perfluorinated chemicals, and triphenyl phosphate, among others. The commonalities between environmental and human health have not escaped the purview of scientists in the field, who are now defining the similarities and developing unified approaches. One international attempt to do so funded by the European Union goes under the acronym ERGO (Endocrine Guideline Optimization) and is focused on thyroid-related endpoints across vertebrate classes. Much work is being

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focused on other endocrine pathways to integrate novel molecular technologies into risk assessment paradigms. There are still many research gaps that are difficult to address with current technology. For example, most contaminants are present as mixtures. While we are finding out important information for each contaminant alone, the contaminants may behave differently as constituents of mixtures, especially when agonists and antagonists are mixed. Another issue is species extrapolation. Novel methods targeting amino acid homologies within active sites among receptors for different species show great promise (LaLone et al., 2016), but there are still many issues including species-specific absorption and metabolism rates that may influence species sensitivity. Lastly, there is still the challenge for the regulatory community to make clear decisions on what to do if a chemical test is positive in a high-throughput assay. Many Tier 2 assays have not been validated and there is still ongoing discussion about their use.

3. MECHANISMS OF ENDOCRINE DISRUPTION Several environmental compounds with EDC activity described by early studies in this field induced their actions, at least in part, by binding to and signaling through estrogen receptors (ESR1 and ESR2). Compounds such as DDT and its breakdown product dichlorodiphenyldichloroethylene (DDE), estrogenic compounds in clover, and various estrogenic pesticides and fungicides induce similar transcriptional changes in target cells as those induced by endogenous estrogens. Further work has identified chemicals that exhibited antiestrogenic activity (e.g., certain PCB congeners). Other reports indicate that a wide variety of processes related to hormone production, transport, and action can be altered by EDCs (Figure 12.1). Subsequent work showed that EDCs can affect other steroid and nonsteroid signaling pathways. The list of mechanisms by which EDCs can act is still expanding, but there are examples of EDCs that affect gene expression in target cells by altering coactivator levels that are critical for both steroid hormone and thyroid hormone signaling. An underexplored mechanism of EDC effects is that they can act as allosteric modulators. This entails them binding to a different site on a receptor than the natural

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and endogenous ligands (agonists), but in so doing, EDCs can alter the shape of the receptor such that it impacts their affinity and efficacy to bind other substances, including their cognate hormones. EDCs may act as positive (lead to increase binding affinity/efficacy of agonistic compounds for the receptor), neutral (no effect on affinity or efficacy), or negative (reduced binding affinity/efficacy of agonistic compounds for the receptor) modulators. For instance, genistein and other flavonoids appear to act as positive allosteric modulators for a7 nicotinic receptors, which may be useful in treating neurodegenerative, inflammatory, and cognitive disorders (Nielsen et al., 2019). There are numerous examples of EDCs that alter hormone receptor concentrations as a result of altering receptor synthesis or degradation, resulting in endocrine disruption. Other mechanisms of EDC action are the inhibition or stimulation of enzymes involved in steroidogenesis and alterations in hormone metabolism and signaling. The enzyme aromatase, which aromatizes androgens to produce estrogens, is a common target of various types of EDCs. For example, the herbicide atrazine as well as the pesticides chlordane and methoxychlor are all capable of stimulating aromatase and increasing estrogen biosynthesis. New EDC regulators of aromatase continue to be discovered, including the commonly used pediatric human anesthetic sevoflurane, whose endocrinedisrupting actions involving stimulation of aromatase activity are described later in this chapter.

4. EXAMPLES OF DISRUPTION OF ENDOCRINE PATHWAYS BY SOME ENVIRONMENTAL CONTAMINANTS AND EMERGING ENDOCRINE DISRUPTORS The study of EDC actions is a dynamic and still evolving field. In some cases, new mechanisms of endocrine disruption are being revealed for chemicals that have other well-established EDC actions. In other cases, recent evidence has suggested that chemicals long suspected of having EDC activity do indeed function as endocrine disruptors. Finally, novel and frequently surprising EDC actions continue to be documented for widely used chemicals, where no previous literature exists which suggests endocrine-disrupting activity. Three examples that exemplify these points are discussed in the sections below.

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4.1. Phthalates Disrupt Several Endocrine Pathways In recent years, it has become clear that some environmental chemicals such as phthalates are capable of disrupting several endocrine pathways, and the EDC activity of these chemicals will be discussed to highlight the broad and complex effects on hormonal signaling by some chemicals. Phthalate plasticizers or esters of phthalic anhydride are of continuing concern for environmental exposures and are a broad ubiquitous class of EDC with complex actions. Phthalates are used in polyvinyl chloride and as additives and softeners for plastic products because they increase flexibility, durability, and longevity of the product. The global population is continuously exposed to phthalates due to their use in plastic toys, personal care products (e.g., lotions and make-up), medical devices, and plastic food packaging. In general, phthalates are not covalently linked to plastic polymers and can therefore leach from products over time. Exposure to phthalates can occur through ingestion of food containing leached plasticizers, dermal contact, and/or inhalation. Some of the more widely used phthalates are di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate, but there are different subcategories with a diversity of chemical modifications and properties (e.g., butyl benzyl phthalate, dibutyl phthalate, di-n-octyl phthalate, dipentyl phthalate, and dicyclohexyl phthalate, among many others). Phthalates exert reproductive and developmental effects in vertebrates, acting as antiandrogenic chemicals to impair both the female and male reproductive systems (Hannon and Flaws, 2015; Pallotti et al., 2020). More recently, phthalates have been shown to activate peroxisome proliferator–activated receptors (PPARs), which are transcription factors that regulate genes involved in lipid homeostasis. As such, phthalates have been designated “obesogens” because of their relatively high binding affinity for PPARs (Goodman et al., 2014). Studies suggest that fatty acids and eicosanoids are natural ligands for PPARs, and reports have shown that phthalates have a propensity to mimic these molecules to cause inappropriate signaling in the metabolic axis. This mechanism of endocrine disruption has significant implications for hormonal regulation of the feeding axis. For example, PPARs regulate neurohormones in the central nervous system that control feeding behavior (e.g., agouti-related protein and neuropeptide Y). Phthalates in the blood have

also been correlated to higher body weight in children and to circulating levels of leptin, a hormone released from adipocytes to regulate food intake. While phthalates have been identified as EDCs of concern for some time, novel mechanisms for endocrine disruption continue to be revealed. This has high global relevance for the growing obesity epidemic.

4.2. Emerging Endocrine Disruptors: Glyphosate Although EDC activity of many compounds has been known for decades, important additions to our list of EDCs are still occurring. One of these with significant human health implications is glyphosate, widely sold under the Roundup brand. Glyphosate was first used in 1974 and has become the most widely used herbicide in the world, with estimates of 8.6 billion kg applied worldwide from 1974 to 2020. Its original uses on genetically modified (GMO) crops have expanded dramatically into areas not originally envisioned by Monsanto, the company that first developed and marketed this compound. It is widely used in noncommercial settings by home gardeners, for example, who use it for weed control on lawns and in gardens. It is also used in various other farming applications, such as sugar cane production, where its use has been associated with a 20% increase in sugar production. The classification of glyphosate as an EDC has been controversial in the field. It was classified as a probable carcinogen in 2015 by the International Agency for Research on Cancer but this was disputed by others, including the European Food Safety Agency that claimed that it was neither a carcinogen nor an EDC (see Agrochemicals, Vol 3, Chap 11). However, a recent study in mice (Pham et al., 2019) suggests it functions as an EDC and that it can affect spermatogenesis. In this study, pregnant mice were treated with glyphosate at three low doses, each differing from the previous one by a factor of 10 and ranging from the acceptable daily intake for humans of 0.5 mg/kg/day to 50 mg/kg/day (the no-observed-adverse-effect level, NOAEL). The treatment was in the drinking water and started in utero from embryonic day 10.5 to weaning at about 20 days postpartum, which corresponded to the last day of lactation. Control and treated mice were sacrificed and dissected at 5, 20, and 35 days old. In 35-day-old mice, there was no change in body weight, but the investigators found the testis weight to be significantly lower in the 0.5 and

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5 mg/kg/day group and they also found significantly lower numbers of spermatozoa per epididymis but this was not found for the 50 mg/kg/ day group. They also found serum testosterone levels to be significantly lower in the 0.5 and 50 mg/kg/day groups but the reduction was not significant for the 5 mg/kg/day group. The work was supported by histology and analysis of alterations of expression for genes involved in spermatogonia differentiation. This study is supported by other published studies showing that exposures to glyphosate or its commercial formulation in utero affect serum testosterone levels and spermatozoa in male pups (Dallegrave et al., 2007; Romano et al., 2010) but the conclusions continue to be controversial as other published studies using high doses of glyphosate (500 mg/kg/day) appear to refute the findings (Dai et al., 2016; Johansson et al., 2018).

4.3. Emerging Endocrine Disruptors: General Anesthetics as Endocrine Disruptors Mounting evidence indicates that general anesthetics (GAs), the most frequently used pharmacological agents in clinical practice, can be added to the growing list of chemicals that interfere with endocrine signaling. It has been increasingly recognized that the effects of GAs are not completely reversible upon anesthesia withdrawal. Clinical studies report learning disabilities, long-term memory impairment, and attention-deficit/hyperactivity disorder in those who had early-life procedures requiring anesthesia, especially in those who had multiple procedures. Laboratory studies in healthy animals that were exposed just to GAs in the absence of surgical procedures support the hypothesis that GAs themselves can induce neurodevelopmental abnormalities. Based primarily on such laboratory findings, the US Food and Drug Administration (FDA) advises that when possible, general anesthesia should be avoided by pregnant women and children under 3 years of age. The full range of early-life anesthesia-induced abnormalities and their underlying mechanisms remain poorly understood (Martynyuk et al., 2020). Laboratory findings indicate that GAs, whose mechanisms of actions include positive modulation of GABA type A receptor (GABAAR)

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signaling, may induce neurodevelopmental abnormalities, at least in part, by acting as potent stressors and endocrine disruptors. Among such GAs are the inhalational anesthetic sevoflurane, which is widely used and whose polyvalent actions include enhancement of GABAAR activity, and propofol, an intravenous GA with a relatively selective GABAAR-mediated action. These GAs increase circulating corticosterone concentrations and electroencephalography (EEG)-detectable seizures in neonatal rats at the time of anesthesia. Adult rats neonatally exposed to sevoflurane exhibit behavioral abnormalities and exacerbated hypothalamic–pituitary–adrenal axis responses to stress (Li et al., 2020; Wang et al., 2020; Zhang et al., 2016). Sevoflurane-induced exacerbation of E2signaling pathways at the time of anesthesia may play an important mediating role in acute and long-term developmental effects of the anesthetic. Exposure to sevoflurane has been shown to increase serum levels of E2 and hypothalamic mRNA levels for aromatase and Esr1, but not Esr2, in neonatal rats. These sevofluranemediated effects, including long-term behavioral abnormalities and exacerbated HPA axis responses to stress, were alleviated by an aromatase inhibitor that blocks conversion of androgens to estrogens. Importantly, exogenous E2 further potentiated EEG-detectable sevofluraneinduced seizures and heightened serum corticosterone concentrations, while blocking ESR1, but not ESR2, signaling reduced sevoflurane-caused seizures (Li et al., 2020; Wang et al., 2020; Zhang et al., 2016). Dysregulated neuroendocrine responses to stress, programmed by adverse experiences especially early in life, are an underlying feature of many neurodevelopmental and neuropsychiatric disorders. Recent studies suggest that the sevoflurane-induced increase in corticosterone levels at the time of anesthesia can also be involved in initiating intergenerational neurobehavioral effects of the anesthetic. In these studies, sevoflurane was administered to neonatal or young adult rats. Rats neonatally exposed to sevoflurane were mated in adulthood to generate offspring, while rats exposed to sevoflurane in young adulthood were mated 25 days after anesthetic exposure. Sevoflurane increased serum corticosterone at the time of anesthesia in neonatal and young adult male and female rats.

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The findings of these studies suggest that sevoflurane-induced acute corticosterone secretion is involved in the anesthetic-induced epigenetic reprogramming of parental germ cells, leading to epigenomic, transcriptomic, and neurobehavioral abnormalities in progeny (Ju et al., 2018, 2019). The EDC effects of sevoflurane and other GABAergic anesthetics, which may include an epigenetic component, require further investigation, as such effects may have long-term health consequences for more than one generation. The epigenetic effects of EDCs are discussed in Section 5. Interestingly, sevoflurane does appear on the EPA ToxCast Dashboard, but it had no activity in the 79 assays reported, indicating the need in further research of the EDC effects of sevoflurane and other anesthetics.

5. EPIGENETIC EFFECTS OF EDCS Endocrine signaling can be altered by EDCs to produce short-term physiological changes in target organs. For example, a weak environmental estrogen may bind to nuclear and membrane estrogen receptors to stimulate cellular alterations, such as uterine epithelial proliferation, and work on EDCs initially focused on their physiological actions. Over the past two decades, it has become abundantly clear that in addition to short-term effects, EDCs can also induce permanent and even transgenerational effects due to epigenetic changes. An epigenetic change is one that through mitotic or meiotic propagation causes heritable changes in gene expression patterns that may result in phenotypic alterations but does so without altering the DNA sequence itself. This area is currently one of the most topical in the EDC field. Several epigenetic mechanisms have been characterized. The most widely studied is DNA methylation, which occurs when a methyl group (CH3) is attached to a cytosine nucleotide preceding a guanine nucleotide (CpG); this type of cytosine methylation, frequently in promoter regions of genes, is associated with transcriptional repression. Epigenetic effects of EDCs can also be produced through chromatin remodeling. Histones, the proteins that DNA wraps around to form chromatin, are also

involved in epigenetic regulation as they can bind less or more tightly to DNA and thereby affect the ability of transcription factors to access the promoter region and initiate transcription. Histones can undergo various types of modifications, such as methylation, acetylation, phosphorylation, ubiquitylation, and other alterations. The precise effect of these modifications depends on the nature of the modification (e.g., methylation, acetylation), and also on the specific amino acid site in the histones where that modification occurs, with methylation of some histone sites always having an inhibitory effect of gene transcription (H3K27) while other methylations (e.g., H3K4) stimulate gene transcription. Small noncoding RNA (ncRNA) can also be involved in epigenetic effects. These ncRNAs are functional RNAs that alter the expression of specific genes by binding to mRNA to block or promote degradation, thus inhibiting translation into protein. This mechanism, first discovered in 1993, may be a critical aspect of transgenerational inheritance, and it has been suggested that microRNAs induced by EDCs may be involved in this process. Epigenetic modifications can target specific genes or alter global gene expression patterns. For example, DNA methylation targets specific sites located near promoter regions of genes; as nucleotides become methylated, the biomolecular changes block transcription factors from accessing their promoter binding sites and transcription of those genes is subsequently repressed. Epigenetic factors also regulate spatial conformation of chromatin, regulating gene expression more globally. Some noncoding RNAs participate in the condensation of chromatin, as well as enzyme complexes that chemically alter the histones of the chromosomes and change their configuration, thus making it more or less accessible for transcription. Our understanding of long-term epigenetic effects and the impacts they could have on an organism have been driven in part from an increased understanding of how adverse events during development can exert delayed effects during adulthood. In the 1990s, the late British epidemiologist Sir David Barker discovered that a baby’s birthweight correlated decades later with the risk of these adults developing cardiovascular disease and other metabolic abnormalities, and this concept was originally termed the

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Barker hypothesis (Barker, 1995). This idea, now called developmental origins of health and disease (DOHaD), links early environmental insults to disease in adults. It has been widely adopted to explain many EDC-mediated epigenetic effects in developing animals and their long-term health consequences. This term though also recognizes that in utero conditions may promote later offspring health, suggestive that adoption of health habits, such as eating well and exercising, by mothers (and fathers) may potentially mitigate harmful effects induced by early exposure to EDCs. The initial link between EDCs and epigenetic changes was provided by the report that exposing neonatal mice to high concentrations of the synthetic estrogen diethylstilbestrol (DES) caused altered methylation of the lactoferrin gene promoter and affected gene activity (Li et al., 1997). Exposure to the xenoestrogen DES or BPA, an EDC that binds to estrogen receptors but also acts as an antiandrogen, in utero caused a greater than two-fold increase in mammary tissue expression of Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase that produces epigenetic alterations in gene expression and has known associations with tumorigenesis. These types of effects have also been seen with EDCs that disrupt androgen signaling. Administration of the antiandrogenic fungicide vinclozolin to rodent fetuses resulted in adult-onset reproductive and other abnormalities (Anway et al., 2005), and these effects were observed in subsequent generations, suggesting epigenetic changes. Jirtle and coworkers (Dolinoy et al., 2007) showed that exposing fetal mice to BPA altered DNA methylation of an endogenous retroviral promoter sequence called an intracisternal A particle (IAP) embedded in the agouti gene. This IAP contains its own promoter site that usurps control of transcription of the agouti gene from its normal promoter region. As it is a viral particle, it is not regulated by normal transcriptional factor regulation. Instead, methylation of this IAP promoter site silences this region and allows for the agouti gene to be once again regulated by its own promoter region. In the demethylated state induced by BPA, the IAP promoter results in constitutive and systemic expression, leading to mice with

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yellow coat colors that are susceptible to metabolic disorders as the agouti signaling protein is expressed in the hypothalamus (resulting in hyperphagia), pancreas, and adipose tissues. However, fetal coexposure to BPA and genistein results in methylation/silencing of this IAP promoter site and mice that have a brown coat color remain healthy (Dolinoy et al., 2007). Although initial work on the epigenetic effects of a variety of EDCs focused on actions involving disruption of reproductive hormone signaling and endpoints, EDCs can act on a wide number of cell types and gene targets through epigenetic mechanisms. For example, arsenic, a known endocrine disruptor, has been shown to produce hypermethylation in the promoter region of a tumor suppressor gene in leukocytes of both the mother and fetus. Additionally, arsenic exposure of pregnant women in Bangladesh, where arsenic contamination of groundwater is extensive, produced epigenetic changes in the placenta and notably decreased PR/SET Domain 6 (Prdm6) expression, a histone methyltransferase associated with congenital heart defects (Rosenfeld, 2017). Prenatal administration of synthetic glucocorticoids given to women to prevent preterm birth increased DNA methylation and also changed histone acetylation in the offspring, and this effect was transmitted to subsequent generations. Additionally, animal studies show that fetal exposure to synthetic glucocorticoids increases the risk of behavioral, endocrine, and metabolic abnormalities in the offspring through epigenetic mechanisms. Exposure to an EDC can produce epigenetic alterations resulting in phenotypic changes for the duration of that individual’s lifetime or even transgenerational effects. This latter type of effect results from epigenetic modifications in the germline capable of being passed to subsequent generations. In situations where there have been transgenerational effects, the epigenetic change in subsequent generations can be independent of the original EDC that caused the original epigenetic change. In other words, even if descendants are not directly exposed to a given EDC, they can be at risk due to ancestral exposure. This germline-dependent mechanism of inheritance results in individuals with epigenetic changes resulting from exposure of an ancestor to an EDC, even though they were never exposed

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to this agent. An example of this type of transgenerational effect has been observed with BPA exposure. Progeny of pregnant dams supplemented with 5 mg BPA/kg diet were evaluated for social behavior and neural expression of proteins related to endocrine signaling in the F1, F2, and F4 generations. An effect on social behavior was observed extending through the F4 generation. Expression of the vasopressin and oxytocin genes was lower across generations, while expression of estrogen receptors was reduced only in generations exposed directly to BPA (Wolstenholme et al., 2012). The precise mechanism of transgenerational epigenetic effects induced by EDCs or other agents is not clearly understood, but likely involves noncoding RNA, as gametes undergo a genomic reset where primordial germ cells are stripped of epigenetic marks during the transmission through generations.

6. FROM REACTIVE TO PROACTIVE ENDOCRINE DISRUPTOR ANALYSIS Over the last three decades, the number of known EDCs has risen dramatically. However, the most critical question related to the human and animal health consequences resulting from EDC exposure is still being debated. Human and animal populations are continuously exposed to a wide and increasing variety of potential EDCs, and further clarity is needed regarding the potential health implications of these compounds now and in the future. A significant challenge to overcome is that EDC activities have historically been described after a compound is in widespread use, and it is indeed the extensive consumer use that typically drives research to establish potential EDC activity of a chemical or group of chemicals. When endocrine-disrupting activity is identified, this then leads to sometimes difficult and often contentious cost–benefit analyses where the benefit of withdrawing a compound from use is weighed against the risks of continuing to use this compound. A classic example of this is the plasticizer BPA, which was widely used for years before the extent of its endocrine-disrupting activity became clear. Other earlier examples are the insecticide DDT and the PCBs, which

were used extensively for years, but following the identification of endocrine-disrupting activity, these have been largely banned. In some cases, compounds introduced as a substitute for DDT, such as methoxychlor, were themselves found to have endocrine-disrupting activity and were subsequently prohibited. However, some of these compounds such as DDT and PCBs are notoriously persistent and measurable terrestrial and aquatic concentrations of these compounds are still evident many years after they have been discontinued, clearly illustrating the shortcomings of establishing potential endocrine disruptor activity after a compound is in broad use. Clearly, a different strategy is needed to identify new and existing chemicals that may have EDC actions. To proactively assess potential EDC activity of the many thousands of new chemicals that enter our environment each year, there is increasing need to adopt novel approaches for testing, in addition to retrospective techniques that have been historically used for decades. These efforts require new model systems for testing EDC activity that will provide the combination of high-throughput capabilities at a reasonable cost. The availability of omic technology and state-of-the-art bioinformatics and computational methods, especially machine learning, will be powerful tools that should facilitate efforts to proactively identify endocrine disruptors. Finally, machine learning and the emerging field of deep learning may provide new ways to predictively identify compounds with endocrine-disrupting activities. As such, the remaining sections of this chapter focus on the techniques predicted to transform identification of EDC compounds in coming decades.

7. EMERGING MODELS IN EDC RESEARCH 7.1. Zebrafish Model New animal models and gene editing approaches are advancing EDC research at an impressive rate. Zebrafish (Danio rerio) have become a significant alternative vertebrate model for environmental toxicology and for studying human diseases involving the

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endocrine system (see Animal Models in Toxicologic Research: Nonmammalian, Vol 1, Chap 22). The zebrafish genome is well annotated and arranged in 25 chromosomes with a size of 1.7
109 base pairs, which is about half the size of the human genome but with many genes orthologous to human counterparts. Because zebrafish are vertebrates, EDCs target pathways conserved in higher mammals. This animal model is gaining wide acceptance at NIH and globally because this model is inexpensive and compatible with high-throughput designs. Zebrafish are also a powerful species for studying EDCs because of their sensitivity to estrogens, in particular for brain aromatase and vitellogenin. An example of zebrafish as a biosensor for EDCs effects is given below. Using traditional molecular biology techniques, Brion and collaborators (2012) developed a unique transgenic zebrafish as a bioindicator for estrogens. In mammals, the aromatase enzyme that converts testosterone into E2 is expressed in the gonads, brain, placenta, adipose tissue, skin, and endometrium and its expression is controlled by tissue-specific promoters. In zebrafish, because of a complete gene duplication event that occurred 350 million years ago in the fish lineage, there are two aromatase genes. One, exclusively expressed in brain, is called brain aromatase, while the other is found in multiple tissues. Brion et al. (2012) placed the green fluorescent protein (GFP) behind the brain aromatase promoter, which is highly sensitive to estrogens in zebrafish. These zebrafish embryos expressed GFP in the presence of estrogenic EDCs. This fish model has been used in a high-throughput manner to detect estrogenic contaminants in waterways in Europe.

7.2. CRISPR Screening In addition to the zebrafish model, another approach that promises to revolutionize EDC research is that of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR-associated protein-9 nuclease) mediated gene editing. This method can be used to extend our understanding of gene–environment interaction through CRISPR screening of EDCs. In principle, any model organism can have specific genes removed from (or added to)

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the genome. The efficiency and precision of these gene editing tools facilitate their use in a wide array of organisms used in toxicology studies. Briefly, short fragments of RNA or single guide RNA (sgRNA) are added to the cell to signal site-specific DNA recognition by Cas9 nuclease. The Cas9 enzyme complexes with the guide at the site to cleave the double-stranded DNA, which is then subsequently repaired by the nonhomologous end joining pathway. There is a diversity of modified CRISPR systems, each with individual strengths and weaknesses depending upon their application. The method can be used to (1) modify gene transcription, (2) determine the role of epigenetic marks via point mutations or gene silencing, (3) label a gene in situ for microscopy, and (4) induce genome rearrangements to understand evolution or coexpression networks. New applications for CRISPR are being rapidly developed. Detailed discussions on CRISPR technologies are beyond the scope of this chapter but individuals are encouraged to seek resources on these applications (Lino et al., 2018). Similar to the example above with aromatase, CRISPR gene editing has been used to modify the zebrafish genome to create a biosensor for EDCs. Using CRISPR “knock-in”, Abdelmoneim and colleagues (2020) generated an in vivo sensor for estrogenic chemicals. A gene construct that contained enhanced GFP, under the control of the regulatory region of vitellogenin 1 (vtg1), was inserted into the zebrafish genome. Zebrafish larvae exposed to xenoestrogens, such as BPA, were shown to fluoresce as activated ESRs bound the regulatory region and activated the expression of the gene. The authors demonstrated proof of concept that CRISPR knock-in in biological systems may be useful as highthroughput screening tools to test estrogenicity in water samples and effluents. Similar approaches can be used for other EDC biomarkers, for example, spiggin, a male reproductive hormone in fish, which is a bioindicator for androgenic compounds. Fertilized zebrafish embryos can be injected easily with sgRNA and Cas9 at the one cell stage to edit specific genes. Genetic screening using zebrafish and other animal models are expected to reveal new mechanisms underlying EDCs. Another application for gene editing includes genome-wide CRISPR screens for toxicants and

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EDCs. Cells are engineered to contain a single gene knockout via a specific sgRNA, and these individual cells can be pooled together to create a knockout library of the genome. The cells can then be challenged by a suspected or known EDC and the resulting phenotype measured. Over time, the chemical exposure will alter the representation/proportions of the genetic pool as cells multiply and divide. Sequencing the transcriptome of the resulting pool of cells allows one to elucidate which genes are associated with resistance or sensitivity to the chemical, revealing which genes are important for a phenotype. Functional genomics can therefore identify new pathways related to EDCs. A CRISPR/Cas9 genome-wide screen in human neuronal cells was conducted for the legacy pesticide dieldrin (Russo et al., 2020). This organochlorine pesticide has EDC effects and can activate estrogenic pathways. On the horizon is a novel approach called Perturb-seq, which combines both single-cell RNA sequencing (RNA-seq) and CRISPR-based perturbations to build integrated gene models that can be used to understand how chemicals perturb endocrine signaling. Moving forward, this approach may be highly valuable for studying endocrine cells, using endocrine disruptors to modify, for example, ovarian follicular cells or testicular Leydig cells to understand which genes are responsive to xenoestrogen perturbation.

8. OMICS TECHNOLOGIES TO EVALUATE ENDOCRINE DISRUPTION 8.1. Transcriptomics and Proteomics Applications for transcriptomics, the study of the complete set of transcripts within a cell or tissue, in toxicology has increased exponentially over the past decade. RNA sequencing is now readily accessible to researchers and these molecular tools have yielded novel insights into adverse effects related to EDCs. EDCs have diverse effects on multiple hormone systems, binding with varying degrees of affinity to an array of nuclear hormone receptors which transduce signals to the genome. Once these transcription factors bind their hormone response elements within DNA promoters, a suite of genes

are activated or inhibited downstream to maintain physiological homeostasis. Elucidating how the transcriptome responds to EDCs is vital to understand the cellular phenotypic response to a chemical. Processes underlying key phenomena in toxicology have been partially revealed with transcriptomics, such as the effects of low-dose exposures to EDCs and their molecular mechanisms. For example, nonmonotonic dose–response effects have been elucidated for BPA and mechanisms underlying different endocrine disruptors (e.g., triclosan, BPA, and fluorene-9-bisphenol) have been revealed using RNA-seq, suggesting that EDCs can perturb lipid metabolism in vivo. This information can be relevant for understanding impacts on steroid hormone biosynthesis. One major objective for regulatory toxicology is to integrate omics data into the adverse outcome pathway (AOP) framework. Integration of omics data is expected to increase predictive capacity, uncover new EDCs, identify points of departure for low level effects, and prioritize chemical testing. The idea is that the transcriptome can elucidate molecular initiating events (MIEs) and key events (KEs) that improve the annotation of AOPs. For example, a transcriptome experiment that identifies altered transcript levels of genes related to thyroid hormone biosynthesis or signaling may suggest direct interactions of an EDC with thyroid hormone receptors. These efforts have recently resulted in AOP development for EDCs such as the nonionic surfactant nonylphenol. As with any emerging science, incorporating omics into regulatory frameworks and risk assessment is not without challenges. Transcriptome responses are highly complex, time- and dose-specific, and subject to biological variability. Thus, much discussion and research remain for standardization, implementation, and interpretation (see Toxicogenomics: A Practical Primer for the Toxicologic Pathologist, Vol 1, Chap 15). New sequencing strategies that include singlecell RNA-seq (scRNA-seq) are poised to characterize transcriptomes in cells of the endocrine system. Kanaya and colleagues (2019) investigated the reorganization of the mammary gland in response to E2 and flame retardants (polybrominated diphenyl ethers, PBDEs). The data indicated that PBDEs, together with E2, modulated

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the immune system in the mammary gland and regulated a number of cytokines and inflammatory markers. One might envision similar studies in endocrine cells of organisms exposed to EDCs. For example, scRNA-seq may reveal novel signaling pathways in the pituitary when focused on a specific cell type such as gonadotrophs or thyrotrophs. The recently released Spatial Transcriptomics analyses method from 10
Genomics that permits examination of gene expression patterns in the context of whole tissue will permit fine-tuned assessments on how EDCs affect individual cell populations and how neighboring cells and tissue might influence each other. A final point to make is that mechanisms of transcriptional regulation are highly complex, and posttranscriptional modifications can include microRNAs (miRNAs) and long noncoding RNA molecules, as mentioned above. Small RNA-seq analyses can be used to screen how exposure to EDCs affect miRNAs and other small RNAs, such as was recently done in the hypothalamus of California mice (Peromyscus californicus) developmentally exposed to BPA and/or genistein (Kaur et al., 2021). Target mRNAs of differentially expressed miRs can then be determined. However, transcriptomics offers but a glimpse into hormone action. One must also keep in mind that proteins are the functional unit of the cell, and act to conduct enzymatic reactions or to function as receptors, signaling molecules or membrane transporters to maintain ionic balance across cellular membranes. Different methodologies to measure the proteome, or the complete protein complement of the cells, include both label free (e.g., spectral counting) and peptide labeling methods (e.g., isobaric tagging for relative and absolute quantitation or iTRAQ) to facilitate relative quantitation of proteins using mass spectrometry. A detailed discussion on the use of proteomics in toxicology is beyond the scope of this chapter but proteomics has been used successfully to identify candidate biomarkers related to endocrine disruptors. Moving forward, there are exciting bioinformatics and computational approaches that can integrate omics datasets into meaningful outputs to improve mechanistic understanding of EDCs. Some of these approaches are described below.

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8.2. Lipidomics and Metabolomics Small molecule omics disciplines, namely lipidomics and metabolomics, have been increasingly used in EDC research in recent years. Lipids (i.e., nonpolar metabolites) and watersoluble metabolites such as sugars, amino acids, nucleotides, vitamins, hormones, and various reaction intermediates are ubiquitously expressed across tissues, individuals, and species for both eukaryotes and prokaryotes. These vital biomolecules are involved in a myriad of diverse physiological and pathophysiological processes and are highly conserved across living organisms. As such, they have been increasingly recognized as not only important end-products of biochemical reactions and mechanisms such as cell signaling, membrane structure, and energy storage, but also as potential biomarkers of health and disease. Lipidomics and metabolomics are complementary fields. In studies investigating effects of environmental or experimental perturbations on normal cellular function (e.g., chemical/ contaminant exposure, radiation, environmental stress, or other stimuli), the integration of lipidomics and metabolomics data has been shown to contribute orthogonal perspectives, thus providing a more comprehensive and representative snapshot of cellular dyshomeostasis. This includes identification of multiple related cellular mechanisms that are affected in parallel and the release of endocrine, paracrine, or autocrine signals that elicit a cascade of responses in lipid and/or metabolite pathways such as inflammation, as well as in disease etiology through the interpretation of these data within epidemiological studies. Lipids and metabolites are often measured using mass spectrometry (MS) due to its high sensitivity and selectivity, as well as its ability to monitor hundreds to thousands of compounds in a wide range of tissues and matrices. MS-based lipidomics and metabolomics applications can be broadly divided into two categories (targeted and nontargeted) based on the research question that guides the analytical strategy, both of which can be applied to comparative studies or for cross-sectional profiling. In targeted studies, there is generally a suspected list of biomolecules that are anticipated to change in response to the experimental

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treatment (i.e., exposure). These can be referred to as hypothesis-driven studies. Conversely, in hypothesis-driving studies, there is an interest in capturing data from as many molecules as possible to discover novel pathways or compounds. Nontargeted data present a particular analytical challenge as compared with targeted data since the nontargeted readouts contain mass spectral information (i.e., massto-charge ratios (m/z) of precursor and fragment ions) in addition to abundance (i.e., ion intensities or chromatographic peak areas) for thousands of ion features. Automated software must be used to deconvolute, integrate, identify, and annotate lipid and metabolite structures. Publicly and commercially available databases currently contain spectra for hundreds of thousands of structures, and many are amenable to process data from multiple MS platforms, vendors, and data formats (e.g., datadependent and data-independent acquisition). However, in nontargeted analyses, compound identifications are predominantly putative (determined using precursor accurate mass, fragmentation behavior, and chromatographic retention time) resulting in an increased propensity for false positives (i.e., incorrect identifications) and false negatives (i.e., misannotations) as compared with targeted assays. Moreover, out of the thousands of ion features that are detected using nontargeted workflows, only a small fraction of these (typically less than 10%) are successfully identified and annotated. This is largely due to the fact that most current databases are built in silico using structures from human and rodent samples, as well as other model species (many of which are mammals). Nevertheless, lipid and metabolite libraries are still expanding to include structures from nonmodel species such as aquatic animals, reptiles, amphibians, and various bacterial cell lines. This is expected to increase the coverage of analyte identifications in future studies, while enabling the retrospective interrogation of previously acquired nontargeted data. In addition to such developments and improvements in bioinformatics resources, there has been increasing adoption of advanced statistical strategies and data handling tools to mine through and interpret nontargeted lipidomics and metabolomics results. Nontargeted

integrative multiomics data (including metabolomics, lipidomics, transcriptomics, proteomics, and genomics) can be finally harmonized and interpreted within a systems biology perspective through pathway analysis. This provides the most comprehensive and unified representation of the state of a cell, tissue, or organism. Targeted and nontargeted lipidomics/metabolomics are generally applied in EDC research through three primary approaches. Firstly, for measurement of biomolecules known to be affected by common EDCs. Secondly, for elucidation of novel pathways affected by known EDCs. Thirdly, for characterization of effects of novel/ emerging EDCs on EDC-associated pathways. Much of the research on the effects of EDCs like xenoestrogens on lipid/metabolite homeostasis has traditionally focused on the former two, with a particular interest on applying these tools to study mechanisms such as the cross talk between nuclear hormone receptors. Initial studies indicated, for example, cross talk between ESR1 and the aryl hydrocarbon receptor. One mechanism was attributed to overlapping binding sites for receptors in promoters of susceptible genes. However, the cross talk goes beyond simple steric hindrance. For example, in cross talk between estrogen signaling and lipid biosynthesis, it is now evident from multiple transcriptomics studies that estrogen upregulates expression of genes involved in lipid transport and in lipid biosynthesis. One of the genes upregulated by E2 is sphingosine kinase 1, which catalyzes phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). This is an important lipid mediator that regulates cell proliferation and activation of NF-kb and, thus, links estrogen signaling to inflammation and the immune system. Another important cross talk is between ER signaling and PPAR-g, where it has been observed that PPAR-g-specific ligands inhibit signaling in human leiomyomas. These examples demonstrate that the endocrine system is intricately coordinated and that endocrine disruptors can have pleiotropic effects at various points through this web of interactions. Beyond EDCs, the novel concept of metabolism-disrupting chemicals (MDCs) has been recently introduced. The MDC hypothesis expands on the scope of a traditional EDC (i.e., substances that interfere with hormone biosynthesis, metabolism, or action) by proposing that

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environmental chemicals (many of which have also historically been considered EDCs) can affect metabolism in a broader sense. This includes obesogens such as nicotine, BPA, and polycyclic aromatic hydrocarbons, insulindesensitizing agents such as phthalates and tetrachlorodibenzo-p-dioxin, as well as other chemicals like persistent organic pollutants that lead to the onset and progression of metabolic syndrome. As many of these chemicals are also known EDCs, their roles have previously been examined from an EDC standpoint. However, as the fields of lipidomics and metabolomics continue to grow, systemic effects of MDCs can be better understood, and therapeutic strategies can be more effectively designed. Furthermore, as technological advancements continue to be made in analytical chemistry, targeted and nontargeted workflows can be applied to identify and screen for novel contaminants of increasing health concern.

8.3. Microbiome The microbiome is broadly defined as the complete repertoire of symbiotic and pathogenic microorganisms within a tissue or host. Skewing of microbiota populations to pathogenic bacteria is considered dysbiosis. However, it should be kept in mind that most bacteria are considered commensal and even provide beneficial functions, such as synthesis of many B vitamins and other factors that humans and other animals cannot produce alone. Microbiota are present, for example, on skin and in the gastrointestinal and reproductive systems and oral-nasal cavities, among other tissues. These communities serve important functions within the host, regulating all aspects of organismal physiology. Each tissue houses a unique microbiota and the local environment can modify microbe diversity and richness. The primary routes of exposure for chemical toxicants include ingestion and absorption through the skin. As such, the gastrointestinal and epidermal microbiota are presumably sensitive to environmental chemicals. It is important to note that the relationship between microbiota and chemicals is reciprocal, and while chemicals can exert toxicity to beneficial microbiota, they can also be metabolized by resident bacteria into beneficial or harmful metabolites to the host (see Digestive System, Vol 4, Chap 1)

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The microbiome has been investigated in different animal models exposed to EDCs such as triclosan and perfluorinated compounds. For the purposes of brevity, we focus on estrogenic and antiandrogenic chemicals below when giving examples and discuss recent experiments in fish and mammals. Microbiota studies are relevant across taxa as many endocrine disruptors such as BPA and phthalates are detected in aquatic ecosystems, as well as indoor/outdoor environments. A number of excellent reviews provide an overview on the state of the science for understanding how the microbiota are perturbed by environmental toxicants and endocrine disruptors (Adamovsky et al., 2018; Rosenfeld, 2017). The effects of exogenous estrogens, BPA, and DEHP treatments have been assessed in zebrafish recently. Male zebrafish reared with E2 (2 mg/L) or BPA (2 mg/L) have unique microbiota compared to control males and females and microbiota of fish given estrogenic chemicals were more similar to each other (Liu et al., 2016) and thus fish gastrointestinal microbiota are sensitive to EDC. Other experiments showed that BPA altered the zebrafish gastrointestinal microbiota (Table 12.1). Dietary exposure to the antiandrogenic plasticizer DEHP leads to adverse responses in the zebrafish gastrointestinal system related to adaptive immunity and more specifically T cell activation and signaling (Adamovsky et al., 2018; Buerger et al., 2020). This dietary exposure was also accompanied by relative increases in Fusobacteriia and Betaproteobacteria and revealed significant interplay between host-microbiota during dietary exposure. In a second study, it was revealed that DEHP disrupts microbial community networks, which may be detrimental to gastrointestinal function (Buerger et al., 2020). The ability of microbial communities to metabolize plasticizers is currently unknown but is a significant knowledge gap. Developmental and adult exposure to EDCs affects the gut microbiome in various mammalian species. In studies done in the Rosenfeld laboratory, developmental and adult exposure to BPA and EE2 affected the microbiome of California mice (P. californicus) parents and their offspring, but different bacteria were altered in each generation (Javurek et al., 2016). Select bacteria, including Bacteroides, Mollicutes, Prevotellaceae, Erysipelotrichaceae, Akkermansia,

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Changes in the Microbiome After Exposure to Plastics or Plasticizers That Act as EDCs. For Each Study, the Species and Sex Used is Given (M, Males; F, females), along with Life Stage Examined, Sample Type Analyzed for Microbial Profiles, Chemical Exposure (BPA, Bisphenol A; DEHP, Di(2-Ethylhexyl) Phthalate; EE2, 17a-Ethinylestradiol; GEN, Genistein), Dose, Duration, Effects on the Gut Microbiota, and Any Host Effects if Measured.

Species

Sex

Stage

Sample

Chemical

Dose

Duration

Effects on Microbiome

Zebrafish (Danio rerio)

M/F

Adult

Fecal

BPA

0.2e2 mg/L in water

3 months

• Increased abundance of pathogenic bacteria of bacteria related to denitrification systems

• Oxidative damage

Reference Chen et al. (2018)

and inflammation

• Dose- and sex-dependent responses

• Altered composition of microbial communities II. SELECTED TOXICANT CLASSES

Zebrafish

M/F

Larvae Adult

Larvae

BPA Others

Doseeresponse

10 days postfertilization

• Decrease in community structure

• Chemicals had differential effects on microbial composition Zebrafish

M

Adult

Fecal

BPA

2 mg/L in water

5 weeks

• Gut dysbiosis and altered communities

Zebrafish

M/F

Adult

Fecal

DEHP

w3 mg/kg in feed

60 days

• Turnover of microbes related to immunomodulation

• Inverse relationship bet-

Catron et al. (2019)

ween host toxicity and microbiota composition

• No behavioral effects

• Increased muscle

Liu et al. (2016)

triglycerides

• No detectable gut

Adamovsky et al. (2020)

pathology

• Induction of gene networks related to inflammation

Zebrafish

M/F

Larvae Adult

Fecal

DEHP

0e100 mg/L in water

3.5 months

• Shift in microbial communities

• Sex-specific responses of microbial communities Zebrafish

M/F

Adult

Fecal

DEHP

w4 mg/kg in feed

60 days

• Chemical fragmented community structure of microbiota

• Immune genes altered

Jia et al. (2021)

in the gut

• Goblet cell number decreased

• Sex differences noted • Not assessed

Buerger et al. (2020)

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• Decreased abundance

Effects on Host (If Measured)

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TABLE 12.1

California mice (Peromyscus californicus)

M/F

Adult weaning

Fecal

BPA EE2

50 mg/kg feed weight for BPA and 0.1 ppb for EE2

• Adultsddirect • Gut dysbiosis with exposure, periconception through postnatal period

• Not assessed

Javurek et al. (2016)

• Gut microbiota

Kaur et al. (2020), Marshall et al. (2019)

specific bacterial changes

• Generational- and sexdependent differences

• Offspringdpreand postnatal exposure M/F

Adult weaning

Fecal

BPA GEN

5 and 50 mg/kg feed weight for BPA and 250 mg/kg feed weight for GEN

• Adultsddirect • Gut exposure, periconception through postnatal period

dysbiosis specific bacterial changes

with

correlated with changes in fecal metabolites and neurobehavioral alterations

• Age- and sexdependent differences

• OffspringdpreII. SELECTED TOXICANT CLASSES

and postnatal exposure Mice (Mus musculus)

M/F

Adult

Fecal

GEN

20 mg/kg body weight

Dams: Gestational day 7 to PND 21

PND 90 Assessment

• Femalesd bacterial signature pattern associated with proinflammatory response

• Females showed

Huang et al. (2018)

enhanced inflammation

• Males exposed to GEN showed reduced inflammation

• Malesdmicrobial profile typical of antiinflammatory response Rabbits (Oryctolagus cuniculus domesticus)

M/F

Weaning

Fecal

BPA

200 mg/kg of body weight/ day to dose

Dose: Gestation day 15 (midgestation) through postnatal day 7

• BPA exposure leads to

• BPA reduced short-

unique reductions in select bacteria in the dose compared to offspring

chain fatty acid levels Increased systemic lipopolysaccharide levels

•

Reddivari et al. (2017)

• Increased intestinal permeability Dogs (Canis lupus familiaris)

M/F

Adult

Fecal

BPA

No direct exposure

Two weeks of eating one of two canned dog foods

• BPA in blood related to bacterial shifts in the gut

• Increasing

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California mice

Koestel et al. (2017)

concentrations of circulating BPA

• Host clinical chemistry altered

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Methanobrevibacter, and Sutterella, whose proportions increase with exposure to BPA (50 mg/kg feed weight) or EE2 (0.1 parts per billion) in one of the generations, are associated with host intestinal and metabolic diseases. Intestinal flora alterations were also linked to changes in various metabolic and other Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Such changes in bacterial metabolites might be one means by which EDC-induced gut dysbiosis impacts the host. Further work with these same mice has shown that developmental exposure to genistein (250 mg/kg feed weight) and BPA (5–50 mg/kg feed weight) can lead to distinct gut bacterial changes. Through use of informatics tools, such as mixOmics analysis, it has been shown that altered gut flora is associated with gut metabolite and neurobehavioral alterations (Kaur et al., 2020; Marshall et al., 2019). When initial studies in California mice were performed, little was known of how EDCs might affect gut microbiota. Subsequently, several other mammalian studies have shown one or more EDCs can affect intestinal bacteria. For example, soy formula diets in pigs are associated with intestinal epithelial lining and resident microbe changes, along with antiinflammatory markers (Yeruva et al., 2016). Mouse model studies using direct pre- or postnatal dietary genistein exposure support the notion that this phytoestrogen alters the gut microbiome, and such alterations are correlated with changes in the metabolome and cognitive functioning (Huang et al., 2018; Lopez et al., 2018; Zhou et al., 2018). Rabbits perinatally exposed to BPA (200 mg/kg body weight orally administered to pregnant does from gestational day 15 through postnatal day 15) have reduced Ruminococcaceae and Oscillospira spp. in the case of those directly exposed to BPA or Odoribacter spp. in BPA-exposed offspring (Reddivari et al., 2017). In dogs, consumption of increasing amounts of BPA in canned food correlates with gut bacterial shifts that in turn are associated with host blood chemistry changes (Koestel et al., 2017). These studies are summarized in Table 12.1. With their trillions of collective genes, gut bacteria can dramatically impact host health and physiology (see Digestive System, Vol 4, Chap 1). Nowhere is this perhaps more true than in the brain. While it was initially shown

that the brain through the vagal nerve could impact gut motility and thereby bacteria collecting in the organ, it is now apparent that this is bidirectional communication, which has given rise to the term the microbiome–gut–brain axis. It implies cross talk between gut microbiota and brain, with intestinal bacteria potentially modulating neurobehavioral responses. How bacteria influence brain and other organs is summarized in Figure 12.3. Some potential mechanisms involve shifts in bacterial metabolites and production of neurotransmitters by these microorganisms, affecting intestinal permeability that could affect bacterial penetration into the bloodstream or vagal nerve and ultimately the brain, enhancing inflammatory cells and their cytokine production, and inducing host epigenetic changes, similar to those detailed above. Multiple factors govern whether bacterial changes ultimately lead to beneficial or detrimental host effects. These include species, sex, age and duration of exposure, other concurrent dietary factors, and specific host responses. The aforementioned studies in fish and mammals only reveal potential associations between gut microbiota/bacterial metabolite change and host effects. Actual causation studies are needed, such as transplanting the fecal microbiome from EDC-exposed individuals into germ-free mice or other species that lack a gut microbiome and then assessing whether similar phenotypic outcomes occur in these animals as were identified in donors directly exposed to the EDC. Such studies are methodologically challenging as germ-free mice or other species must be maintained in specialized housing to prevent any contamination.

8.4. Exposomics Much like lipidomics and metabolomics, exposomics is an emerging field. The “exposome” is defined as the cumulative set of environmental exposures across an individual’s lifespan, beginning in utero. Thus, exposome examinations aim to apply various analytical techniques such as small molecule omics, genomics, and proteomics, among others, in addition to the measurement of chemicals and contaminants in order to better understand the relationship between exposure and health outcomes. Exposomic

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FIGURE 12.3 Model of how EDC-induced changes in gut microbiota can affect host health, including the brain and metabolic tissues. Such extensive bidirectional communication exists between gut flora and the brain that it has given rise to the term the microbiota–gut–brain axis. The figure illustrates that changes in gut microbiota can lead to host effects by a plethora of mechanisms. These include producing factors that induce epigenetic changes in the host, producing harmful factors, with a prime example being lipopolysaccharide, producing metabolites that can affect the host function. Changes in gut microbiota can also trigger host inflammation, causing gut lining cells to separate (otherwise considered increasing “gut leakiness”), allowing pathogenic bacteria to cross this border and access to underlying blood vessels that permit them to circulate throughout the body. Finally, changes in microbiota can also lead to gut bacteria transiting to the brain via the vagal nerve, and altered metabolism that results in more potent forms (e.g., daidzein to S-equol) of some chemicals being produced.

assessments also integrate multiple demographic, geographic, and chronologic tools in addition to various metadata to assess exposure and/or risk. Within the endocrine disruption research framework, exposure to many EDCs is known to

fluctuate across environments (both individual and shared), thus modulating exposomes differently between organisms at different stages of life. The measurement of the totality of one’s exposure is therefore difficult to ascertain, and

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as such, many investigations aim to simplify the number of variables by, for example, focusing on specific stages of development or populations in order to provide more parsimonious discussions. Presently, community initiatives such as the EXPOsOMICS project aim to integrate multiple omic measurements and contaminant analysis to assess individual and population exposure (i.e., characterizing the exposome), to integrate experimental and epidemiological human studies across all life stages, and to apply novel statistical models to interpret internal (i.e., xenobiotics, metabolites) and external exposure measures (i.e., omics) to assess exposure to air pollution and water contamination. Through continued growth of the field, prospective studies on EDCs will focus on characterizing the contributions of both legacy contaminants (e.g., heavy metals, PCBs) and chemicals of increasing concern (e.g., per- and polyfluoroalkyl substances) to the exposome. People are exposed to chemicals on a daily basis, for example, food additives and preservatives, pesticides and fungicides on vegetables and fruits, antibiotics in meat, PFAS and phthalates in cosmetics and personal care products, lubricants, solvents, and plastics, among other numerous examples. These exposures are usually (but not always) at low doses that do not produce immediate health effects, but chronic exposures to these mixtures over years (or a lifetime) may result in disease. While there are many examples of exposures to individual chemicals that can result over time in disease, the idea of interrogating the whole exposome is a relatively novel concept and few concrete examples exist associating the exposome to disease. What is particularly distinct about exposomics is that traditional toxicology data are incorporated with all the morphometric data, geographic data, socioeconomical data, etc., that often are ignored to provide a more complete/comprehensive view of toxicity and its relation to health. Single studies devoted to measuring biomarkers of effects and the chemicals themselves are rapidly growing and studies like these, that blend different data systems/ streams together, will allow us to get a more holistic view of how chemical exposures lead to disease. New toxicological studies that test combinations of chemicals at low doses, below their NOAEL, mimick real-life scenarios to

better simulate actual risk in humans. Some human diseases which may be affected by a lifetime exposure to EDCs include fertility disturbances, obesity, cancer, and diabetes, among others. The effects of chemical mixtures with endocrine activities, each with diverse modes of action, are unpredictable regarding human health. For example, a study showed that EE2 and the pesticide trans-nonachlor were not able to individually activate the pregnane X receptor; however, both together activated the receptor, suggesting these chemicals interacted with each other to ligand with the receptor. Many mixtures contain both agonists and antagonists for sex steroid receptors (and other receptors), and it is unclear how the biological system responds to their combination. Computational methods are being developed to be able to handle the large diversity of chemicals that are found in blood and urine and these studies are being aided by specific mixture studies in vivo and in vitro, to be able to get a handle on how they may work in humans.

9. NEW FRONTIERS IN BIOINFORMATICS AND INTEGRATIVE AND FUNCTIONAL ENRICHMENT OMICS APPROACHES The above studies provide solid evidence that EDCs can induce global changes in the gut microbiome, metabolome, lipidome, transcriptome, and proteome in a variety of species. While such findings are important, it is essential to link them with phenotypic or pathological changes to understand the etiological basis of how such chemicals cause disease. This section provides a brief summary of several programs developed in recent years for addressing this issue and how they have been and may be used going forward for EDC studies.

9.1. Integrative Correlation Analyses To integrate various omics and phenotypic data, one of the most useful programs is the mixOmics R package (Rohart et al., 2017). This approach has been utilized to integrate the effects of BPA and genistein on gut microbiota

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10. MACHINE LEARNING AND EDCS

and fecal metabolome or gene expression changes and host neurobehavioral responses. This program was also used to link BPAinduced changes in placental gene expression, metabolome, neurotransmitter concentrations, and histopathological changes to show that reductions in placental serotonin seen in the BPA-exposed mouse placenta correlate with reductions in certain trophoblast cells, specifically parietal trophoblast giant cells (GCs) that are the most invasive cells in the mouse placenta and contact the underlying uterine myometrium (Figure 12.4) (Mao et al., 2020). An example of mixOmics results from a recent publication (Kaur et al., 2020) that links the effects of BPA on the gut microbiome and metabolome and neurobehavioral responses in California mice (P. californicus) is shown in Figure 12.5. Recently, this approach was extended to identify correlations in BPA responses across organs collected from the same or comparable individual rats within the same CLARITY-BPA consortium study that included 13 independent investigators within the United States who collaborated with the FDA and NCTR (Heindel et al., 2020). In so doing, this approach expanded the findings to a systems biology level to reveal strong organismal relationships at different doses and three different ages ranging from weaning (21 days of age) to older adults (6 months of age). This study demonstrated that mixOmics analyses can be used to integrate various combinations of available multiomics and phenotypic datasets generated as part of EDC studies to examine positive and negative associations. As with any correlation analysis, however, it cannot determine the sequence of changes or establish causation. Even so, follow-up studies can be designed to pinpoint and define the order of biomolecular and phenotypic changes induced by EDCs.

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one needs to go above and beyond such analysis. The IMPRes algorithm and tool is one such tool that allows users to utilize multiomics datasets collected following exposure to endocrine disruptors to conduct in silico hypothesis generation for further testing and validation (Jiang et al., 2018). The IMPRes method utilizes transcriptomics, proteomics, and metabolomics data individually and/or in any combination as evidence and allows users to input genes/ proteins and metabolites based on the investigator’s interest and experience or utilize differential expression lists. It then uses a stepwise active pathway detection method using a pathway detection algorithm that identifies network interactions between proteins and >250 pathways from 7 KEGG pathway categories and protein–protein interactions (PPIs) as a background network. The method narrows down the pathways within the background network and generates resulting output as a tree structure of the most significant and connected genes/proteins/metabolites for different conditions being studied as shown in Figure 12.6. This result depicts the cascade of signal from starting genes/proteins/metabolites of interest to others of significance in the various pathways including those previously unidentified as being significant and ultimately helps build new hypotheses. The IMPRes tool is publicly available at http://digbio.missouri.edu/impres, where users can bring in their own multiomics datasets and run analyses and generate new hypotheses via an interactive website.

10. MACHINE LEARNING AND EDCS Besides data analysis on existing omics approaches as detailed above, another emerging topic in EDC research is predicting unknown EDC properties by utilizing advanced machine learning techniques.

9.2. Integrative MultiOmics Pathway Resolution

10.1. How Machine Learning Works

The above mentioned technique for multiomics data integration is mainly aimed at identifying key changes identified in multiomics datasets. However, to understand the molecular mechanisms at play in studies including endocrine disruptors and to generate new hypotheses,

With the data explosion and computational advancements of recent years, advanced computational approaches such as machine learning have arisen in many research fields such as image processing and natural language processing. “Supervised learning,” a subtype of

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FIGURE 12.4 Histological images of placentas (gestational age 12.5) from control, BPA-exposed, and BPSexposed mice. Panels (A–C) show the three main regions of the fetal mouse placenta: labyrinth (LA), spongiotrophoblast (spongioTB), and GC. The areas of each these regions were determined, as well the ratio of the areas to each other. D) BPA or BPS exposure reduced the spongioTB to GC ratio, suggestive that these trophoblast lineages are vulnerable to BPA/BPS exposure. *(P  .05). N ¼ 10–16 individuals (males and females combined) per group. Adapted under the exclusive license to publish from Mao J, Jain A, Denslow ND, et al.: Bisphenol A and bisphenol S disruptions of the mouse placenta and potential effects on the placenta-brain axis, Proc Natl Acad Sci U S A 117:4642–4652, 2020, Figure 12.4, page 4647.

machine learning, can be applied broadly in EDC studies to predict prospectively chemical toxicity and endocrine-disrupting activity. Machine learning builds a mathematical model of relationships between data and their observed

labels. Usually, the model is initially provided with a dataset with known labels (“Training Set’). The model can then be adjusted by specific rules to optimize performance, making it possible to distinguish labels on these known

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FIGURE 12.5 An example of circos plot correlation figure generated with mixOmics analyses that shows the interrelationships between gut bacterial changes, fecal metabolome alterations, and behavioral parameters in female California mice exposed to a high dose of BPA versus AIN (AIN93G control diet) females. Red lines in the center indicate a positive correlation. In contrast, blue lines indicate a negative correlation. Results for AIN females are indicated with a blue line outside of the circle. Orange line indicates results for upper dose BPA females. The color of the line further from the circle indicates the treatment group where these results are greater. The inset shows a magnified view of GCMS metabolites that were effected by BPA exposure and some of their relationships to bacterial and host phenotypic changes. EPM, elevated plus maze; GCMS, gas chromatography–mass spectrometry analysis; LCMS, liquid chromatography with tandem mass spectrometry analysis. Adapted with permission from Supplementary figure 13 in Kaur S, Sarma SJ, Marshall BL, et al.: Developmental exposure of California mice to endocrine disrupting chemicals and potential effects on the microbiome-gut-brain axis at adulthood, Sci Rep 10:10902, 2020.

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FIGURE 12.6 Workflow for integrative multiomics pathway resolution (IMPRes), showing steps in the methodology along with input datasets and output results.

data by the process of “Training.” The trained model can then be applied to another group of data with a similar set of “features” with unknown labels (“Test Set’). Because the model is built on known experiences of the data–label relationships, it can predict the unknown label in the given Test set. In this process, the machine “learns” the rules and “predicts” the unknown. There are numbers of machine learning methods, including the latest cutting-edge technology called “Deep Learning,” which applies

stacked neural networks for prediction. In EDC studies, machine learning can be utilized to model chemicals with known toxicity (“Training Set’), and then it can predict toxicity of related chemicals based on their structure or other properties (“Test Set’). For example, input data could include all that is known about BPA in terms of physiochemical and receptor binding properties, and then the machine could be asked to predict toxicity of BPA analogues such as bisphenol S. Thus, the major limitation of machine learning

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is that extensive training periods are required with data acquired from well-characterized chemicals before it can be utilized to predict potential chemical toxicity of related chemicals. Even with this limitation, it holds significant promise in the endocrine disruptor field. The next section considers a few machine learning and toxicological programs already in use.

10.2. Examples of Current Deep Learning Programs for Toxicology Numbers of deep learning programs devoted to toxicology have greatly expanded in the past 5 years. In this section, we consider some example programs and how the input data are used to determine potential chemical toxicity. The main ones include eToxPred, chemTox, ProTox, DS TOPKAT, and DeepTox. Of these, the strengths and weaknesses of the first and last will be considered. The eToxPred program (https://github.com/ pulimeng/etoxpred) uses several machine learning algorithms together to predict chemical toxicity. The input data to render such projections include multiple datasets with known compounds, potentially hazardous chemicals, natural products, and synthetic bioactive compounds. According to the program developers, its accuracy of prediction is up to 72% by using a generic model to estimate toxicity directly from the molecular fingerprints of chemical compounds. While eToxPred and similar programs have some utility, they do not assimilate all of the existing data known for a given chemical, including chemical structure, pharmacokinetic activity, binding properties, and known in vivo and in vitro responses, to provide a complete picture of a given chemical’s potential toxicity. Moreover, this and related programs do not consider potential dosedependent and combinatorial effects. How exposure at various ages may influence toxicological responses induced by various chemicals is also not considered. To address this latter possibility, it would require reference data on metabolism of the chemical or related chemical at different ages. To bridge some of these areas, the Tox21 Data Challenge 2014, as detailed previously, was

initiated with the mission of assessing a number of methods to predict the pathobiological effects of a number (12,707) of compounds. Current software approaches were employed to determine those environmental and industrial chemicals that posed the greatest health concerns to humans. In this Challenge, DeepTox (http:// www.bioinf.jku.at/research/DeepTox/) was considered one of the best programs overall based on its predictive performance in several in silico assays. This program creates several chemical descriptors based on normalized chemical representations for the compound(s) interrogated, and these are then used as input data for machine learning. After evaluating several trained models, deep learning is then used to integrate those that are the most accurate, which are then used to build ensembles that can predict the toxicity of other new compounds submitted to DeepTox. This area of research is rapidly evolving, with novel algorithms being developed by machine learning and bioinformatics groups across the world, and thus great strides in this area are predicted in the coming years.

11. CONCLUSIONS From early research on estrogenic chemicals in food and the environment to more recent mechanistic work on ubiquitous and persistent organic pollutants, it is evident that despite our advancing understanding of endocrine disruption pathways, further efforts are needed to characterize effects of many legacy chemicals and novel contaminants on humans, animals, and the environment. New analytical technologies, as well as integration of complementary methodologies such as various omics, advanced molecular biology and gene editing, contaminant analysis, and biochemical/physiological assays, will fuel the growth of EDC research well into the 21st century. Furthermore, evolving computational and machine learning approaches hold the promise of advancing our knowledge of EDCs to the point in the foreseeable future that even before a potential chemical is mass produced and animals and humans are exposed to it, we can predict its potential endocrinedisrupting activity.

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ABBREVIATIONS AC50 Concentration at 50% of maximum activity AMPA a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AOP Adverse outcome pathway AR Androgen receptor BPA Bisphenol A Cas9 CRISPR-associated protein-9 nuclease CEBPb CCAAT enhancer binding protein beta CRISPR Clustered Regularly Interspaced Short Palindromic Repeats DDE Dichlorodiphenyldichloroethylene DEHP Di(2-ethylhexyl) phthalate DES Diethylstilbestrol DOHaD Developmental origins of health and disease E Embryonic day E2 17b-Estradiol EAT Estrogen, androgen, and thyroid hormones EDCs Endocrine-disrupting chemicals EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee EE2 17a-Ethinylestradiol EEG Electroencephalography EFSA European Food Safety Agency EPA Environmental Protection Agency ER Estrogen receptor ERGO Endocrine Guideline Optimization ESR1 and ESR2 Estrogen receptor 1 and 2 (also known as ERa and b) EZH2 Enhancer of Zeste homolog 2 FDA Food and Drug Administration GABAAR GABA type A receptor Gas General anesthetics GFP Green fluorescent protein GMO Genetically modified organisms GOLIATH Generation of Novel, Integrated and Internationally Harmonized Approaches for Testing Metabolism Disrupting Chemicals IAP Intracisternal A particle IARC International Agency for Research on Cancer IL-6 Interleukin-6 IMPRes Integrative multiomics pathway resolution iTRAQ Isobaric tagging for relative and absolute quantitation KCC2 Kþ-2Cl- Cl exporter KE Key event LORR Loss of righting reflex m/z Mass-to-charge ratio MDC Metabolism-disrupting chemical MIE Molecular initiating event MS Mass spectrometry ncRNA Small noncoding RNA NCTR National Center for Toxicological Research NF-kb Nuclear factor-kappa beta NKCC1 Naþ-Kþ-Cl- Cl importer NMDA N-methyl-D-aspartate receptor NOAEL No-observed-adverse-effect level OECD Organization for Economic Cooperation and Development PBDE Polybrominated diphenyl ethers PCBs Polychlorinated biphenyls PPAR Peroxisome proliferator–activated receptor Prdm6 PR/SET Domain 6 REACH Registration, Evaluation, Authorisation and Restriction of Chemicals

S1P Sphingosine-1-phosphate scRNA-seq Single-cell RNA-seq sgRNA Single guide RNA SPEED Strategic Programs on Endocrine Disruptors vtg1 Vitellogenin 1

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bisphenol A: implications for host health in zebrafish, Environ Pollut 234:307–317, 2018. Colborn T: Epidemiology of great lakes bald eagles, J Toxicol Environ Health 33:395–453, 1991. Colborn T, vom Saal FS, Soto AM: Developmental effects of endocrine-disrupting chemicals in wildlife and humans, Environ Health Perspect 101:378–384, 1993. Cooke P, Simon L, Denslow ND: Endocrine disruptors. In Haschek WM, Rousseaux CG, Wallig MA, et al., editors: Haschek and Rousseaux’s Handbook of Toxicologic Pathology, 2013, Academic Press. Dai P, Hu P, Tang J, et al.: Effect of glyphosate on reproductive organs in male rat, Acta Histochem 118:519–526, 2016. Dallegrave E, Mantese FD, Oliveira RT, et al.: Pre-and postnatal toxicity of the commercial glyphosate formulation in Wistar rats, Arch Toxicol 81:665–673, 2007. Dolinoy DC, Huang D, Jirtle RL: Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development, Proc Natl Acad Sci U S A 104:13056–13061, 2007. EDSTAC: Endocrine disruptor screening and testing advisory committee (EDSTAC) final report, Washington, DC, 1998, U.S. Environmental Protection Agency. http://www.epa.gov/endocrinedisruption/endocrine-disruptor-screening-and-testing- adviso ry-committee-edstac-final (Accessed December 2022). Fent K: Progestins as endocrine disrupters in aquatic ecosystems: concentrations, effects and risk assessment, Environ Int 84:115–130, 2015. Goodman M, LaKind JS, Mattison DR: Do phthalates act as obesogens in humans? A systematic review of the epidemiological literature, Crit Rev Toxicol 44:151–175, 2014. Guillette Jr LJ, Edwards TM, Moore BC: Alligators, contaminants and steroid hormones, Environ Sci 14:331–347, 2007. Hannon PR, Flaws JA: The effects of phthalates on the ovary, Front Endocrinol 6:8, 2015. Heindel JJ, Belcher S, Flaws JA, et al.: Data integration, analysis, and interpretation of eight academic CLARITY-BPA studies, Reprod Toxicol 98:29–60, 2020. Henley DV, Korach KS: Physiological effects and mechanisms of action of endocrine disrupting chemicals that alter estrogen signaling, Hormones 9:191–205, 2010. Huang G, Xu J, Cai D, et al.: Exacerbation of type 1 diabetes in perinatally genistein exposed female oon-obese diabetic (NOD) mouse is associated with alterations of gut microbiota and immune homeostasis, Toxicol Sci 165:291–301, 2018. Javurek AB, Spollen WG, Johnson SA, et al.: Effects of exposure to bisphenol A and ethinyl estradiol on the gut microbiota of parents and their offspring in a rodent model, Gut Microb 7:471–485, 2016. Jia PP, Junaid M, Xin GY, Wang Y, Ma YB, Pei DS: Disruption of Intestinal Homeostasis Through Altered Responses of the Microbial Community, Energy Metabolites, and Immune System in Zebrafish After Chronic Exposure to DEHP, Front Microbiol 12:729530, 2021. Jiang Y, Wang D, Xu D, et al.: Integrating gene expression data and pathway knowledge for in silico hypothesis generation

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Lino CA, Harper JC, Carney JP, et al.: Delivering CRISPR: a review of the challenges and approaches, Drug Deliv 25: 1234–1257, 2018. Liu Y, Yao Y, Li H, et al.: Influence of endogenous and exogenous estrogenic endocrine on intestinal microbiota in zebrafish, PLoS One 11:e0163895, 2016. Lopez P, Sanchez M, Perez-Cruz C, et al.: Long-term genistein consumption modifies gut microbiota, improving glucose metabolism, metabolic endotoxemia, and cognitive function in mice fed a high-fat diet, Mol Nutr Food Res 62: e1800313, 2018. Mao J, Jain A, Denslow ND, et al.: Bisphenol A and bisphenol S disruptions of the mouse placenta and potential effects on the placenta-brain axis, Proc Natl Acad Sci U S A 117:4642– 4652, 2020. Marshall BL, Liu Y, Farrington MJ, et al.: Early genistein exposure of California mice and effects on the gut microbiota-brain axis, J Endocrinol 242:139–157, 2019. Martynyuk AE, Ju L-S, Morey TE, et al.: Neuroendocrine, epigenetic, and intergenerational effects of general anesthetics, World J Psychiatr 10:81–94, 2020. Monosson E, Kelce WR, Lambright C, et al.: Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat, Toxicol Ind Health 15:65–79, 1999. Nielsen BE, Bermudez I, Bouzat C: Flavonoids as positive allosteric modulators of a7 nicotinic receptors, Neuropharmacology 160:107794, 2019. Pallotti F, Pelloni M, Gianfrilli D, et al.: Mechanisms of testicular disruption from exposure to bisphenol A and phtalates, J Clin Med 9:471, 2020. Pham TH, Derian L, Kervarrec C, et al.: Perinatal exposure to glyphosate and a glyphosate-based herbicide affect spermatogenesis in mice, Toxicol Sci 169:260–271, 2019. Reddivari L, Veeramachaneni DNR, Walters WA, et al.: Perinatal bisphenol A exposure induces chronic inflammation in rabbit offspring via modulation of gut bacteria and their metabolites, mSystems 2:e00093–17, 2017. Rohart F, Gautier B, Singh A, et al.: mixOmics: an R package for ‘omics feature selection and multiple data integration, PLoS Comput Biol 13:e1005752, 2017. Romano R, Romano M, Bernardi M, et al.: Prepubertal exposure to commercial formulation of the herbicide glyphosate

alters testosterone levels and testicular morphology, Arch Toxicol 84:309–317, 2010. Rosenfeld CS: Gut dysbiosis in animals due to environmental chemical exposures, Front Cell Infect Microbio 7:396, 2017. Runnalls TJ, Beresford N, Losty E, et al.: Several synthetic progestins with different potencies adversely affect reproduction of fish, Environ Sci Technol 47:2077–2084, 2013. Russo M, Sobh A, Zhang P, et al.: Functional pathway identification with crispr/cas9 genome wide gene disruption in human dopaminergic neuronal cells following chronic treatment with dieldrin, Toxicol Sci 176:366–381, 2020. Smith L, Lavelle C, Silva-Sanchez C, et al.: Early phosphoproteomic changes for adverse outcome pathway development in the fathead minnow (Pimephales promelas) brain, Sci Rep 8: 1–14, 2018. Vera-Chang MN, St-Jacques AD, Gagne´ R, et al.: Transgenerational hypocortisolism and behavioral disruption are induced by the antidepressant fluoxetine in male zebrafish Danio rerio, Proc Natl Acad Sci U S A 115:E12435– E12442, 2018. Wang J, Yang B, Ju L, et al.: The estradiol synthesis inhibitor formestane diminishes the ability of sevoflurane to induce neurodevelopmental abnormalities in male rats, Front Syst Neurosci 14, 2020. Williams RJ, Keller VDJ, Johnson AC, et al.: A national risk assessment for intersex in fish arising from steroid estrogens, Environ Toxicol Chem 28:220–230, 2009. Wolstenholme JT, Edwards M, Shetty SRJ, et al.: Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression, Endocrinology 153:3828– 3838, 2012. Yeruva L, Spencer NE, Saraf MK, et al.: Formula diet alters small intestine morphology, microbial abundance and reduces VE-cadherin and IL-10 expression in neonatal porcine model, BMC Gastroenterol 16:40, 2016. Zhang J, Xu C, Puentes DL, et al.: Role of steroids in hyperexcitatory adverse and anesthetic effects of sevoflurane in neonatal rats, Neuroendocrinology 103:440–451, 2016. Zhou L, Xiao X, Zhang Q, et al.: Improved glucose and lipid metabolism in the early life of female offspring by maternal dietary genistein is associated with alterations in the gut microbiota, Front Endocrinol 9:516, 2018.

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13 Nanoparticulates Ann F. Hubbs, Dale W. Porter, Robert R. Mercer, Vincent Castranova, Linda M. Sargent, Krishnan Sriram NIOSH, Centers for Disease Control and Prevention, Morgantown, WV, United States

O U T L I N E 1. Background 1.1. Definitions 1.2. Historical Perspective 1.3. Development of Nanotechnology 1.4. Current and Future Nanotechnology Applications 1.5. Human Exposures

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1. BACKGROUND 1.1. Definitions The National Nanotechnology Initiative (NNI) coordinates federal nanotechnology activities in the United States. The Organization for Economic Cooperation and Development (OECD) is an international organization involved in nanotechnology through their Programme on Manufactured Nanomaterials. In addition, the International Organization for Standardization (ISO) is an international organization with a technical committee involved in standardization of nanotechnologies, Technical Committee 229 Nanotechnologies. The NNI defines nanotechnology as “the understanding and control of matter at dimensions

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between approximately 1 and 100 nm, where unique phenomena enable novel applications. Encompassing nanoscale science, engineering, and technology, nanotechnology involves imaging, measuring, modeling and manipulating matter at this length scale.” This definition of nanotechnology introduces the terminology and the important concept that nanoscale products can accomplish many things not previously possible. Before addressing the potential benefits of nanotechnology, additional definitions may be helpful. The prefix nano commonly confers a meaning of very small or a billionth (Flexner and Hauck, 1993). However, within nanotechnology and related disciplines, the prefix nano is often used to refer to dimensions from 1 to 100 nm. Thus, nanomedicine is the medical

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application of nanotechnology, and nanotoxicology is the study of the toxicology of the products of nanotechnology. The terminology for products of nanotechnology is still evolving and the definitions may affect how specific products are regulated. Thus, precise definitions of common terms such as nanoparticle, nanoparticulate (NP), nanomaterial, nanotechnology, and nano-enabled are subject to debate. The ISO defines the nanoscale as “length range from approximately 1 nm to 100 nm” and defines a nanomaterial as a “material with any external dimension in the nanoscale or having an internal structure or surface structure in the nanoscale” (International Organization for Standardization, 2017). The Scientific Committee on Emerging and Newly Identified Health Risks has proposed that a nanoparticle should be defined as a “discrete entity which has three dimensions of the order of 100 nm or less” (Scientific Committee on Emerging and Newly Identified Health Risks (EU) SCENIHR, 2008). Similarly, the ISO defines a nanoparticle as “a nano-object with all external dimensions in the nanoscale where the lengths of the longest and the shortest axes of the nano-object do not differ significantly” (International Organization for Standardization, 2017). In this chapter, the term NP will be used for a particulate with at least one dimension in the nanoscale range from 1 to 100 nm. As used here, NPs will encompass both solid and liquid NPs.

1.2. Historical Perspective NPs have been present in the human environment for centuries. The National Nanotechnology Initiative website (http://www.nano. gov) maintains a Nanotechnology Timeline (http://www.nano.gov/timeline) which begins with the use of colloidal gold and silver in making dichroic glass in the 4th century. It is the ability to engineer in nanoscale dimensions that is a recent phenomenon and led to the new field known as nanotechnology. This is reflected in the rapid increase in PubMed indexed nanotechnology publications during the past decade. Nanotoxicology and nanomedicine have received increasing attention during the past 15 years, but nanotoxicology publications lag far behind nanotechnology publications (Figure 13.1). Several earlier key discoveries made nanotechnology possible. Among these discoveries were discovery of the electron microscope and the scanning tunneling microscope that led to the 1986 Nobel Prize in physics for Ernst Ruska, Gerd Binnig, and Heinrich Rohrer (Robinson, 1986). The electron microscope revealed ultrastructural details previously unseen by scientists while the scanning tunneling microscope produced atomic scale maps of the surface of biological and inorganic samples and could determine their atomic composition (Binnig and Rohrer, 1987).

FIGURE 13.1 Publications in nanotechnology (dashed black line) began about a decade prior to publications in nanomedicine (dashed blue line). Nanotoxicology publications (dashed purple line) have lagged in both number and time. Figure modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.1, p. 1374, with permission.

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In this same time frame, other research groups were working on a new form of carbon that would soon revolutionize nanoscale threedimensional engineering. In 1981, a group of researchers from Rice University discovered buckminsterfullerene, now also known as the buckyball (Kroto et al., 1985). As indicated by its chemical formula, C60, buckminsterfullerene is comprised solely of carbon. Buckminsterfullerene is shaped like a soccer ball with a sheet of carbon atoms analogous to the leather surface of the soccer ball, so that a cagelike internal structure is formed which is suitable for enclosing specific atoms (Curl and Smalley, 1988). The carbon–carbon bonds are the seams of this molecular soccer ball, and like a soccer ball, regular hexagons and pentagons contribute to its three-dimensional symmetry. As with other carbon compounds, each carbon molecule in C60 forms four bonds with adjacent carbon molecules. This means that some of the carbon molecules are linked by double bonds. Double bonds are near single bonds and other double bonds, so that sites of double bonds between carbons are destabilized. Thus, the hexagons and pentagons that comprised C60 are actually aromatic; a shell of delocalized p electrons surround the internal and external surfaces of C60 and add stability to the structure (Figure 13.2A) (Kroto et al., 1985). C60 was the first characterized member of an amazing new class of carbon compounds devoid of hydrogen and known as fullerenes. This class of carbon compounds is as different from previously described carbon compounds as the carbon compound, coal, is from the carbon compound, diamond. The fullerenes form a surface which is one atom thick. That one atom thick carbon surface can form threedimensional structures of varying shapes. Robert F. Curl, Sir Harold Kroto, and Richard E. Smalley received the 1996 Nobel Prize for this discovery (Curl, 1997; Kroto, 1997; Smalley, 1997). The group of new carbon compounds soon expanded to include carbon nanotubes, which form rolled sheets of carbon hexagons in the shape of single- or multi-walled tubes with a diameter in the nanoscale and a much greater length (Figure 13.2B) (Iijima, 1991; Iijima and Ichihashi, 1993). Like C60, carbon nanotubes have electrons that are used for double bonds

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FIGURE 13.2 NPs are a large and diverse group of particulates sharing the common feature of sizedat least one dimension in the range of 1–100 nm. The top image is a drawing of one of the early products of nanotechnology, a buckyball, which is comprised solely of carbon arranged into pentagons and hexagons to form a sphere that is surrounded by a cloud of electrons which illustrate the aromatic nature of the buckyball. The middle image is an image of another early product of nanotechnology, a single-walled carbon nanotube, which is principally comprised of carbon molecules arranged into hexagons but also contains variable amounts of metals used as catalysts during synthesis. Single-walled carbon nanotubes tend to behave as polyalkenes, which allow chemical additions to the carbon wall. The bottom image is a hypothetical NP intended to illustrate the concept that modern products of nanotechnology can be complex structures made from many different compounds such as a drug core (shown in red) surrounded by a protective coating (shown in gray), a tracer for imaging (shown in blue), and a ligand for cellular receptors to target the delivery. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.2, p. 1376, with permission.

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and can have aromaticity (Lu and Chen, 2005). The chemistry needed to produce the tubelike shape of the carbon nanotubes also used transition metal catalysts, such as iron, which were a variable contaminant of the carbon sheet which comprised the nanotube (Iijima and Ichihashi, 1993; Charlier et al., 1997). Some considered nanotubes to be fullerenes while others considered nanotubes to be a separate group of carbon-based NP (Smalley, 1997; Government Accountability Office as edited by the journal, 2010). As the understanding of C60, carbon nanotubes, and related carbon NPs evolved, so developed the realization that the curved structure and the presence of five- and four-membered carbon rings made some carbon-based NPs more reactive than most aromatic compounds; chemically many carbon-based NPs behaved more like alkenes than aromatic compounds (Taylor and Walton, 1993). As a group, the carbon-based NPs demonstrate nanoscale dimensions, chemical and physical properties that include durability, an ability to conduct electricity, and exhibit a diversity of potential shapes which can modify the chemical and physical properties and have the potential for many commercial applications (Law et al., 2004). However, toxicologists and toxicologic pathologists were at first generally unaware of the emergence of these new engineered materials or the unique properties of some products of nanotechnology. Thus, the investigation of their potential toxicity was not initially a major topic of discussion.

1.3. Development of Nanotechnology The earliest nanotechnology products are known as the first-generation products of nanotechnology and were generally passive structures. The first-generation NPs included C60 and the carbon nanotubes but also included other products that demonstrated the principal of engineering in nanoscale dimensions. These included nanotubes formed from cyclodextrins, nanotubes formed from cyclic polypeptides, DNA stick figures, and DNA arranged in a cube. Additional close relatives of the firstgeneration products of nanotechnology came later but included commercially important products such as modified carbon nanotubes (including carbon nanoribbons and

functionalized carbon nanotubes) and nanotubes made of additional elements (such as silicon). By the early 1990s, a vision of nanotechnology emerged within the scientific community. In November of 1991, the journal Science included a special section called Engineering a Small World: From Atomic Manipulation to Microfabrication with related articles (Appenzeller, 1991; Brauman, 1991; Stroscio and Eigler, 1991; Sundaram et al., 1991). Material science, quantum chemistry, and physics had evolved to the point where control of atomic arrangements and variety of molecular devices were envisioned. The similarity in size between synthetic nanostructures and biological structures, such as viruses, proteins, nucleic acids, and cellular organelles, was recognized. It also became apparent that the noncovalent bonds of proteins and nucleic acids played an important role in determining the three-dimensional structure and selfassembly of these natural biological NPs and that these protein and nucleic acid bonds had a potential role in synthesizing engineered NPs (Whitesides et al., 1991). As noted previously, carbon, DNA, and peptides were among the earliest building blocks for nanotechnology products. The biological precedent indeed demonstrated the feasibility of these noncovalent interactions but can now be viewed as an early indicator that the manufactured NPs might interact with biological structures in unintended ways that could produce toxicity. These early visions of nanotechnology primarily were directed at developing nanodevices. Electronic devices were among the earliest nanodevices. Movement of electrical charge is the basic feature of electrical conductors and movement of electrons is one way to move electrical charge. As mentioned above, the p electrons in C60 were destabilized and it is, with some limitations, an aromatic compound. In single-walled carbon nanotubes (SWCNTs), the mobility of the p electrons and the semiconductor properties depend upon the tube diameter and helicity (Hamada et al., 1992; Mintmire et al., 1992; Smalley, 1997; Ajayan et al., 1999). Thus, SWCNTs could behave as semiconductors or as metallic compounds. This was important because electron flow could be improved by connecting electron-rich with electron-deficient semiconductors (Saito et al., 1992). The role

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of shape in determining the semiconductor properties also meant that this important property could be modified. In 1997, by combining some of the greatest scientific advances of the 1980s, the scanning tunneling microscope was used to micromanipulate and measure the electrical properties of SWCNTs to describe an early nanodevice, an SWCNT nanodiode (Collins et al., 1997).

1.4. Current and Future Nanotechnology Applications By 2004, nanotechnology was already in widespread use in the computer and electronics industries (Service, 2004). In 2007, the Environmental Protection Agency (EPA) noted that nanotechnology products predominantly fell into four groups (Environmental Protection Agency, 2007): (1) Carbon-based materials, such as C60 and the carbon nanotubes (2) Metal-based materials, which are nanomaterials principally comprised of metals (3) Dendrimers, which are NPs formed by branched polymers (4) Composites, which contain different NPs or combinations of NPs and larger materials Within the United States, federal support for research on nanotechnology was $10.5 billion total from fiscal years 2001 through 2009 (Zhu et al., 2003). In 2009, Lux Research, Inc., reported that the automotive industry had the greatest use of “nano-enabled” products with the construction, electronics, healthcare, environment, and energy sectors also using nanotechnology (Lux Research Inc., 2009). The US Patent and Trademark Office reported a 20% annual growth rate for nanotechnology patents between 1985 and 2005. According to StatNano, in 2020, nanotechnology patent applications were 8.5% of the total applications to the US Patent and Trademark Office. Unfortunately, statistics on the use of specific nanotechnology products are incomplete. There are several reasons for that. Under the Toxic Substances Control Act (TSCA), manufacturers of new chemical substances must provide information to Environmental Protection Agency

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(EPA) before the chemical substances enter commerce. The majority of the publicly available information is for high production volume chemicals. In an industry, such as nanotechnology, that is rapidly increasing production and where millions of particles can be in a gram of material, existing reporting has undoubtedly missed most of the activity. Reporting requirements have only recently changed to address some of the products of nanotechnology. In 2017, EPA established reporting requirements that specifically address the production of many solid nanoscale materials. The new requirements are for nanoscale materials that at standard atmospheric pressure are solids at 25
C, have 1% of their mass with one dimension between 1 and 100 nm, and have properties not seen in larger particles of the same composition that are a reason for manufacturing or processing at that size. The United States Government Accountability Office has noted the challenges for federal agencies and Congress when trying to ensure safety of any rapidly evolving technology. Nanomedicine, as mentioned above, is the medical application of nanotechnology. More specifically, as defined by Bawa and coworkers (Bawa et al., 2005), it is “the application of nanoscale technologies to the practice of medicine, namely, for diagnosis, prevention, and treatment of disease and to gain an increased understanding of the complex underlying disease mechanisms.” While a search of PubMed revealed only a handful of publications prior to 2005 (Figure 13.1), a more detailed search for all publications and patents revealed that the earliest nanomedicine publications appeared in the 1990s and that a sharp increase in nanomedicine patents began about a decade later. The reason for the interest is obvious, the potential to improve patient outcomes. Nanoengineering of pharmaceuticals can improve solubility and stability, target delivery, or decrease drug toxicity (also see Pathology in Nonclinical Drug Safety Assessment, Vol 2, Chap 4). For example, NPs often have greater solubility than larger particles. Consistent with that greater solubility, the albumin-conjugated nanoparticle form of paclitaxel, Abraxane (Abraxis, Los Angeles, CA), has greater solubility than previous paclitaxel formulations and does not require organic solvents that can cause

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hypersensitivity reactions in some patients. In addition to greater solubility, the albumin in Abraxane may be targeting the caveolae. Caveolae are invaginations of the vascular endothelium that are present in very high numbers, react with specific target molecules (such as albumin), and form trafficking vesicles that move the material through the endothelium and into the target tissue (Figure 13.3). This is just one example of how nanotechnology has been used to overcome toxicity and improve delivery. Other examples include NPs designed to scavenge b-amyloid within the vasculature as a potential Alzheimer’s disease therapy. Additional new nanomedical concepts and products are rapidly developing. The development of novel nanomaterials with unique physicochemical characteristics suggests the potential use of some nanomaterials in medical imaging (see In Vivo Small Animal Imaging, Vol 1, Chap 13). The small size and large surface area of nanomaterials can facilitate translocation across biological barriers and may enhance cellular and intracellular interactions, features that can be valuable for imaging as well as drug development (Klein et al., 2021). Entire issues of scientific journals have been devoted to therapeutic and diagnostic (“theranostic”) nanomedicine. The surface and the core of the NP can each be engineered to contain multiple components (Figure 13.2C). Each of the components can have important properties. Thus, it is possible to construct NPs that target delivery, are contrast agents, and/or contain a therapeutic payload. The development of NPs designed to cross the blood–brain barrier (BBB) can potentially deliver drugs to treat devastating neurologic disorders. However, there is very little published information available on how these new nanomedical

products will be degraded within cells, such as neurons, that may not have previously been reached by their therapeutic payload or other by-products of nanopharmaceuticals. In other words, drug delivery to the brain should involve traversing the BBB to deliver the drug and safe elimination of the drug. This is also true of any other target tissue or subcellular organelle uniquely being reached by nanopharmaceuticals. As has been noted in recent reviews, nanomedicine has the potential to overcome some of the greatest medical challenges of our time. Nanotechnology is already providing innovative new products for a variety of nonmedical applications. The challenge is trying to harness this promise as safely as possible. The NPs of today can be made from a virtually infinite number of different compounds and combination of compounds. Part of meeting the challenge for safe development of nanotechnology is understanding how nanosizing alters the toxic effects of particulates. This chapter helps to address some of those issues. However, an important aspect of nanomedicine products is that they should be designed and tested for biocompatibility and potential toxicity before their mass production. The findings from such studies can be proprietary and many more nanomedicine products are in development than have reached the market. Data on nanomedicine safety and toxicologic pathology will undoubtedly increase in the future when more is known about the nanomedicine products that do, and those that do not, reach the market. Thus, much of the available data and risk assessment on human exposures focuses on workplace and consumer NP exposures. However, it is important to understand what is currently known about human exposures to NPs.

FIGURE 13.3 Caveolae are invaginations of the vascular endothelium that react with target molecules such as albumin and form trafficking vesicles. This permits NPs with surface target molecules (shown here in orange) to move across the vascular endothelium. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.3, p. 1378, with permission.

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1.5. Human Exposures For workers and consumers, inhalation, dermal exposure, and ingestion are major routes of NP exposure. Inhalation exposures can occur when NPs are aerosolized within the workplace, home, or other environments. In addition to inhalation, aerosolized nanoparticles can be deposited on surfaces, which in turn can result in dermal or even oral exposures. Some consumer spray products can release NPs into the breathing zone of consumers. Once inhaled, NPs can reach the nervous system via the olfactory nerves, sensory nerves of the nose and airway, or the vasculature. Additional tissues, such as the spleen, gastrointestinal tract, and liver, can be exposed to NPs that initially enter the respiratory tract and are translocated to other sites. The known pathways for translocation include the lymphatics, the blood vasculature, neuronal transport, and the gastrointestinal tract (Choi et al., 2010; Hubbs et al., 2013; Mercer et al., 2013). However, the magnitude and frequency of parenteral exposure after inhalation remain controversial and may well depend upon the physiochemical characteristics of the NP. The lung itself is a major target of injury from first-generation NPs, including singlewalled (SWCNT) and multi-walled carbon nanotubes (MWCNTs). Dermal exposure to engineered NPs can occur during workplace exposure, after environmental contamination, or through topical application of NPs in products such as cosmetics, sun protection lotions, or antibacterial lotions. If NPs are released into the environment, they can be incorporated into food and water, and the gastrointestinal tract then becomes a route of exposure. The gastrointestinal tract can also be exposed through incorporation into foods, since nanomaterials are used for food packaging, in food processing and in nutritional supplements (see Food and Toxicologic Pathology, Vol 2, Chap 19). The eyes can also become a route of NP exposure. NP exposures are difficult to measure. Since the lung is a major target for NP toxicity, NP concentrations in air need to be measured. Standard measurements of particulate exposures in air are based upon mass and may then be divided into size classifications. Thus, for environmental exposures, the concentrations usually measured are for total particulates, particulate

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matter less than 10 micron (PM10), and/or particulate matter less than 2.5 micron (PM2.5). For occupational exposures to aerosolized particulates not otherwise regulated (PNOR), the Occupational Safety and Health Administration distinguishes particles less than 5 micron from total PNOR. For PNOR, for an 8 h average, the permissible exposure limit (PEL) for total particles is 15 mg/m3 and for particles less than 5 micron the PEL is 5 mg/m3. Traditionally, occupational and environmental exposure limits for particulates are based upon mass and have been used for particles in a size range of 1–5 microns or greater. NPs are much smaller, with at least one dimension less than 0.1 micron (100 nm). With most studies of ambient workplace particulates, the percentage of the particles which were NPs is not reported. In the NP size range, collecting the particles to measure them by mass, surface area, particle number, or any other measure becomes a challenge requiring specialized instrumentation. Recent technical improvements enable measurements of aerosolized nanoparticles using area and personal samplers. These technical improvements greatly facilitate studies of occupational nanoparticle exposures. Carbon nanotubes (CNTs) and carbon nanofibers (CNFs) can be released into the air (aerosolized) within workplaces if sufficiently agitated. Activities causing aerosolization of MWCNT include oven opening, preparation, weighing, transferring, blending, spraying, and sonication. Diameters of MWCNT aerosolized into workplace air depend on the manufacturing process, with mode diameters ranging from 20 to 30 nm for catalyst preparation and 120–300 nm for ultrasonic dispersion. CNF can be aerosolized during weighing, mixing, handling, transfer, and bagging of dry CNF as well as during wet sawing or grinding of CNF composites. Additional NPs have also recently been demonstrated to produce aerosols under workplace conditions. In studies of workers exposed to NPs, recent data suggest that some NPs may cause lung function alterations, modify resting heart rate, and alter biomarkers, suggesting potential adverse effects. However, current data are limited to studies of a few types of NPs. In response to the rapid expansion of nanotechnology products in workplaces, exposure

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banding and other categorical approaches to risk assessment are being used to evaluate potential worker risks. Local exhaust ventilation and HEPA filters have been reported to be effective in controlling nanoparticle exposures in at least one MWCNT laboratory (Han et al., 2008).

2. EXPERIMENTAL TOXICOLOGIC PATHOLOGY OF NPS In 2005, the ILSI Research Foundation/Risk Science Institute Nanomaterial Toxicity Screening Working Group identified the key elements for toxicity screening of NPs as “Physicochemical Characteristics, In Vitro Assays (cellular and noncellular), and In Vivo Assays” (Oberdorster et al., 2005). Determining the physicochemical characteristics of NPs is essential if the study is to produce data that can be interpreted for risk assessment purposes. The chemical composition, size and size distribution, shape, agglomeration, surface properties, porosity, and a biologically relevant measure of exposure dose are each important to understanding the relevance of NP toxicology studies. Some common features of NPs that can influence toxicologic pathology are surface area, solubility, quantum chemistry, and size.

2.1. Enhanced Toxicity of Nanoscale Particulates Surface Area Several studies have compared the bioactivity of fine versus ultrafine carbon black or fine versus ultrafine TiO2 after pulmonary exposure. On an equal mass basis, ultrafine carbon black or TiO2 were found to be more inflammatory than fine particles of the same chemical composition. However, when dose was converted to total particulate surface area delivered to the lung, the bioactivities of fine versus ultrafine carbon black or TiO2 were similar. NPs have the tendency to agglomerate. If particulate surface area influences pulmonary response, then the agglomeration state of NPs should have a significant effect on bioactivity. Indeed, Shvedova et al. (2007) have shown that well-dispersed nano-carbon black (dispersed in diluted lung

lining fluid) was more inflammatory after intratracheal instillation in rats than an equal mass of poorly dispersed nano-carbon black (suspended in phosphate-buffered saline) (Shvedova et al., 2007). The influence of dispersion was confirmed in a more extensive study which reported that on an equal mass basis, welldispersed ultrafine carbon black was 65-fold more inflammatory and cytotoxic in the lung than fine carbon black. Similarly, Sager et al. reported that well-dispersed ultrafine TiO2 was 42fold more inflammatory and cytotoxic than fine TiO2 (Sager et al., 2008). When exposure doses were equalized on a basis of total particulate surface area instilled into the lung, no significant difference in potency of fine versus nano-carbon black or TiO2 was noted. Solubility For metals, particulate surface area is also a major determination of solubility. As noted above, for a given mass of particulates, the surface area goes up as particle size goes down. Thus, the solubility of dilute suspensions of organic-coated silver NPs increases on a mass basis as particle diameter decreases (Ma et al., 2012). This is important because dissolution of certain nano-metallic particles and the formation of toxic metal ions have been proposed as an important mechanism determining bioactivity. For example, nano-ZnO has been shown to exert toxicity in a cell culture system via the formation of Zn2þ ions and the resultant generation of reactive oxygen species (George et al., 2010). Doping of ZnO with iron (10%) has been shown to decrease dissolution by 93% (Xia et al., 2011). This decrease in Zn2þ formation was associated with a striking reduction in pulmonary inflammation and lung damage in a rat model after intratracheal instillation of nano-ZnO. Dissolution of nanoparticles would also affect the translocation of metals from the lung to systemic organs. Indeed, the lung burden of Zn rapidly declines 24 h after exposure to ZnO with a concomitant rise in Zn levels in systemic organs over this time (Sager et al., 2010). Quantum Chemistry NPs can have different chemical and physical properties than larger particles with the same chemical properties. This difference is attributed

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to changes that occur in the nanoscale where quantum phenomena predominate, particularly in the size range of 10–50 nm (Hakkinen et al., 2003; Roco, 2011). These effects are described in quantum theory in physics and occur when particle size becomes similar in size to physical and chemical phenomena such as wavelengths (Roco, 2011). Even thermodynamic properties can be different in the nanoscale as opposed to bulk materials of the same composition. Further, different nanoparticles with the same chemical composition can differ in their thermodynamic properties because particle volume within the nanoscale size range influences quantum mechanical behavior (Volokitin et al., 1996). Thus, quantum phenomena are very important in the nanoscale and can markedly alter the properties of compounds that are relatively inert when larger. Fortunately, pathologists do not need to understand quantum theory to understand nanotoxicology. However, it is important that toxicologic pathologists understand that fundamental properties of compounds can change in the nanoscale and that these altered properties can change the toxicity profile. For an in depth understanding of the quantum realm and altered properties in NPs, collaborators in other scientific fields are particularly important members of many nanotoxicology research teams. Size Size affects the properties of surface area, solubility, and quantum chemistry. However, size also influences the ability of a particulate to translocate within the body, enter cells, and interact with subcellular structures. Even the ability of a pathologist to find the particulate in a tissue section is dependent upon particulate size.

2.2. Visualizing NPs in Tissue Some NPs can be very similar in appearance to normal subcellular components (Figure 13.4). The general principles of identifying NPs in light microscopic and ultrastructural tissue sections include familiarity with (1) the appearance of the NP, (2) the appearance of normal and diseased tissue, and (3) a means for clearly distinguishing between the NPs and changes that may be associated with the NPs. For example, eosinophil granules containing tubular structures

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within their granules and eosinophils can be a component of an inflammatory response. However, the eosinophil granules are not engineered nanoparticles (Figure 13.4A). Thus, structures in NP-exposed animals will not necessarily be the NP, even when similar in appearance to the test article and absent in controls. It is essential that the evaluation of tissues from NPexposed animals for intracellular distribution (1) be conducted by someone familiar with the spectrum of pathologic responses in the exposed tissues, (2) include a means for clear distinction between the NP and cellular responses to the NP, and (3) be conducted with an understanding of the dilution effect and detection limits for NPs in tissue. Failure to identify NPs in an organelle is most likely evidence that the technique is not sensitive enough to detect them rather than evidence that they are not there. In cultured cells and in tissue sections, NPs may be imaged by using the intrinsic optical properties of the nanoparticle or by labeling. For example, many nanoparticles block light, which allows them to be seen in standard H&Estained sections (Figure 13.5). Factors which Limit the Ability to Identify NPs in Tissue Sections When preparing to examine NPs in tissue, it is critical that the NPs occur frequently enough for the sampling strategy. The frequency of occurrence of nanoparticles within the tissue must be sufficiently high that NPs are likely to be in each field of view. Given the high number of NPs present in even a microgram of NP material, visualization of NPs might, at first, appear to be a routine microscopy exercise. For instance, a 1 mg lung burden of well-dispersed MWCNTs in the mouse lung could easily distribute into 200 million or more nanotubes throughout the lungs. Given that the mouse lung has on the order of 4 million alveoli (Mercer et al., 1994), this would, on average, yield approximately 50 or more nanotubes per alveolus. For light microscopy of paraffin sections this would yield 10 nanotubes in a typical alveolar profile (a 5-micron section would be approximately one-fifth of the 25 micron alveolar diameter in the mouse). Thus, the frequency of occurrence is sufficiently high that NPs would likely be in the field of view. However, given the limited resolution of a conventional light microscope (0.2 microns), NPs may not be

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FIGURE 13.4 The image in the upper left is a normal eosinophil in the lung of an SWCNT-exposed mouse. The eosinophil granules contain normal variations in staining intensity that can give the appearance of fiberlike particles, but these are normal structures that should not be confused with SWCNTs even when absent in controls. The image on the upper right is a granuloma containing SWCNTs (asterisk) that are being walled off by epithelioid macrophages. The SWCNTs are very similar in size and shape to the collagen fibers (solid red arrows) within the granuloma. The rectangle is a region that was photographed at higher resolution as shown in the lower panel. (Bottom) At this higher magnification, the SWCNTs (asterisk) can be distinguished from the collagen fibers, which have a more distinct fiber shape. At even higher magnification, the gold label that had been attached to the SWCNTs confirmed that these were the SWCNTs. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.4, p. 1382, with permission.

detectable in conventional light microscopy. An alternative would be to consider examination with TEM. However, examining the same

tissues/burden under TEM would be prohibitively difficult at this dose. A typical TEM section might contain 20 alveolar profiles but would be

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FIGURE 13.5 In this H&E-stained section from an MWCNT-exposed mouse, transmitted light is blocked by the MWCNTs which show up as black structures (arrows) in macrophages, giant cells, and alveolar epithelial cells. Bar is 20 microns. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.5, p. 1383, with permission.

only 60 nm thick. Therefore, on average one would need to examine the entire TEM section to find one or two NPs. The above example illustrates that both the inherent visibility and the frequency of occurrence of NPs must be considered to successfully evaluate NPs in tissue sections. Numerous other factors work in concert to reduce the possibility of detecting NPs in section. These include: (1) Lack of adequate dispersion. (2) Limitations in visualization due to narrow depth of field. (3) The small fraction of the section area covered by the NPs. (4) The lack of contrast between the biologic tissue and the nanomaterial. 1. Lack of adequate dispersion. Although NPs are characterized by the dimensions of single particles, the individual NPs often agglomerate to form functionally larger particulates, particularly when suspended in aqueous solutions. Failure to adequately disperse NPs is one of the major problems in preparation, administration, and detection of

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NPs. Many of the NPs which are of interest for health risk evaluations have a high selfaffinity and require treatment with special dispersants to prevent agglomeration into micrometer dimensions (Sager et al., 2007; Mercer et al., 2008; Porter et al., 2010). In the absence of such treatment, as much as 80% of SWCNTs may remain agglomerated into micrometer-sized clumps. 2. Limitations in visualization due to narrow depth of field. Due to their small dimensions, examination of well-dispersed NPs requires high magnification objectives or electron microscopy which typically also have a limited depth of field. Because of the limitations of the depth of field at high numerical aperture only a fraction of the particles in the section will be in focus at one time. For example, a 100 high numerical aperture lens may have a depth of field of only 0.2 micron. Thus only 1/25 of the thickness of a 5-micron section would be in optimal focus at one time. For larger fibers such as asbestos this does not pose a significant problem as the out-of-focus regions of the large fibers are still detectable. 3. The small fraction of the section’s area covered by the NPs. To be detectable in a microscopic section the nanomaterial must cover a sufficient area of the section to alter the light path. In a ½ cm2 tissue section of a mouse lung exposed to 50 mg of MWCNTs, there may be as many as 2 million MWCNTs each being 50 nm in diameter by approximately 5 micron long. Even if these fibers were maximally aligned side to side into a sheet parallel to the section, the fibers would cover less than 1% of the ½ cm2 tissue section. This combined with the lack of contrast makes individual and small clumps of NPs difficult to detect in microscopic sections. 4. Lack of contrast between the biologic tissue and the nanomaterial. Many NPs, such as carbon nanotubes, were developed as structural materials and as such are relatively unreactive to conventional biologic stains. Furthermore, the dimensions of the NPs are frequently less than the visible wavelengths of light which further diminishes the likelihood of detection. Carbon nanotubes only give the appearance of being differentiated in the sections because there is sufficient mass of nanomaterial in the light path to block light. Detection of these difficult nanomaterials requires one or more of

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the special techniques for detection described in subsequent sections. These and other factors frequently limit the ability to detect and identify NPs in tissue sections. Specialized instruments and techniques have been developed to overcome these problems. The techniques include labeling of the NPs, use of Field Emission Scanning Electron Microscope (FESEM) in thick sections, and enhanced darkfield microscopy. Labeled NPs Labeling of NPs with a fluorescent indicator or some conveniently detected particle such as colloidal gold is one possible solution to make NPs easily visible in sections. Functionalization such as the oxidation of the carbon–carbon bonds may be used to label the CNTs with colloidal gold. Labeling with colloidal gold allows the application of a variety of established techniques developed for immunohistochemistry and other fields. These techniques can be used to allow detection in microscopic sections by silver enhancement and to aid in identification in TEM/FESEM observation (Mercer et al., 2008). High-Resolution FESEM Conventional scanning electron microscopes (SEMs) that are used for biologic specimens have been applied with great success to imaging of micrometer-dimensioned inhaled particles which were studied prior to the advent of nanomaterials. For NPs, the conventional SEM does not have the submicrometer resolution necessary to resolve or identify NPs. Difficulties in imaging NPs with an SEM are further complicated by the fact that many NPs, such as carbon nanotubes, have no significant difference in secondary electron or backscatter emissions from the organic carbon in which they are immersed. Introduction of the FESEM has significantly improved the resolution and applicability of the SEM to examination of NPs in tissue. The unique “cold” cathode design of the FESEM produces high-quality, low-voltage images with significantly lower electrical charging that can be used to identify NPs in tissues at levels of resolution not previously available with the conventional SEM. The high-resolution capability of the FESEM greatly facilitates the imaging of MWCNT interactions with cells and tissues

of the lung. The FESEM images in Figure 13.6 show the penetrating nature of MWCNT 28 days after exposure. To image NPs at high magnification with the FESEM, some consideration of the methods of specimen preparation are necessary to obtain a stable image. Use of thin sections from paraffin embedded tissue has been found to be preferable to large, unevenly cut blocks because it provided a uniform thickness of organic material on a conductive carbon planchet. At 5–8 micron of thickness, paraffin sections are thick enough to convey three-dimensional information and less likely to charge or undergo physical shifts when examined at the high magnifications necessary to study nanomaterials. Enhanced Darkfield Microscopy Traditionally, darkfield has been used to examine larger fine-sized particles in tissue sections. Darkfield microscopy suffers from lack of resolution, in part, because the transmitted light is not blocked from the image path. Newer, enhanced darkfield systems have modified optics that virtually eliminate transmitted light from the image. This greatly enhances the contrast between sectioned tissue (a poor source of scattered light) and NPs as can be seen in the enhanced darkfield image of Figure 13.6. The value of enhanced darkfield is that the vast majority of nanomaterials efficiently scatter light while normal tissue sections do not. Nanomaterials, such as carbon nanotubes, have many of the characteristics which produce Rayleigh scattering of light. These include dimensions less than the wavelength of light, a close and ordered alignment of atoms, and typically have a refractive index significantly different from that of biologic tissues and/or mounting medium. Normal preparation of mounted tissue is designed to minimize scattered light. For instance, the refractive index of glass, mounting medium, and tissue are all closely matched (1.47–1.55). The refractive indices of nanomaterials are much higher being 2.2 for cerium oxide (nanometer-sized diesel fuel catalyst), 2.6 for zinc sulfide (component of quantum dot), and 3.6 for crystalline silica (nanosilica). Together these characteristics produce significantly greater scattering of light by nanoparticles than by the surrounding tissues. The enhanced

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darkfield optical system images light scattered in the section and, thus, nanomaterials in the section stand-out from the surrounding tissues with high contrast. Using this method of imaging, large areas can be easily scanned at relatively low magnification to identify NPs that would not be detected by other means. Although the enhanced darkfield technology is relatively new, in our laboratory we have found the technique useful to detect a wide variety of NPs in tissue sections. These have included cerium oxide, titanium oxide, diesel exhaust, welding fumes, SWCNT, MWCNT, silver nanowires, silicon nanowires, nanosilica, quantum dots, colloidal gold, and others (Mercer et al., 2018).

2.3. Cytopathology Cytoplasmic Membrane Damage NPs can be produced in almost any shape. The aspect ratio of a particle is the ratio of the longest dimension to the shortest dimension of a particle (Figure 13.7A) (Carter and Yan, 2005). Fibers are the classic particulates with a high aspect ratio. Asbestos fibers are naturally occurring carcinogenic mineral fibers, and some asbestos fibers have diameters in nanoscale dimensions. In 1981, Mearl Stanton and colleagues noted that experimental pathology studies indicated: “The probability of pleural sarcoma correlated best with the number of fibers that measured 0.25 mm or less in diameter and more than 8 mm in length .” (Stanton et al., 1981). Some asbestos fibers, and, by definition, all nanotubes, have diameters less than 0.25 mm. Therefore, a great deal of concern has been expressed regarding the similarities between asbestos fibers and

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FIGURE 13.6 The top image demonstrates MWCNTs within a subpleural lymphatic using FESEM. In the middle image, FESEM is used to demonstrate penetration of the visceral pleura by MWCNT. In the lower

image, enhanced darkfield imaging demonstrates MWCNTs within macrophages, the interstitium and pleura (arrow). Reprinted with permission from Mercer RR, Hubbs AF, Scabilloni JF, Wang L, Battelli LA, SchweglerBerry D, Castranova V, Porter DW: Distribution and persistence of pleural penetrations by multi-walled carbon nanotubes. Part Fibre Toxicol 7: 28, 2010. Mercer RR, Hubbs AF, Scabilloni JF, Wang L, Battelli LA, Friend S, Castranova V, Porter DW: Pulmonary fibrotic response to aspiration of multi-walled carbon nanotubes. Part Fibre Toxicol 8:21, 2011.

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some of the nanotubes (Pacurari et al., 2010). The similarities in some cases include a high aspect ratio, durability, surface reactivity, the inflammatory reaction in the exposed lung, an ability to translocate through the pleura, and incomplete phagocytosis (Shvedova et al., 2005, 2008; Mercer et al., 2010; Porter et al., 2010). It is not just nanotubes that can have these properties; NPs now include nanofibers, nanowires, nanobelts, and many other high aspect ratio particulates with nanoscale dimensions. Discussions of the potential carcinogenicity of biologically persistent particles with high aspect ratios often focus on incomplete phagocytosis. In normal particle phagocytosis, macrophages and neutrophils phagocytize particles and are then carried out of the lung by mucociliary clearance or lymphatics (Harmsen et al., 1985, 1987; Moller et al., 2004). In some cases, high aspect ratio particles appear to undergo phagocytosis and fusion with the lysosome to form a phagolysosome (Figure 13.7B). Incomplete phagocytosis is the failure to completely internalize a fibrous particulate within the cytoplasm of a phagocytic cell. Classically, phagocytosis is incomplete when the length of the fibrous particle exceeds the dimensions of the phagocytic cells (Archer, 1979; Donaldson et al., 2010). When phagocytosis is incomplete, the phagocytic vacuole may still be open to the exterior of the cell when it fuses with the lysosome to form the phagolysosome (Figure 13.7C). Lysosomal enzymes include enzymes that produce free radicals for microbial killing and enzymes capable of digesting cells. Release of those enzymes outside of the phagolysosome causes cell injury through damage to the cytoplasmic membrane and inflammation (Donaldson et al., 2010). Human macrophages are larger than rodent macrophages (Krombach et al., 1997). Human macrophages often show less fiber-induced incomplete phagocytosis and less cytotoxicity than rodent macrophages, consistent with a role for incomplete phagocytosis in the pathogenesis of fiber-induced lung disease (ZeidlerErdely et al., 2006). However, the asbestos fibers most associated with pleural sarcomas are longer than 8 micron, while 8 micron is less than the length of an average macrophage and certainly less than the length of a giant cell (Haley et al., 1991; Krombach et al., 1997). This suggests that incomplete phagocytosis is not

FIGURE 13.7 High aspect ratio particles: (A) A high aspect ratio particle is a particle with a much greater length than width; (B) complete phagocytosis; (C) incomplete phagocytosis; (D) membrane penetration after phagocytosis. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.7, p. 1386, with permission.

the only pathogenic mechanism for fiberinduced cell injury. Studies with NPs are providing data that may help pathologists understand the pathogenesis of diseases such as mesothelioma and asbestosis that have long been associated with durable particles with a high aspect ratio. Some of these NP-induced changes are also manifested as cytoplasmic membrane damage. MWCNTs are NPs with a high aspect ratio and can be engineered within narrow dimensional

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ranges that may permit a greater understanding of fiber toxicology. MWCNTs block transmitted light which makes them relatively easily identified by light microscopy where they are seen as black rodlike structures within the lighttransmitting cytoplasm. Even better visualization of MWCNTs is possible with enhanced darkfield microscopy. In the lungs of mice exposed to MWCNTs (median length of 3.86 micron and a median width of 49 nm), incomplete phagocytosis is observed and would be anticipated to release lysosomal enzymes and cause cell membrane damage. Importantly, incomplete phagocytosis or partial engulfment is seen with tangled mats of MWCNTs as well as with long MWCNTs (Porter et al., 2010). In addition, MWCNTs appear to migrate within the lung much like a nanoscale version of a splinter might migrate through tissue. Thus, MWCNTs are seen penetrating the visceral pleura of the lung, extending from alveolar septa and within lymphatics (Figure 13.6) (Mercer et al., 2010, 2011; Porter et al., 2010). MWCNTs can also sometimes penetrate the nuclear envelope of macrophages and other cells (Figure 13.7D) (Porter et al., 2013). Within the cytoplasm, MWCNTs are frequently outside of vacuoles, suggesting that they either enter cells by means other than phagocytosis or do not stay in the phagolysosome (Mercer et al., 2011). Functionalized MWCNTs have been demonstrated to enter cells without phagocytic capabilities and to escape the phagolysosomes of phagocytic cells, suggesting that both mechanisms play a role in the location of MWCNTs within cells (Al-Jamal et al., 2011). Since the cytoplasmic membrane is basically a protein-containing lipid bilayer, the ability of a thin tube with high tensile strength to migrate through the membrane(s) of mobile cells such as macrophages and into additional cells is not surprising. Nor is it surprising that the migration may continue through additional cells in a tissue such as the lung, which moves and undergoes pressure changes with every breath. In the past decade several endocytic processes have been identified as being able to bring NPs into cells. Those processes are described late in the chapter and are not limited to phagocytic cells and can be influenced by the physiochemical properties of the NPs. Thus, data support penetration of the cytoplasmic and nuclear membranes by NPs

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with high aspect ratios, and this may be explained by multiple potential processes including: (1) classic incomplete phagocytosis of a high aspect ratio particle that exceeds the length of the cell, (2) incomplete endocytosis that is not part of the phagocytic process, and (3) migration of the NP out of the phagolysome and/or through other cell membranes. In addition, recent studies indicate that high aspect ratio and spherical particles can fundamentally differ in how they enter and traffic through cells, and how they interact with subcellular structures (Zhao and Stenzel, 2018). Mitotic Spindle Interactions Toxicologic anatomic pathologists rarely evaluate changes involving the mitotic spindle. However, it is very important that toxicologic pathologists recognize changes in histopathology that are outside of the spectrum of possible changes in normal tissue sections. For example, during the evaluation of histopathology in the lungs of mice inhaling SWCNTs, the toxicologic pathologist noted in a single dividing cell SWCNTs that were gathered at the site of the spindle pole, chromatin streaming from the one chromatin bundle toward the other, SWCNTs attached to the streaming chromatin, and the other chromatin bundle unusually condensed (Figure 13.8). This strongly suggested that SWCNTs could interfere with the specialized system responsible for sending the correct genetic material to the daughter cells during cell division, the mitotic spindle (Shvedova et al., 2008). A summary of some of the most critical information on the mitotic spindle and the genotoxicity of NPs is included below. OVERVIEW OF THE MITOTIC SPINDLE

The mitotic spindle is a structure that forms during cell division and separates duplicated chromosomes. In eukaryotic cells, the mitotic apparatus is composed of two centrosomes and spindle microtubules (Figure 13.9). The centrosome as well as the microtubules determine the shape of the cell as well as the mitotic spindle apparatus. In eukaryotic cells, the polymerization of microtubules from alpha and beta tubulin is initiated at the centrosome to form the mitotic spindle and the structure for cytokinesis (Yeates and Padilla, 2002; Doxsey et al., 2005). During cell division the

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FIGURE 13.8 On the left is a 60 oil field examined by the pathologist and on the right is a high resolution enlarged digital image from a 100 objective showing material consistent with SWCNTs near the spindle poles (dashed arrows) and attached to streaming chromatin (solid arrow). Identifying this unanticipated change required a detailed histopathologic evaluation and the use of high magnification. Photographing the interaction took a highresolution digital camera and cropping of the image. Changes such as these are at the limits of resolution of light microscopy. Subsequent studies of SWCNT-exposed cells detected high levels of aneuploidy as well as interactions between SWCNTs and the mitotic spindle. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.8, p. 1388, with permission.

microtubules continue to polymerize, and the mitotic apparatus elongates until metaphase when the chromosomes line up at the center of the cell at the metaphase plate (Figure 13.10A). As the cell cycle progresses, the chromosomes are separated by the mitotic apparatus as the microtubules of the mitotic apparatus shorten through depolymerization (Figure 13.10B). When mitosis is completed, a furrow is formed between the two dividing daughter cells (Figure 13.10C). The furrow between the dividing cells (midbody) contains microtubules from each pole of the mitosis (Mullins and McIntosh, 1982). Disruption of centrosome number or structure or of the microtubule assembly is common in most cancers and results in aberrant mitotic spindles, failure of cell separation, and errors in chromosome number (aneuploidy) (Pihan et al., 1998; Salisbury et al., 2004; Lingle et al., 2005; Hornick et al., 2008; Salisbury, 2008).

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SWCNTs have been shown to fragment the centrosome resulting in multipolar mitotic spindles and dramatic aneuploidy while exposure to some MWCNTs results in monopolar mitotic spindles as well as aneuploidy (Sargent et al., 2009; Siegrist et al., 2014). In both cases, the SWCNT and MWCNT materials were strongly associated with the centrosome. Threedimensional reconstructions of mitotic figures from SWCNT-dosed respiratory epithelial cells have shown SWCNTs located inside the centrosome structure (Sargent et al., 2012). SWCNTs have also been shown incorporated into the microtubules of mitotic cells (Sargent et al., 2009). MICROTUBULE INTERACTIONS

SWCNTs have been observed within the nucleus and in association with cellular and

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FIGURE 13.9 Drawing of a normal mitotic spindle apparatus. The green stained centrosomes are indicated by white arrows, the microtubules are in red, and the DNA is in blue. The cell is in metaphase stage of cell division with the chromosomes lined up in the middle of the mitotic spindle. The figure was reproduced with permission from Sargent LM, Reynolds SH, Castranova V: Potential pulmonary effects of engineered carbon nanotubes: in vitro genotoxic effects. Nanotoxicology 2010, 4:396–408.

mitotic tubulin, in the bridge separating dividing daughter cells (midbody) as well as in the DNA, potentially disrupting the normal mitotic process as shown in Figure 13.11 (Sargent et al., 2009). The basis of the incorporation of carbon nanotubes into the mitotic apparatus may be due to several mechanisms. In laboratory studies, carbon nanotubes have been shown to form functional hybrid molecules with tubulin (Dinu et al., 2009). The carbon nanotube/microtubule hybrid molecules are transported by the spindle motor kinesin that is essential for normal cell division; however, the hybrids are transported with less efficiency than the cellular microtubules. In addition, spherical nanoparticles less than 40 nm in diameter inhibit the activity of the kinesin motor, further indicating the potential for the disruption of mitosis by nanomaterials (Bachand et al., 2005). Inhibition of kinesin motor activity has been shown to result in mitotic spindle disruption (Ochi, 2002). Carbon nanotubes and microtubules have many physical properties in common including high tensile strength (Pampaloni and Florin, 2008). Carbon nanotubes are five times stronger than steel, and microtubules are 100 times stronger than any other cellular cytoskeletal fibers; however,

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their strength is 100 times less than that of carbon nanotubes (Dalton et al., 2003; Pampaloni and Florin, 2008). Although there are many physical properties in common, there are also some distinct chemical differences between microtubules and carbon nanotubes. Carbon nanotubes are composed of covalently bound carbon molecules rolled into a tube while microtubules are polymers of alpha and beta tubulin subunits that are bound by noncovalent hydrogen bonds. The microtubules are dynamic structures that polymerize and depolymerize within the cell during cell division (Yeates and Padilla, 2002). Once they are synthesized, individual carbon nanotubes are static in size. The similar size and shape of the carbon nanotubes to the microtubules may make it possible for the nanotubes to displace microtubules at critical cellular targets including the centrosome (Figure 13.12). Alternatively, the nanotubes have also been shown to be incorporated into the microtubules as well as the centrosome. The incorporation of the strong carbon nanotubes into the cellular structures may be responsible for the fragmenting of the centrosome as well as cytokinesis failure during cell division. Fragmented centrosomes and cytokinesis failure have been shown in other systems to result in multipolar mitotic spindles. CHROMOSOMAL INTERACTIONS

Carbon nanotubes have a high affinity for DNA. SWCNTs have the highest affinity for DNA of GC-rich DNA sequences in the chromosomes and have been shown to bind to the GCrich regions of the chromosome ends (telomeric DNA) (Li et al., 2006a,b). The DNA intercalation of the nanotubes results in a conformational change which can be stabilized by carboxyl modification of the SWCNT by acid treatment (Li et al., 2006a,b). Intercalating agents can induce chromosome breakage and instability. The damaging effects of carbon nanotubes may be induced by a variety of mechanisms linked in part to the physical and chemical properties of nanotubes. DNA damage and increases in multinucleated cells have been observed following in vitro exposure to SWCNT and MWCNT (Kisin et al., 2007; Lindberg et al., 2009; Yang et al., 2009; Pacurari et al., 2010). In addition, in some, but not all studies of SWCNT-and MWCNT-exposed cells, there is

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FIGURE 13.10 Composite of mitotic figures. (A) Cell in metaphase. The duplicated centrosomes have formed two mitotic spindle poles. (B) The mitotic spindle apparatus has elongated separating the chromosomes. (C) The cell has progressed through mitosis and a bridge of cytokinesis or midbody separates the cells. The high-resolution confocal images of dividing cells are courtesy of Jeffrey L Salisbury, Department of Biochemistry and Molecular Biology, Tumor Biology Program, Mayo Clinic, Rochester, Minnesota. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013.

evidence of lactate dehydrogenase leakage from cells as well as depletion of the oxidant protective enzymes (glutathione and superoxide dismutase) indicating reactive oxygen species generation. The generation of reactive oxygen species can damage cell membranes, proteins, and DNA. Oxidant-induced DNA damage has been reported in vivo in both mice and rats exposed to iron-contaminated MWCNT and SWCNT (Folkmann et al., 2009; Jacobsen et al., 2009). In recent investigations, the more rigid 49 nm Mitsui-7 MWNCT was shown to fragment the center of the chromosome, the centromere. The fragmenting of the chromosome (centromere) resulted in chromosomal translocations and more dramatic aneuploidy than was observed following exposure to SWCNT or a narrower (10–15 nm) MWCNT. The extreme chromosomal damage was not prevented by either heat treatment or nitrogen doping of the Mitsui-7 material

(Siegrist et al., 2014, 2019). Additionally, the Mitsui-7 materials were observed in the nucleus and associated with the centrosome, which controls the mitotic spindle, as well as with the microtubules of the mitotic apparatus. Genomic instability can result from damage to the DNA or damage to the mitotic spindle apparatus. The loss of whole chromosomes has been reported in established cancer cell lines indicating a disruption of the mitotic spindle (Muller et al., 2008; Doak et al., 2009; Lindberg et al., 2009). Exposures of rodents to MWCNT have demonstrated micronuclei in primary mouse type II epithelial cells 3 days following intratracheal administration of 1 mg/kg body weight (Muller et al., 2008). Micronuclei indicate either a high level of chromosomal breakage or mitotic spindle disruption. Two in vitro investigations have shown dramatic errors in chromosome number after treatment of primary small airway epithelial cells and immortalized bronchial

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FIGURE 13.11 Three-dimensional reconstruction of a mitotic spindle with three mitotic spindle poles (tripolar mitosis). The mitosis was isolated from a cell exposed to SWCNT for 24 h. The DNA was detected with DAPI and is blue. The tubulin and centrosomes were detected using immunohistochemical methods. The tubulin is red, and the centrosomes are green. Differential interference contrast imaging images the nanotubes. The nanotubes block the light and produce a black image. The cell was imaged using confocal microscopy. Serial optical sections of 0.1 micron in depth were used to construct a three-dimensional reconstructed image of the tripolar mitosis. The reconstructed image shows nanotubes inside the cell in association with each centrosome fragment. The white arrow indicates association with one of the centrosome fragments. Nanotubes are also integrated with the microtubules and the DNA. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 43.11, p. 1391, with permission.

epithelial cells with 0.024–96 mg/cm2 SWCNT (Sargent et al., 2009, 2012). A more recent investigation demonstrated even greater chromosomal damage following exposure of immortalized and primary small airway cells epithelial cells to 0.0024–24 mg/cm2 MWCNT (Siegrist et al., 2014). As indicated previously, the chromosome errors were attributed to disruption of the mitotic spindle. The SWCNTs were observed within the nucleus, the DNA, in association with cellular and mitotic tubulin, and in the bridge separating dividing daughter cells (midbody). The association of the nanotubes disrupted the normal mitotic process as shown in Figure 13.11. Additionally, the more

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FIGURE 13.12 The drawing demonstrates a proposed model for the interaction of carbon nanotubes with subcellular structures. The carbon nanotubes in this drawing are attached to the centrosome and displace microtubules at the centrosome. The figure was reproduced with permission from Sargent LM, Reynolds SH, Castranova V: Potential pulmonary effects of engineered carbon nanotubes: in vitro genotoxic effects. Nanotoxicology 2010, 4:396–408.

rigid Mitsui-7 MWCNT caused further damage by fragmenting the center of the chromosome (Siegrist et al., 2019). ADDITIONAL CYTOPATHOLOGIC INTERACTIONS

The findings noted above demonstrate that some NPs cause important alterations in cytoplasmic membranes and the mitotic spindle. These are among the best described cytopathologic effects of NP exposure. However, the size range of NPs (30 Gy. Other studies examined the critical factors in the genesis of radiationinduced heart disease in breast cancer patients. These noted that the total volume of heart exposed, age of patient, and radiation dose were the most significant factors.

FIGURE 14.37 Skeletal muscle, leg: Fisher 344 rat. This animal was part of a depleted uranium study. The photomicrograph shows a malignant rhabdomyosarcoma. The tumor arose around the implanted fragment. H&E stain. (A) Bar ¼ 100 mm, (B) Bar ¼ 30 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.37, p. 1473, with permission.

therapeutic thoracic irradiation for breast cancer, lymphomas (Hodgkin’s disease in particular), and peptic ulcer therapy, cardiac toxicity associated with irradiation has become apparent, contradicting the previous dogma. Frequently these late effects are associated with insult to the pericardium and the microvasculature, and have bystander effects which result in regional hypoxia or even ischemia, eventually resulting in interstitial cardiac fibrosis years after the irradiation. An extensive retrospective study evaluated 2231 survivors of Hodgkin’s disease, all of whom had

Coronary Arteries Studies of the development of coronary artery disease in younger patients examined cohorts who received radiation therapy in comparison with retrospective studies from Chernobyl in which victims otherwise lacked risk factors for atherosclerosis. In both populations, a highly significant correlation between radiation and development of arterial disease was noted. Histopathological findings are consistent with atherosclerosis, including adventitial scarring and medial smooth muscle atrophy with severe intimal pathology consisting of necrosis, fibrosis, mineralization, and endothelial cell loss (Figure 14.38). The aorta responds to irradiation with intimal proliferation consisting of subendothelial nodular masses or plaques of collagenous connective tissue admixed with smooth muscle and elastin fibers. Adventitial fibrosis is another characteristic finding. The intimal proliferation increases with increased doses given in 2- to 3Gy fractions. When given in single doses, the intimal proliferation increases up to 15 Gy and then progressively decreases with higher doses. Three or more years after single doses of 35 Gy or more, the affected aortas often show almost complete occlusion of the aortic lumen by organizing thrombi, and disruption of the elastic fibers of the tunica media with medial fibrinoid necrosis and hyalinization. Furthermore, dissecting aneurysms are frequently associated with the lesions. Early endothelial cell damage by irradiation probably leads to the intimal proliferation. A variety of growth factors, such as basic fibroblast growth factor, platelet derived growth factor, and TGF-b, are involved in the pathogenesis of the

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Chronic restrictive pericarditis was noted in up to 5% of human patients who underwent irradiation for lung and breast cancers, but increased to 10% in those treated for lymphoma. The disease typically presents with pericardial effusion and cardiac tamponade.

FIGURE 14.38 Heart, coronary artery: Hartley guinea pig that received 5 Gy gamma radiation at 1 Gy/min (cobalt-60 source), 30 days prior. The vessel is affected by marked circumferential adventitial fibrosis, with mononuclear inflammation and mild atherosclerotic mural changes. H&E stain. Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.38, p. 1474, with permission.

vascular injury. Damage to the vasa vasorum with vascular leakage may lead to the progressive adventitial fibrosis. The combination of intimal proliferation, fibrosis of the adventitia, and damage to the vasa vasorum may compromise the nutrient and oxygen supply to the media and result in medial necrosis. These would predispose the aorta to thrombosis and disruption. Smaller arteries and arterioles show lesions similar to that of the aorta, but they appear at earlier times after irradiation and at lower doses. Pericardium The pericardium is the primary heart tissue affected, presenting frequently as an acute pericarditis with associated pericardial effusion or fibrosis leading to restrictive cardiac function. While the pathogenesis is poorly understood, it is theorized to be the result of insult to the microvasculature as discussed earlier in the chapter. The acute lesion can lead to chronic constrictive pericarditis, myocarditis, conduction abnormalities, coronary arteritis, and valvular dysfunction.

Heart While the myocardium is less affected than the pericardium, the literature is replete with evidence of significant myocardial disease associated with irradiation. Recent reports from the World Health Organization, examining survivors of atomic bombing in Japan who received single whole-body doses ranging from 0 to 4 Gy, showed that cardiovascular disease risk was dose-related and increased by 14% per Gy. As with radiation insult in other tissues, the pathology observed is due mainly to the microvascular damage and response, and less to the damage to the postmitotic cardiomyocytes. Injury more commonly presents in the left ventricle than the right due to its position in fields of thoracic irradiation. Studies of the effects of irradiation on the hearts of rats, rabbits, and dogs have been reported. One month after fractionated doses of 36–52 Gy, there is a transient increase in heart weight and wall thickness primarily due to a mild, diffuse myocarditis and myocardial edema. Within 3 months, there is significant mesothelial hypertrophy and marked changes in the microvasculature, characterized by endothelial cell swelling, cytoplasmic blebbing, loss of organelles, formation of lamellar bodies, obliteration of the capillary lumens, microrupture, platelet dysfunction, hemorrhage, and occasional thrombosis. Between 9 months and 1 year subsequent to injury, thinning of the heart wall and large focal areas of myofiber loss are seen. Empty sarcolemmal sheaths and a mild influx of macrophages characterize these areas, but fibrosis is variable and speciesdependent. Cardiomyocytes frequently are degenerate with sarcoplasmic vacuolization (Figure 14.39A). With increased chronicity, the myocardium undergoes progressive, perivascular, subendocardial, and subepicardial fibrosis. Often, the microscopic lesions consist of fibrotic plaques which replace cardiomyocytes downstream from affected coronary arteries. This is suggestive of a chronic reparative process

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interstitial fibrosis, another species peculiarity is the apparent extreme radiosensitivity of the right atrial wall of the canine heart. Within 1 month of irradiation, the right atrium is characterized by a hemorrhagic myocarditis. By 1-year postinsult, the atrium is shrunken, and cardiomyocytes are degenerate and necrotic and are replaced by fibrosis and chronic hemorrhage. Hemangiosarcomas have been reported to develop in the irradiated tissue after 2 or more years. While the left atrial wall lesions are similar, the changes are much less severe. The pathogenesis of this sensitivity is not fully understood and has not been seen in other species. Interestingly, the right atrium is also the primary predilection site for spontaneous hemangiosarcomas in dogs. While pigs display a similar right atrial predisposition for injury with certain chemicals, studies of whole-body irradiation at various doses in Gottingen minipigs have not found any regional cardiac predisposition in swine. The endocardium is affected in much the same manner as the myocardium, with loss of cardiomyocytes and marked infiltration of interstitial fibrosis.

4.10. Urinary System FIGURE 14.39 Heart, myocardium: Irradiation with a single high dose of ionizing radiation. (A) Sarcoplasmic degenerative changes with vacuolation, fragmentation, and pigment deposition. Additionally, the interstitium contains edema, fibrin, and rare fibroblasts. (B) Multifocally the cardiomyocytes are atrophic, characterized by rounded, shrunken sarcoplasm, replaced by adipocytes and loose fibrous connective tissue, reactive fibroblasts, and lymphoplasmocytic and histiocytic inflammation. H&E stain. (A) Bar ¼ 30 mm, (B) Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.39, p. 1475, with permission.

postinfarction (Figure 14.39B). Interstitial fibrosis can be severe in the hearts of rabbits and humans, but not in dogs or rats (Figure 14.40). Along with differences in radiation-induced

General Reaction to Ionizing Radiation Injury The effects of renal irradiation have been studied extensively in animal models, but less so in humans even though the effects of therapeutic pelvic and abdominal irradiation can result in significant long-term pathology. The kidney is among the more radiosensitive organs in humans, with late effects occurring months to years after single doses as low as 4.5–6.0 Gy. In addition to clinical irradiation, both early and late renal pathology has been reported in 29% of radiation accident victims exposed to doses high enough to cause severe hematological syndrome. Furthermore, radiation-induced multi-organ failure included renal failure in two victims of the Tokai-mura criticality accident. In humans, radiation nephropathy may develop months to years after irradiation and manifest clinically as severe anemia, proteinuria,

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FIGURE 14.40 Heart, myocardium: Gottingen minipig irradiated with a single whole-body dose of 1.7 Gy, 30 days prior. The heart contains multifocal to coalescing areas of fibrous connective tissue (white areas) infiltrating the ventricular wall, consistent with repair to areas of infarct. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.40, p. 1476, with permission.

oliguria, azotemia, the presence of casts in the urine, and a consistent hypertension. The anemia is considered to be significantly worse than that normally correlated to the degree of azotemia. Renal tubular epithelium and glomerular endothelial cells appear to be the most radiosensitive cell types in the kidney; however, other regions of the urinary system can also develop lesions. Radiation damage to the ureter has been reported occasionally to result in stricture formation with subsequent hydronephrosis, and radiation damage to the urinary bladder can cause fibrosis and loss of function. Kidney (See also Kidney, Vol 5, Chap 2). In both humans and animal models of radiation nephropathy the gold standard is histopathology, in spite of studies demonstrating a strong correlation between blood urea nitrogen (BUN) and the severity of radiation-induced microscopic lesions. Significant debate continues in that the primary target cell of radiationinduced nephropathy has not been definitively determined, and neither has the pathogenesis

of injury been identified. The literature provides examples of both the parenchymal cells and the vasculature as being the primary target tissue, while others argue for the glomeruli or the tubular epithelium. Irradiation doses of 10 Gy or less have been reported to cause chronic renal injury in a variety of species. In experimental studies in pigs, clinical renal abnormalities have been seen by 3 months postirradiation after a single dose of 7.8 Gy. In rats, single radiation doses of 9.5 Gy cause radiation-induced renal failure within 8 months, while clinical abnormalities are observed within 7 months at 7.2 Gy. Nonhuman primates had evidence of radiation nephropathy greater than 6 years following single total body doses of 7.2–8.5 Gy. The pathogenesis of radiation nephropathy in dogs was studied by sequential renal biopsy every 2 weeks after a single dose of 15 Gy. At 3 weeks after irradiation, there was a mild vacuolar change in renal tubular epithelial cells. This lesion progressed to degeneration and a reduction of total tubular epithelial cell volume by 50% at 9 weeks after exposure. Evidence of tubular regeneration, characterized by enlarged basophilic cells lining tubules, piling of epithelium, and increased mitotic activity, was evident 5 weeks after irradiation and resulted in recovery of tubular epithelium to 80% of normal volume by 11 weeks. However, by 13 weeks the renal tubular epithelium volume had started to decrease again and reached a level of 15% of normal volume by 24 weeks. As the percentage of renal tubular epithelium decreased there was a corresponding increase in interstitial connective tissue. Blood vessel lesions were characterized by an early thickening of the medial and intimal layers of small arteries and arterioles. Fibrinoid necrosis and fibrosis of the media accompanied progressive perivascular fibrosis. The glomerular volume remained unchanged, but marked thickening of the juxtaglomerular arteries was associated with loss of some glomeruli as early as 3 weeks after irradiation. Glomerular and periglomerular sclerosis was seen at later times (Figure 14.41A). These results suggest that irradiation affects both vasculature and tubular epithelium. The earliest loss of tubular epithelium may be due to a direct effect of irradiation.

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FIGURE 14.41 Kidney: B6D2F1/J mouse irradiated with two whole-body doses of 9.25 Gy, 1 month apart. (A) Membranous glomerulonephropathy with periglomerular inflammation and minimal mesangial necrosis. Note the complete sclerosis of the glomerular tuft with replacement by a homogenous, eosinophilic matrix causing narrowing and loss of the capillary loops, and rare synechiae. (B) This kidney contains a mild, multifocal to coalescing lymphoplasmocytic and histiocytic interstitial nephritis, with tubular regeneration characterized by epithelial piling, high nuclear to cytoplasmic ratio, and increased mitoses. Additionally, there is mild to moderate thickening of the glomerular mesangium by fibrillar eosinophilic material and occasional mesangial necrosis. H&E stain. (A) Bar ¼ 30 mm, (B) Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.41, p. 1477, with permission.

After epithelial proliferation, the second wave of depopulation may be related to a decreased vascular supply.

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In contrast, in mice, renal tubular damage was thought to be due to a slow loss of irradiated parenchymal cells rather than to injury to blood vessels. The thickening of the tunica media and intima of arteries was felt to be secondary to the tubular epithelial damage. Still other investigators have suggested that the initial lesions of radiation damage occur in the glomeruli. Irradiation of the kidneys of mice, pigs, and monkeys produced early thickening of glomerular capillary walls beginning at 6 weeks. These changes progressed to replacement of the glomerular tuft by amorphous eosinophilic material consisting of basement membrane fragments, collagen, and fibrin thrombi in capillary lumens at 9 and 12 months after irradiation. In contrast, tubular atrophy and stromal fibrosis was not seen before 4 months and was not as severe as the glomerular lesions (Figure 14.41B). In pigs, after a 9.8 Gy single dose there was early leukocyte adhesion to glomerular capillaries, followed by endothelial cell swelling, microthrombi formation, and increased capillary permeability. At 12 weeks, there was exudation, mesangial proliferation, and sclerosis with a resultant decreased glomerular filtration rate. Most evidence suggests a key role for the microvasculature in both glomerular and tubule damage. There is also a role for TGFb, as this has been shown to be increased in mesangium after irradiation. Radiation-induced renal neoplasia is described in studies examining the epidemiology of Japanese atomic bomb survivors as well as in retrospective studies associated with patients receiving abdominal and pelvic radiotherapy. In the former study, which examined a total population of approximately 105,000 people, 247 cases of renal neoplasia were diagnosed as late effects of the bombings in Hiroshima and Nagasaki. Of these, almost 70% were restricted to the renal parenchyma, while the remainder were distributed between the renal pelvis and the ureter. In the latter case, the renal pelvis and ureter showed a stronger statistical association with targeted irradiation oncogenesis than the renal parenchyma. The predilection correlates with the fact that transitional cells within the renal pelvis and ureters are more susceptible to injury, and as such have a higher likelihood of malignant transformation than renal parenchyma. The main tumor type in the kidney was

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renal cell carcinoma, and in the renal pelvis and ureter was renal pelvis carcinoma. Additionally, there is evidence from the Chernobyl nuclear accident that the relative risk for renal neoplasia almost doubled in the population of regions contaminated with cesium-137, which is excreted through the kidneys (Figure 14.42). Ureter (See also Lower Urinary Tract, Vol 5, Chap 3). Histological lesions after irradiation of the ureter in the rat and dog consist of early thinning and degeneration of the urothelium which progresses to ulceration and inflammation by 2– 6 months after irradiation. Later lesions consist of focal areas of urothelial thinning and cystic degeneration alternating with areas of hyperplasia and polypoid proliferation. Varying degrees of fibrosis of the lamina propria and adventitia can extend into the muscle wall. There is sometimes muscular hypertrophy associated with strictures due to fibrosis. Ureteral stricture can lead to hydronephrosis. Vascular lesions consist of adventitial fibrosis, fibrinoid necrosis of the media, intimal proliferation, and decreased microvascular volume. These changes

FIGURE 14.42 Kidney: Fisher rat exposed to lowdose irradiation. Renal cell carcinoma, clear cell variant. The renal parenchyma is replaced and effaced by islands and trabeculae of polygonal neoplastic cells with clear cytoplasm. H&E stain. Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.42, p. 1478, with permission.

occur at single doses of 30 Gy and above. The volume or length of ureter irradiated affects the dose required to cause strictures. Urinary Bladder (See also Lower Urinary Tract, Vol 5, Chap 3). Radiation damage to the urinary bladder has been studied in mice and dogs. Transitional epithelium of the urinary bladder normally has a slow turnover rate which increases after injury. In humans, acute radiation cystitis is transitory and usually mild. Chronic radiation cystitis develops months to years later and can result in severe hemorrhage and blood loss (Figure 14.43). After doses of 30 Gy to the canine urinary bladder, initial submucosal edema, petechiae, and lymphocytic infiltration are followed after 3–6 months by vacuolization of the epithelial cells, multifocal ulceration, submucosal and smooth muscle fibrosis, and fibrinoid degeneration and intimal fibrosis of submucosal arterioles. Squamous metaplasia of the epithelium

FIGURE 14.43 Urinary bladder: Gottingen minipig irradiated with a single dose of 1.9 Gy. Multifocally, severely expanding the subepithelial connective tissue and obscuring the muscularis are large pools of hemorrhage. This animal presented at necropsy with coalescing ecchymoses on all serosal surfaces, but the gastrointestinal tract and the urinary tract were most severely affected. The animal had a severe thrombocytopenia, anemia, and lymphocytopenia at presentation. H&E stain. Bar ¼ 500 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.43, p. 1478, with permission.

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may occur. The submucosal and muscle fibrosis is thought to cause increased urinary frequency and decreased bladder volume that is evident clinically. Urinary frequency and decreased bladder volume are both dose-related changes in mice. Submucosal fibrosis has been proposed to be secondary to surface ulceration, and perhaps related to vascular lesions. Similar lesions are observed in humans after urinary bladder irradiation. Increased risk of urinary bladder malignancies has been extensively studied in the Nagasaki and Hiroshima atomic bomb survivor cohorts, as well as in those populations exposed to highdose irradiation in Chernobyl. Among the former population, there is a doubling of relative risk for development of a urinary tract neoplasm in comparison to the nonaffected population, suggesting a significant correlation between irradiation and urinary bladder neoplasia. Furthermore, retrospective studies in women treated with clinical radiation therapy for cervical cancer showed an increased incidence of bladder cancer of over 57 times that of untreated women, and patients with various ovarian cancers receiving radiation therapy showed a two-fold increase in risk for developing urinary bladder malignancies.

4.11. Fetal Effects As sensitivity of a tissue to radiation insult is directly proportional to its rate of proliferation, the relative risk of the developing fetus to radiation injury would appear to be significant. Since radiation-induced embryonic mutation is a stochastic effect, any dose of radiation could have the potential to cause a phenotypic change. This area of research is one of the more controversial in the assessment of radiation risk, and the literature is full of conflicting data. Japanese atomic bomb survivors who were pregnant at the time of, or soon after, the detonations were monitored for congenital malformations in the newborns. Assessing malformations in isolation from the incidence of leukemias and solid tumors suggests no increased risk associated with radiation. Furthermore, an ongoing population study in Belarus compares pre- and postChernobyl accident data on rates of congenital malformation among aborted fetuses and in newborns. The study examined the incidence

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rate of a variety of congenital malformations, including anencephaly, spina bifida, palatoschisis, polydactyly, limb reduction defects, esophageal and anal atresia and stenosis, and Down’s syndrome. While a progressive increase in congenital malformations has been noted in areas of both high and low radioactive contamination, the increase does not demonstrate a dose–response pattern and the study found that there were significantly fewer congenital abnormalities reported in areas of higher amounts of contamination in comparison with those having received lower doses. The field of diagnostic imaging is acutely concerned with the described and potential risks, and the literature associated with those risks is extensive. The consensus of opinion appears to be that in the first 2 weeks of life, the embryo is considered to be radiosensitive to the lethal effects of radiation; however, doses significantly higher than those used diagnostically are required to cause fetal death. From the third to the eighth week of pregnancy, the embryo is in the period of early embryonic development but is not at increased risk of birth defects, pregnancy loss, or growth retardation unless the exposure is substantially above the 20 rad or 0.2 Gy exposure level. Subsequently, from the eighth to the 15th week of pregnancy, the embryo or fetus is considered to be highly susceptible to injury with regard to the developing central nervous system, but again in this case the dose required to elicit adverse effects has to be very high. The threshold has been estimated to be higher than 30 rad or 0.3 Gy before an effect can be seen on the Intelligence Quotient of the developing embryo. Animal studies in mice which have shown increased rates of p53-dependent apoptosis in the developing embryos do support the concern of embryonic risk.

4.12. Reproductive Tract Testes (See also Male Reproductive System, Vol 5, Chap 9). The effects of irradiation on the testes are most striking in the highly sensitive differentiating spermatogonia which are affected by only 20 cGy in rodents. After irradiation, testicular

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sperm counts remain constant for 20 days and then decrease to a minimum at 29 days. The constant sperm counts until 20 days indicate that the spermatocytes and spermatids are relatively resistant to irradiation. The decrease in sperm at 29 days indicates that spermatogonia have the highest sensitivity, as this is the time they would normally have become sperm. After this time, sperm counts may increase again, indicating recovery of stem cell spermatogonia. However, at fractionated doses above 6 Gy there is often incomplete recovery of sperm production, indicating residual damage to stem cells. Leydig cells and Sertoli cells are much less sensitive to irradiation than spermatogonia. Histologically, irradiated testes lack sperm, have rare spermatogonia, thickened basement membranes of seminiferous tubules, vascular sclerosis, and occasionally giant cell formation (Figure 14.44). In one study which examined male beagles used in chronic studies of the effects of inhaled plutonium, 166 cases of testicular neoplasia were observed in 105 dogs. The tumor types

FIGURE 14.44 Testis, seminiferous tubules: Hartley guinea pig. This animal received 5 Gy gamma radiation at 1 Gy/min (cobalt-60 source), 30 days prior. Diffusely there is atrophy and loss of germ cells with marked decrease in spermatocytes with shedding, rare degeneration and necrosis, complete absence of mature spermatids, and focal giant cell formation. H&E stain. Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.44, p. 1480, with permission.

comprised of 113 interstitial cell tumors, 46 seminomas, and 7 Sertoli cell tumors. Additionally, in a recent study, pregnant, genetically modified mice with a predisposition for testicular germ cell tumors, received two doses of 0.8 Gy. The incidence of tumor development in the offspring increased from 45% to 100%. Finally, retrospective studies of humans who received radiation for testicular cancer noted that 5% of those patients developed a contralateral testicular neoplasm. Ovary (See also Female Reproductive System, Vol 5, Chap 10). Radiation damage to the ovary is dependent on the age of the ovum and the development of the granulosa cell layers. Oocytes with few layers of granulosa cells are more radiosensitive than oocytes with more layers. This finding suggests that injury to the oocyte may be indirect with the granulosa cells being the target, possibly because the granulosa cells play a supportive role for the oocyte. In some species this may not be true. Radiation-induced ovarian fibrosis and progressive vascular sclerosis may also contribute to ovum death. Traditionally it was felt that the mature ovary could tolerate doses of 20 Gy before permanent sterility is observed; however, recent studies have determined that the radiation dose required to destroy 50% of primordial follicles (LD50) is between 2 and 4 Gy. In laboratory animals, oocyte radiosensitivity differs significantly between species, with the mouse oocyte being reported as approximately 350 times more radiosensitive than that of the monkey (50 Gy). Because all primordial ovarian follicles are formed prior to birth, in utero irradiation can have marked effects on the developing ovary. Doses to the fetus in the 1- to 2-Gy range can cause a marked reduction of follicles in the postnatal animal. Animal studies conducted in rodents noted that subsequent to low-dose irradiation, the mice and rats would be capable of reproduction only so long as mature ovum remained, after which they became sterile since the primordial follicles are highly radiosensitive and would be irrevocably damaged. Doses as low as 0.25 Gy would result in a 50% decrease in fecundity. Histological changes to the rat ovary subjected to a single dose of 7.5 Gy caused

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a significant decrease in follicle number, and surviving follicles were characterized by loss of the primordial follicles, disorganization of the granulosa cell wall, degeneration and necrosis of the remaining intermediate-sized follicles, and lipid degeneration of the corpora lutea. Radiation-induced ovarian neoplasia has been reported in atomic bomb survivors, and runs the gambit of epithelial, sex-cord, and germ cell tumors. The most common were epithelial forms, with a high incidence of malignancy. Germ cell tumors followed in terms of overall frequency, with high numbers of both benign and malignant teratomas being reported. Of the sex-cord stromal tumors, granulosa cell tumors and thecomas were the most commonly diagnosed (Figure 14.45).

FIGURE 14.45 Ovary: granulosa cell tumor. Expanding and replacing the ovarian stroma is a polygonal cell neoplasm forming occasionally rosette-like structures (arrow) containing a wispy eosinophilic hyaline material (Call–Exner bodies) (arrowheads), which stained positively for Periodic acid-Schiff (PAS). B6D2F1 mouse irradiated with a single whole-body dose of 9.25 Gy, 1½ years prior. H&E stain. Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.45, p. 1481, with permission.

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4.13. Integumentary System (See also Integument, Vol 5, Chap 7). General Reaction to Ionizing Radiation Injury Cutaneous radiation injury includes the reaction of the stratified keratinizing epidermis; the underlying dermis with its connective tissue, vascular, and nervous components; the adnexa, including hair follicles, sebaceous, apocrine and merocrine glands; and, to a lesser extent, the deeper subcuticular or hypodermal tissues. The epithelium and some hair follicles are in a constant state of cell turnover as basal cells divide, and undergo maturation and terminal differentiation. This rapidly dividing population of basal cells is highly radiosensitive and is responsible for some of the acute reactions seen during therapeutic irradiation, as well as in instances of both accidental and targeted whole-body irradiation. Late reactions of fibrosis, atrophy, necrosis, telangiectasia and, rarely, neoplastic transformation occur months to years after irradiation. In cases of accidental irradiation, as occurred in Chernobyl, a syndrome of cutaneous pathology has been described which has been associated with over 50% of the radiationassociated mortalities from the nuclear accident. The pathological findings in humans range from a transient erythema with occasional blister formation, desquamation, ulceration, and superficial necrosis to severe erythema, with coalescing bullae, deep ulceration, necrosis, and hemorrhage progressing to rhabdomyocytic and osseous necrosis. Severe cutaneous lesions may compound the effects of the other radiation syndromes, and greatly impede the victims’ ability to heal. This is upheld historically in examples from Hiroshima and Nagasaki, as well as in association with contamination from Chernobyl, in which severe irradiation of the skin and its downstream effects can have a significant accentuating effects on both morbidity and mortality. Understanding of this cutaneous syndrome is critical in that it illustrates the difficulty to the pathologist of considering radiationinduced damage in isolation, rather than in the context of combined injury, especially in situations outside of clinical use of radiation as a treatment modality.

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The early reaction of the skin to irradiation is a transient erythema, which occurs within hours of exposure to fractionated doses of 15–20 Gy. In this early phase, there is rapid production and activation of cytokines released by the irradiated cells. These induce the expression of a series of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and E-selectin, all of which contribute to increased vascular permeability, edema, attraction of inflammatory cells, and transmigration of cells into tissue. Within 1 week of irradiation, a more prolonged erythema occurs. This erythema is dosedependent, and the time to development depends on the species and the site irradiated. During this time, there is depletion of the rapidly proliferating basilar epidermal stem cells. Studies have demonstrated mitotic inhibition of germinal cells within the adnexae and the epithelium occurring at doses as low as 2– 4 Gy, while induction of alopecia has been described in atomic bomb survivors at estimated doses of 0.75 Gy. Having lost the ability to regenerate and maintain the process of keratinocyte turnover, the initial insult can lead to ulceration. In the pig, there is an initial decrease in proliferation in the basal cell layer leading to a decrease in cell density of the epidermis. Degeneration and necrosis of the basal cells may also be seen. Proliferative capability begins to return to normal within 2 weeks following exposure. In the dermis, capillaries may still be dilated and perivascular edema and inflammatory cell infiltrates are common. Additional changes also present at this early stage include endothelial cell swelling, proliferation, necrosis, mononuclear perivascular inflammation, and, occasionally, microthrombosis. In minipigs irradiated with 1.6–2 Gy, the depletion of thrombocytes and microvascular damage frequently present as miliary petechiae and coalescing ecchymoses which, depending upon dose, can be limited to friction points or randomly distributed throughout the integument (Figure 14.46). This first acute skin reaction is the result of a combination of vascular, epithelial, and dermal responses, but the epithelial responses are probably the most significant. Also during this time there is increased TGF-b1 expression in the suprabasal layers of the skin and in the dermis,

FIGURE 14.46 Haired skin: Gottingen minipig irradiated with a single whole-body dose of 1.8 Gy, 30 days prior. Diffusely, there are cutaneous petechiae and ecchymoses along the ventrum. This animal was severely thrombocytopenic, anemic, and lymphopenic at presentation for necropsy. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.46, p. 1482, with permission.

which peaks at 14 days after 20–50 Gy. The increase persists until 30 days after irradiation. TGF-b1 is central to the pathogenesis of radiation-induced injury, upregulating itself and the cell types involved in the acute stages and inducing fibrotic disease in late stages. Studies examining the role of TGF-b1 in pigs subsequent to irradiation with single doses ranging from 16 to 64 Gy noted increased production of TGF-b1 in endothelium, fibroblasts, and keratinocytes. In irradiated mice receiving targeted 50 Gy, TGF-b was found to have three waves of expression. The first occurred rapidly after irradiation (6–12 h), followed by a second wave between 14 and 28 days and, finally, by a third occurring between 6 and 9 months postinsult. The waves correlated with an acute inflammatory response and subsequently with cellular activation and directed remodeling of the injured tissue with fibrosis (Figure 14.47). At lower doses, the acute cutaneous reaction usually regresses and the skin returns to normal. However, if the dose is high enough, a second wave of skin reaction occurs. In the pig, this second reaction occurs 2–4 months after irradiation and is characterized by persistent

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FIGURE 14.47 (A) Haired skin: B6D2F1/J mouse having received a single thoracic dose of 8.25 Gy, 3 months prior. Dermal and subcuticular fibrosis, diffuse, severe with marked acanthosis, hyperkeratosis and atrophy, and loss of adnexa. (B) The use of the Masson’s trichrome highlights the deposition of collagen and the spread of the fibroplasia to incorporate the whole of the dermis, subcutis, and expanding into and replacing the muscularis carnosus. (A) H&E stain. Bar ¼ 200 mm. (B) Masson’s Trichrome stain. Bar ¼ 200 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.47, p. 1482, with permission.

microvascular damage resulting in erythema and hypoxic dermal necrosis. Capillary density in the dermis decreases and focal edema is present. Blood and lymphatic flow are decreased at 9– 12 weeks after irradiation. Late effects are often mediated by microvascular damage and the progressive irradiationinduced fibroplasias. The vasculature may

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exhibit medial necrosis of arterioles and small arteries in the deep dermis, and telangiectasis in the upper dermis. Fibrosis progressively increases within the dermis and the subcuticular tissues. The overlying epidermis may be thinned to 1 cell layer, and undergo either hyper- or hypopigmentation, with loss of adnexa or dysplasia of keratinocytes. In some cases, the epidermis may become completely necrotic and ulcerated due to a combination of epidermal and dermal atrophy and decreased vascularization of the affected tissue. Furthermore, alopecic regions have increased susceptibility to trauma and secondary bacterial infections. Irradiation induces melanocytes in the skin to increase melanosome production, which results in hyperpigmentation. However, high radiation doses can destroy follicular melanocyte stem cells, which then results in hypo- or depigmentation (Figure 14.48). Repigmentation of radiationdamaged coloration depends on available melanocyte stem cells from three possible sources: from the hair follicle itself, which is the principle provider of pigment cells; from the border of the radiation lesion; and from unaffected melanocytes within depigmented areas. Premelanocytic pigment cells in these areas can initiate a perifollicular repigmentation pattern. However, in order for repigmentation to take place, melanocyte tissue stem cells located in the niche at the bulge region of the hair follicle must remain in adequate numbers to provide immature pigment cells which can progress to

FIGURE 14.48 C57BL/6 mouse irradiated with single thoracic dose of 16 Gy. This demonstrates the focalized damage to follicular melanocytic stem cells within the beam of radiation (white thorax). This animal was treated with a radiation countermeasure allowing survival and regrowth of adnexa. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.48, p. 1483, with permission.

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terminal differentiation and start repigmentation of the hair follicles. Langerhans cells, which are derived from bone marrow precursors and take part in immune responses in the epidermis, may decrease in number in mouse skin 8– 12 days after 20 Gy but then return to normal numbers as cells migrate in from the blood. At 19 and 20 months after irradiation, a second decrease in the number of Langerhans cells occurs due to damage to the blood vessels and connective tissue in the dermis inhibiting replacement of cells. Hair follicles react in a manner similar to the epidermis. Early damage to the hair follicles is probably a result of damage to the basal cells, and results in hair loss. As hair follicle proliferative activity is normally cyclic, the extent of radiation damage to the hair and extent of alopecia depends on the stage of cycle the hair follicle was in when irradiated. Later developing, more permanent, damage to the hair follicle may be secondary to changes in dermal microvasculature and progressive fibrosis. Sebaceous glands appear to be quite radiosensitive, and decrease in number within 3–4 weeks of irradiation. Sweat glands are less sensitive, and do not decrease in number until 3–4 months after irradiation. At lower doses, the glands may regenerate. Permanent atrophy of these glands is often accompanied by early periglandular infiltrates of lymphocytes and plasma cells, and fibrosis. The pathogenesis of the effects of irradiation on these glands is not well understood. While the skin is not considered to be a sensitive tissue with respect to ionizing radiationinduced carcinogenesis, both squamous cell and basal cell carcinomas can be induced in humans and animals with high enough doses. Furthermore, a variety of vascular proliferative responses have been described as late effects following targeted irradiation of skin. Both benign and malignant growths can occur in humans, including benign lymphangioendothelioma, benign lymphangiomatous papules, and angiosarcomas. For additional information regarding ionizing radiation see Ainsbury et al. (2016), Altman and Gerber (1983), Baselet et al. (2016), Belinsky et al. (1996), Benjamin et al. (1998), Cappuccini et al. (2011), Casarett (2021), Cerveny et al. (1989), DeGroot (1988), Fajardo et al. (2001), Fajardo (2005), Gourmelon et al. (2005), Hahn and

Lundgren (1992), Hahn et al. (1996), Hill (2005), Hollander et al. (2003), Hubert and Bertin (1993), Little (2009), Mayo et al. (2010), Meineke (2005), Movas et al. (1997), Murro and Jakate (2015), NCRP (1985), Northdurft (2011), Richardson and Hamra (2010), Shrieve and Loeffler (2011), Thompson (1989), Uozaki et al. (2005), Williams (2009), Williams (1991), Yahyapour et al. (2018).

PART II ULTRAVIOLET RADIATION 5. Nature and Action of Ultraviolet Radiation Ultraviolet (UV) radiation includes wavelengths of electromagnetic energy between 10 and 400 nm, thus bridging the gap between ionizing radiation (X-rays) and visible light. By convention, UV radiation is subdivided into extreme UV (10–120 nm), far UV (120–200 nm), UVC (200–280 nm), UVB (280–320 nm), and UVA (320–400 nm) regions. Sunlight includes all UV wavelengths; however, the Earth’s atmosphere attenuates sunlight by processes of absorption and scattering, screening out UV wavelengths shorter than 280 nm. Because of this screening, biologically relevant wavelengths of UV include only the UVA and portions of the UVB regions of the spectrum. Removal of shortwavelength UV is due primarily to stratospheric ozone. Recently, focal thinning of the stratospheric ozone layer has been observed in the spring near the South Pole and attributed to ozone destruction catalyzed by free chlorine released from technogenic chlorofluorocarbons. It is predicted that global decreases in stratospheric ozone will result in increased UV exposure and associated adverse health effects. An epidemiological study which examined white populations in Europe, the United States, Canada, and Australia determined that the average increase of skin cancers other than melanoma (i.e., basal cell carcinomas and squamous cell carcinomas) has been 3%–8% per year since the 1960s. These rising incidence rates are theorized to be associated with increased UV exposure due to lifestyle changes, as well as increased longevity, and global ozone depletion. When UV interacts with matter, it behaves as though composed of particles (termed

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“photons”). The energy of a photon is transferred to the electron of an atom or molecule, resulting in an excited state. The electronic excitation energy is then dissipated by releasing the energy as heat or light, by losing an electron to form a free radical or ion, by using the energy to drive chemical reactions with other molecules, or by undergoing fragmentation. For a molecule or atom to absorb photons of a given wavelength, it must have electrons in appropriate energy levels. Thus, not all molecules absorb all UV wavelengths. A molecule that absorbs a given UV wavelength is a “chromophore” for that wavelength. UV doses are expressed as energy per unit area, typically as Joules/m2. The biological effects of UV depend not only on the total energy of UV absorbed, but also on the wavelength of that energy. The “action spectrum” expresses the functional relationship between biological effect and wavelength for a given UV dose. Action spectroscopy can help identify the UV chromophore ultimately responsible for initiating a given biological response. Studies of the action spectrum for UV-induced skin cancer indicate that the most effective wavelengths are those that can penetrate the skin and damage DNA, suggesting that DNA is a major chromophore for this response.

6. MECHANISMS OF ULTRAVIOLET RADIATION INJURY Biologically important cellular targets for UV include DNA, RNA, proteins, and lipids. Carbohydrates do not absorb light above 230 nm, thus they are unaffected by UV radiation reaching the Earth’s surface. UV effects on biological molecules may be direct or photosensitized. In photosensitivity reactions, UV-activated intermediate compounds such as free radicals actually mediate UV effects. Most UVA-induced cellular damage is due to the formation of activated oxygen species such as peroxides, superoxide anion, and hydroxyl radical. Pyrimidines in nucleic acids (maximum absorption at 260 nm) appear to be more sensitive to direct UV damage than purines. Pyrimidine dimers are the most numerous pyrimidine lesions; (6–4) adducts form much less frequently. Cyclobutane

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pyrimidine dimers can form between any two adjacent pyrimidines, but (6–4) adducts form only at TC, CC, and TT pairs. These DNA photoproducts appear to be responsible for many of the adverse cellular effects of UV, including cell death and mutation. Unlike pyrimidines, purines are fairly insensitive to direct photochemical interactions but do undergo photosensitized reactions. Because of the short half-life and relative abundance of RNA, the effects of RNA photoproducts are difficult to assess. The only amino acids that are excitable by UV are tryptophan, tyrosine, and cysteine, with maximal absorption at 280 nm for tryptophan and tyrosine, and absorption over a broad range of UVB wavelengths for cysteine. Tryptophan is the major chromophore of proteins. When tryptophan absorbs UV, the indole ring is photo-ionized to form a neutral indolyl radical and a hydrated electron. Reactions of the indolyl radical subsequently form a variety of products, including potent photosensitizers. The hydrated electron is scavenged by oxygen to form the superoxide radical ion. Cysteine and tyrosine can undergo direct or sensitized photolysis. In the case of cysteine, interchain disulfide bridges may thus be split. Methionine, histidine, cysteine, and tryptophan can be photo-oxidized to cause protein denaturation. Lipids do not absorb UV above 290 nm, with the exception of the vitamin D precursor 7-dehydrocholesterol, and are thus not susceptible to direct UV damage. However, photosensitized oxidation of lipids is an important mechanism for UV-induced changes in biological membrane function. Photoreactivation, nucleotide excision repair, or postreplication repair may repair DNA photoproducts. Photoreactivation of pyrimidine dimers and (6–4) photoproducts is an error-free repair system catalyzed by specific photolyases that recognize and bind to the appropriate photoproduct, absorb long-wavelength visible light, and use the absorbed energy to drive the enzymatic monomerization of the photoproduct. While most species appear to possess a (6–4) photolyase, placental mammals seem to lack a pyrimidine dimer photolyase. Excision repair acts on a variety of bulky DNA adducts, such as photoproducts, to introduce single-strand nicks on either side of the adduct, to remove a short segment of the DNA strand containing

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the lesion, and to resynthesize the missing DNA using the remaining strand as a template. Adverse cellular effects of UV include cell death (characteristically by apoptosis), cell cycle arrest, mutation, and altered gene expression. DNA damage plays a major role in initiating these responses; however, activation of cytoplasmic signaling pathways also contributes to some UV effects. The protein product of the p53 tumor suppressor gene is an important mediator of UV-induced cell cycle arrest and apoptosis. Exposure to UV results in rapid and prolonged nuclear accumulation of P53 protein. UVinduced G1 arrest requires P53-dependent transcription of the p21WAF1/CIP1 gene. The pathway linking P53 stabilization and apoptosis has not been elucidated and is currently the subject of intense investigation. The “UV response” is an ordered sequence of alterations in gene expression similar to that induced by many growth factors. The genes activated include those for a variety of transcription factors, cytokines and growth factors, and proteases. UV is believed physically to activate growth factor receptors on cell surfaces in a ligand-independent manner. The epithelial growth factor (EGF) receptor appears to be particularly important in mediating UV responses. Receptor stimulation, in turn, leads to activation of cytoplasmic signaling pathways, including the mitogen-activated protein kinase cascade. Both cytoplasmic signaling pathways and DNA damage responses initiated by UV ultimately modulate the activity of transcription factors, particularly AP-1, P53, and NF-kB, which coordinately regulate the expression of many genes. An important area of consideration with regards to UV radiation is the possibility of diverse organic and chemical compounds to become either directly or indirectly phototoxic when exposed to a range of different light sources. When certain pharmacological agents such as psoralen, quinolone-based antibiotics, antiinflammatory drugs, and certain antidepressants or agrochemicals are exposed to light, they have the potential to become toxic in animal models and/or in humans. The pathology which has been described in such cases is most often associated with the integumentary, ocular and gastrointestinal systems. In the skin, erythema, pruritis, and edema, which would be equivalent to

a significant sunburn, are often reported. In the eyes, compounds with tricyclic, heterocyclic or porphyrin ring structures have the potential to be an ocular chromophore. With UV light, there is a risk of lens injury, while, with exposure to visible light, the retina is also under threat of insult. Drugs frequently undergo phototoxicity assessment with exposure to a range of variable radiation sources, to include visible light, infrared light, xenon arc solar simulators, and fluorescent UV radiation. A variety of animal models have been developed to support this area of research, including guinea pigs, mice and rats such as the pigmented Long Evans or albino Sprague Dawley. A review conducted in 2018 evaluated 240 published reports of drug related phototoxicity comprising 1134 cases correlated with 129 different drugs. Excluding cases deemed to be photoallergy, almost a further 90% of the studies were deemed to be based on questionable data. Those medications for which the review determined that there was significant evidence to suggest a correlation with phototoxicity included vemurafenib, nonsteroidal antiinflammatory drugs (NSAIDs), as well as fluoroquinolone and tetracycline antibiotics. Retinal phototoxicity has been reported as occurring through two different mechanisms. Type 1 is defined as caused by direct reactions involving proton or electron transfers and Type 2 is due to reactive oxygen species reactions. Retinal UV damage has been reported in association with a range of commonly prescribed drugs, including certain antibiotics, diuretics, NSAIDs, psychotherapeutic agents (benzodiazepines), and even herbal medicines.

7. RESPONSE TO INJURY INDUCED BY ULTRAVIOLET RADIATION 7.1. Integument (See also Integument, Vol 5, Chap 7). Most incident UVB and UVC radiation is absorbed in the epidermis by keratin of the stratum corneum, epidermal melanin, and keratinocyte DNA. In contrast, UVA can penetrate to and be absorbed in the dermis. Acute skin responses to UV include the edema, erythema, and desquamation that characterize sunburn. Action spectroscopy indicates that

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UVB and UVC are considerably more effective than UVA in eliciting these changes. Edema and erythema are due to vascular changes in the dermis. UVA, UVB, and UVC induce erythema responses with different time courses. UVA induces a short-lived immediate increase in vascular permeability followed by a delayed response after 2–8 h. UVB-induced erythema peaks at 6–24 h after exposure, then fades gradually; a second peak is sometimes seen at about 48 h. Early phase erythema is responsive to nonsteroidal antiinflammatory compounds, but not to antihistamines. Late phase erythema does not respond to either. UVC-induced erythema is maximal at 8 h and fades over the next day or two. Inflammatory mediators released from both the epidermis and dermal mast cells appear to stimulate vascular changes. A transient perivascular inflammatory cell infiltrate is seen in UV-exposed skin. A common measure of skin responsiveness to UV is the minimal erythemal dose (MED). The MED is the lowest UV dose required to stimulate, on previously unexposed skin, just perceptible erythema 24 h after exposure. In experimental animals, measurement of skin thickness (edema) can also be used to determine the MED. UV-induced cell death in the epidermis is maximal at about 24 h after exposure. Cell death occurs by p53-dependent apoptosis, and apoptotic epidermal cells are termed “sunburn cells” (Figure 14.49). In hematoxylin and eosinstained sections, they have pyknotic nuclei and deeply eosinophilic, homogeneously stained cytoplasm. UV exposure also stimulates a later phase of epidermal hyperplasia and parakeratosis with desquamation, peaking a few days to a week after exposure. The epidermal melanocyte synthesizes melanin and donates it to surrounding epithelial cells. In humans, an epidermal melanin unit consists of 1 melanocyte surrounded by approximately 36 keratinocytes. Melanocytes synthesize both brown to black eumelanin and light brown to red pheomelanin. Constitutive skin color is due to genetically determined melanin levels, while facultative skin color is the UV- or pituitary-hormone-induced increase in melanin pigmentation above constitutive skin color. In humans, an increase in epidermal melanin, which absorbs and scatters radiation over a broad

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FIGURE 14.49 Sunburn cell (arrow) in the epidermis of Monodelphis domestica exposed 31 h previously to a UV dose of 500 J/m2 UVB (approximately 0.75 MED). Note the homogeneous appearance of the cytoplasm and the pyknotic and fragmented nucleus of the sunburn cell. Removing pyrimidine dimers by photoreactivation markedly reduced the number of sunburn cells in the UV-exposed skin of this animal, implicating DNA damage as the initiating lesion. H&E stain. Bar ¼ 20 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Vol. 1, Fig. 32, p. 582, with permission.

spectral range, is the major adaptive response to UV exposure. Facultative skin color in response to UV exposure can be due to “immediate” or “delayed” tanning. Immediate tanning (immediate pigment darkening, Mierowsky phenomenon) occurs within minutes of UV exposure, and reaches a maximum in about an hour. UVA wavelengths are more effective than UVC or UVB in producing immediate tanning, although visible light can also induce the phenomenon. Immediate tanning is caused by photo-oxidation of preformed melanin, migration of melanosomes from the cell body to the cell processes of melanocytes, increased transfer of melanosomes from melanocytes to epidermal cells, and changes in the distribution of melanosomes within keratinocytes. There is no change in size of melanosomes or number of melanocytes. In delayed tanning, however, there is an increase in both the number and size of functional melanocytes, accompanied by increased melanosome synthesis, increased size and melanization of melanosomes, and increased transfer of melanosomes from melanocytes to keratinocytes. Delayed tanning becomes visible

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approximately 72 h after UV exposure and increases for several days thereafter. All UV wavelengths, as well as blue visible light, can induce delayed tanning, but UVB is the most effective portion of the spectrum. The skin of the trunk of many nonalbino rodents is unpigmented, with melanocytes restricted to hair follicles; therefore, few rodents tan in response to UV exposure. In humans, chronic exposure to UV causes photoaging and skin cancer. Photoaging is characterized grossly by laxity, roughness, sallowness, irregular hyperpigmentation, and telangiectasia. Microscopic changes include increased deposition and degeneration of elastic fibers (elastosis), decreased insoluble collagen, increased glycosaminoglycans, chronic dermal inflammation, and focal epidermal hyperplasia and dysplasia (solar keratosis). Solar keratosis and elastosis have been reproduced in hairless mice (Figure 14.50). UVB is considerably more effective than UVA in inducing photoaging in this animal model. Photosensitizers are endogenous or exogenous compounds that are readily activated by UV or visible light and, once activated, induce an adverse cutaneous response. UVA is the most effective of the UV spectral ranges in eliciting

FIGURE 14.50 Haired skin: nude mouse. UV exposure resulting in solar elastosis, zonal thinning (arrow) and hyperplasia (arrowhead) of the epithelium, dermal fibrosis, and a moderate necroulcerative dermatitis (asterisk) with edema. H&E stain. Bar ¼ 100 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.50, p. 1487, with permission.

photosensitization reactions. Exogenous photosensitizers may reach the skin by topical or systemic routes. Most photosensitizers are unsaturated tricyclic aromatic rings in linear arrangement, many have a lone pair of electrons not involved in bonding, and most are fluorescent. Efficient photosensitizers include coal tar derivatives, chlorothiazides, porphyrins, phenothiazines, sulfonamides, and tetracyclines. Photosensitivity can manifest as phototoxicity or photoallergy, and many chemicals that are phototoxic may also act as photoallergens. In veterinary medicine, photosensitization diseases can be induced by both exogenous and endogenous routes, and present clinically as cutaneous damage to unpigmented skin which is not covered by heavy wool or hair. These are typically categorized into three types of photosensitization. Type I or primary photosensitization occurs subsequent to the ingestion of preformed photodynamic agents in plants such as St John’s Wort (Hypericum perforatum), buckwheat (Fagopyrum esculentum), Bishop’s weed (Ammi majus),and spring parsley (Cymopterus watsonii), all of which can cause disease in large animals. This form of the disease can also present with ingestion of drugs such as tetracyclines and phenothiazine, as discussed earlier in humans. The second type is associated with abnormal porphyrin metabolism and with congenital enzyme defects which cause abnormal synthesis of uroporphyrin and coprophyrin. Finally, the third type is termed hepatogenous, and is associated with inherited hepatic defects, such as portosystemic shunts, or defects secondary to hepatic injury which reduces the liver’s ability to process, metabolize, and excrete photodynamic agents. Phototoxic reactions are characterized grossly by erythema, and sometimes edema, occurring during or immediately after exposure. Variable degrees of desquamation and hyperpigmentation follow. Microscopically, intracellular edema in the epidermis and necrosis of keratinocytes is seen and there is little dermal involvement. Phototoxicity is believed to be due to the formation of excited triplet states, free radicals, and peroxides. These damage cell membranes to cause release of lysosomal contents and mast cell degranulation. The severity of the reaction is proportional to the dose of photosensitizer or UV. Phototoxicity does not require an allergic response.

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Photoallergy, on the other hand, involves a circulating antibody or a cell-mediated immune response. Thus, photoallergy differs from phototoxicity in requiring an incubation period after first exposure to the photosensitizer and repeated exposure to light or UV in previously sensitized individuals. The response is not linearly dose-related. Photoallergy is characterized clinically by immediate erythema and urticaria or by delayed papular or eczematous dermatitis on sun-exposed skin. There is usually a dense lymphocytic infiltrate in the dermis, and epidermal spongiosis or vesicle formation may occur. The urticaria is associated with degranulation of mast cells at the site of UV exposure, increased numbers of eosinophils, and release of neutrophil chemotactic factors. Several mechanisms for the photoallergic reaction have been proposed. First, absorption of radiant energy by the photosensitizing molecule may lead to formation of a photohapten that binds to a carrier macromolecule in the skin to form a photoantigen. Second, radiation may alter a tissue protein to allow it to serve as a carrier for the photosensitizer or its photoproduct, thus forming an antigenic carrier–hapten complex. Finally, radiation in the presence of a photosensitizer may alter a tissue component to transform it into a tissue antigen. An additional complication associated with photoinjury occurs with the exacerbation of certain critical autoimmune diseases described in veterinary species. Discoid lupus erythematous (DLE), colloquially termed “collie nose,” affects a variety of dog breeds and is typically localized to the skin. The disease is caused by antigen–antibody complexes which deposit in tissue and cause a Type III hypersensitivity reaction. The second disease of import, systemic lupus erythematous (SLE), a more severe form of the autoimmune disease affecting multiple organ systems, has been described in dogs, cats, horses, humans, nonhuman primates, mice, snakes, and iguanas. In both cases, the UV-exacerbated cutaneous lesions are induced through the translocation of antigens to the keratinocyte cell membrane. Damaged keratinocytes then release IL-1, IL-2, IL-6, and TNF-b which further promote the damage. UV irradiation also induces expression of ICAM-1, which is the major ligand for LFA-1, an adhesion molecule found on all leukocytes, and thereby increases the inflammatory response.

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7.2. Eye (See also Special Senses – Eye, Vol 4, Chap 9). UV wavelengths less than 315 nm are largely absorbed in the cornea and conjunctiva. With acute UV exposure, photoconjunctivitis and photokeratitis can develop following a latent period of 30 min to 12 h, and last up to 48 h. Clinical signs include photophobia, lacrimation, blepharospasm, and scleral and conjunctival vasodilation. Microscopically, edema and an acute inflammatory cell infiltrate are seen. In severe cases, blistering and ulceration of corneal and conjunctival epithelium and edema of underlying stroma may occur. Repair of DNA damage and replacement of lost cells in the corneal epithelium is very rapid. With chronic UV exposure in experimental animals, neovascularization of the cornea may develop. In vitro studies in several species, including humans, and in vivo studies in laboratory animals have provided considerable information about UV effects on the lens. The lenses of diurnal animals, like humans, contain yellow pigments that effectively absorb 300- to 400-nm light. In addition, chromophores such as riboflavin and tryptophan can also absorb these wavelengths, leading to the formation of free radicals. These highly reactive compounds can trigger a variety of chemical reactions, including formation of singlet oxygen, lipid peroxidation, protein cross-linking, and enzyme inactivation. The epithelium and outer portion of the lens sustain the major direct damage. Adverse UV effects on the lens include DNA damage to epithelium, decreased sodium–potassium ATPase activity, disruption of actin filaments, aggregation and breakdown of crystallins, and alterations in the oxidation–reduction balance (decreased glutathione levels, increased mixed disulfides, and decreased –SH groups). Swelling, degeneration, and loss of anterior lens epithelium, swelling and disruption of lens fibers, and formation of amorphous protein aggregates is seen microscopically. Grossly, yellowing of the lens and the development of lens opacities is seen, and, ultimately, cataracts are formed. Loss of visual acuity is due both to increased light scattering by and increased autofluorescence of altered lens components. In general, the retina is protected from the effects of UV by the lens. However, in animals

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without yellow lens pigments, in aphakic animals and humans, and in those exposed to extremely high levels of UV, UV radiation in the 300–400 nm range can cause irreversible retinal degeneration and atrophy.

7.3. Immune System (See also Immune System, Vol 5, Chap 6). UV is an immunosuppressive agent. Acute or chronic low-dose UV exposure inhibits both tumor rejection and the development of contact hypersensitivity. UV-induced skin tumors are highly antigenic, and are rejected when transplanted into syngeneic mice. However, if the recipient mice are pretreated with UVB, the transplanted tumors are not rejected. This tolerance is specific for UV-induced tumors; chemically induced tumors and skin allografts are rejected normally. Contact hypersensitivity is a cell-mediated immune response elicited by skin application of a sensitizing dose of hapten followed, days or weeks later, by cutaneous challenge with the hapten. When the sensitizing hapten is applied either to UVB-irradiated or unirradiated skin of mice that were immunized in an irradiated area, little response is seen upon challenge. This suggests that immunosuppression is both local and systemic in nature. As for tumors, hapten tolerance is specific. Both for tumors and for haptens, specific UVinduced tolerance can be adoptively transferred with splenic suppressor T cells, suggesting that maintenance of tolerance is an active process. In all species, the major antigen-presenting cell of the skin is the bone marrow-derived Langerhans cell, a specialized epidermal dendritic cell capable of presenting antigen to Th1 and Th2 helper T lymphocytes. In addition, the mouse has dendritic epidermal T cells and both human and mice have dermal dendritic cells that can present antigen to suppressor T cell populations. Epidermal and dermal dendritic cells capture antigen in the skin and migrate via lymphatics to regional lymph nodes, where they present antigen to the appropriate T lymphocytes. One of the primary targets of UV-induced immune suppression is the Langerhans antigen-presenting cell in the skin. UV irradiation has been shown to impair the function of these antigen-presenting cells both by direct UV effects on the cells themselves and by the

production of soluble mediators that act indirectly on the cells. Upon exposure to even suberythemal doses of UVB radiation, the majority of Langerhans cells either die or migrate from the skin to the regional draining lymph node, while the remaining Langerhans cells at the point of injury appear contracted, with loss of dendritic processes and decreased ATPase and major histocompatibility class II antigen reactivity. Additionally, mast cells have been shown to be involved through UVradiation–induced release of calcitonin gene-related peptide which results in increased mast cell production and release of TNF-a. Furthermore, mast cells have been shown to migrate to regional skin-draining lymph nodes subsequent to UV irradiation, and once there participate in the propagation of the immune suppressive response. At higher doses of UV irradiation, there is increased blood flow to the site, with increased production of mediators released from damaged keratinocytes. Candidate mediators include prostaglandins, cytokines, and urocanic acid, a component of the stratum corneum that isomerizes in response to UV from the trans form to the immunosuppressive cis isomer. These soluble factors attract IL-10 secreting macrophages, mediated by regulatory T cells. This results in an active process of systemic immunosuppression, compounding the local passive damage to the antigen-presenting cells, and the decrease in activated effector T cells. Recent evidence supports that theory that UV does more than physically deplete or functionally inactivate Langerhans cells; rather, the insult converts these cells from immunogenic to tolerogenic antigen-presenting cells that induce anergy. Keratinocytes constitutively express rather low levels of cytokines, neuroendocrine hormones, and other immunomodulatory molecules. However, as shown in Table 14.3, UV exposure dramatically increases the production of a variety of these substances. The role of these molecules in UV-induced immunosuppression is under investigation in a number of laboratories. In the case of contact hypersensitivity, there is considerable evidence to suggest that keratinocyte-derived TNF-a is an important mediator of UVB effects. Furthermore, UVinduced degranulation of dermal mast cells releases interleukin-1, TNF-a, and histamine.

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TABLE 14.3

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Immunomodulatory Molecules Released From UV-Exposed Keratinocytes

Modulator

Effects

Interleukin-1a (IL-1a)

Fever, induction of acute phase proteins; cytokine induction in several cell types; T lymphocyte chemotaxis; costimulation of B lymphocytes; stimulation of mediator production and cytotoxic activity by macrophages; increased ICAM-1 and ELAM-1 expression by vascular endothelium

Interleukin-6 (IL-6)

Fever, induction of acute phase proteins; costimulation of T cell proliferation, stimulation of natural killer and cytotoxic T cell activity; B cell differentiation, proliferation, immunoglobulin production

Interleukin-8 (IL-8)

Neutrophil, basophil, lymphocyte chemotaxis; neutrophil enzyme release

Interleukin-10 (IL-10)

Inhibitor of Th1 effector function and IL-1, IL-2, IFN-g, GM-CSF, TNF-a production; thymocyte and mast cell costimulation; inhibitor of antigen presentation by macrophages and B cells

Tumor necrosis factor-a (TNF-a)

Cachexia, fever, hemorrhagic necrosis of tumors; neutrophil, eosinophil, macrophage, fibroblast activation; ICAM-1 expression by vascular endothelial cells; costimulation of B and T cells; stimulation of MHC I and II antigen expression on various cells

Granulocyte/macrophage colony-stimulating factor (GMCSF)

Nonspecific stimulation of hematopoietic cell proliferation; stimulation of neutrophil, eosinophil, macrophage function; inhibition of neutrophil migration; induction of phagocytosis, eicosanoid production, and antibodydependent cell-mediated cytotoxicity by macrophages

Interleukin 3 (IL-3)

Nonspecific stimulation of hematopoietic cell proliferation; induction of phagocytosis by macrophages

Basic fibroblast growth factor (bFGF)

Proliferation of multiple cell types; angiogenesis

Transforming growth factor a (TGF-a)

Keratinocyte proliferation

Nerve growth factor (NGF)

Poorly characterized effects on hematopoietic cell differentiation and immune cell function

aMelanocyte stimulating hormone (aMSH)

Antagonism of IL-1 and TNF-a effects; stimulation of natural killer cell activity; reduction in MHC class I antigen expression; modulation of IgE synthesis

Adrenocorticotropic hormone (ACTH)

Modulation of IgE synthesis

Prostaglandins (E2, F2a) Tissue inhibitor of metalloproteinase-3 (TIMP3)

Increased vascular permeability, vasodilation; neutrophil chemotaxis Downregulation in keratinocytes leading to upregulation of MMP1, TNF-a, CXCL1, and IL-8 promoted by C/EBP (a CCAAT-enhancer-binding protein) all associated with tissue remodeling and inflammatory signaling pathways Contributes to UV-induced immunosuppression through upregulation of multiple genes UV radiation exposure results in PAF secretion which stimulates mast cells to undergo epigenetic modifications and increased response to CXCR4 agonists Increased ROS production activates multiple signaling pathways involved in the stress response (translocation of AP-1 and NF-kB) UVB activates NLRP3 inflammasome which initiates inflammatory responses through activation of IL-1b and IL-18 and resulting in inflammation-dependent cell death (pyroptosis)

Urocanic acid trans-isomerization (cis-UCA) Platelet activating factor (PAF) Reactive oxygen species (ROS) Inflammasome

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UV exposure can exacerbate or otherwise alter the pathogenesis of a variety of human diseases, including herpes simplex dermatitis and SLE. The immunomodulative effects of sunlight have been known since the late 1800s, with successful therapy of lupus vulgaris, a skin disease caused by Mycobacterium tuberculosis infection. However, as discussed earlier, UV radiation exerts significant suppressive effects on immune responses to UV-induced skin cancer cells, and experimental studies of the inhibition of tumor rejection suggest that this immunosuppression may be adoptively transferred by T lymphocytes in an antigen-specific manner. The field of research examining the phenomenon of UV radiationinduced suppression of immune responses, also called immune tolerance, has given rise to the field of photoimmunology. This systemic immune suppression has been reported in association with certain skin neoplasms, as well as having been illustrated through infections with certain papillomaviruses which subsequently progress to the development of nonmelanoma skin cancer with UV exposure. Additionally, the literature describes the UV-induced modified immune response to certain chemical antigens and also in conjunction with selected infectious agents such as hepatitis C and Candida albicans. It is important to note that bacterial superinfections following extensive UV irradiation injury are rarely reported and are not considered to be a significant risk factor. The fact that UV is so potently immunosuppressive has raised fears concerning ozone depletion and initiated significant research into the potential for increased UV exposure resulting in increased risk of infectious disease worldwide.

7.4. Ultraviolet Radiation Carcinogenesis Epidemiologic Evidence Evidence for the role of sunlight in nonmelanoma skin cancer (NMSC) in humans (basal cell and squamous cell carcinomas) is based on a number of observations. First, people with light skin color are more susceptible to NMSC than those more heavily pigmented. Second, the frequency of NMSC in light-skinned people increases near the Equator, where solar radiation is high. Third, those who spend much of their time outdoors have a higher incidence of

NMSC than those staying mostly indoors. Fourth, NMSC develops predominantly on sun-exposed parts of the body. Fifth, Xeroderma pigmentosum patients unable to repair DNA photoproducts develop NMSC in sun-exposed areas at a frequency much greater than DNA repair-proficient individuals. Finally, NMSC can be produced in mice by chronic UVB irradiation. Although sun exposure contributes to the development of melanoma in humans, its exact role is unclear. In the past few decades, there has been an alarming increase in the number of NMSC and melanomas among light-skinned populations throughout the world, leading some scientists to postulate an emerging epidemic of skin cancer. Animal Models Species and strains vary considerably in their susceptibility to UV-induced skin cancer. These differences are due largely to variables such as pigmentation, hair coat, and thickness of the stratum corneum. Mice appear to be the experimental animals most susceptible to UV carcinogenesis. Hairless SKH-1 mice are widely used for photobiology studies. Advantages of these mice are that they do not require shaving, are unpigmented, and have a relatively normal immune system; however, a limitation is that the mice are not inbred. Furthermore, UV exposure alone does not cause melanomas in mice or other commonly used experimental animals, although a combination of chemical carcinogen and UV is effective. For this reason, a variety of unusual animal models susceptible to UVinduced melanoma, such as the South American opossum (Monodelphis domestica) and swordtail or platy fish (Xiphophorus sp.), have been used to study the relationship between UV exposure and melanomas. In the former case, under experimental conditions, the opossum demonstrated high levels of UV radiation (UVB)induced neoplasms including papillomas, keratoacanthomas, squamous cell carcinomas, basal cell tumors, fibrosarcomas, and both melanocytomas and melanomas. Swordtail platy fish hybrids have been used as a model for UVBinduced melanomas, and have been critical in the debate as to the carcinogenicity of different forms of UV irradiation (Figure 14.51). With the advent of new strains of genetically altered mice, a host of new murine models is becoming

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FIGURE 14.51 Scaled skin: platy fish exposed to UV radiation. Cutaneous melanoma (top left) with invasion of the dorsal fin, elevation, and obliteration of the scales. H&E stain. Bar ¼ 200 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.51, p. 1492, with permission.

available, including mice that are highly susceptible to melanoma, heterozygous and homozygous p53 knockout mice, and murine models of xeroderma pigmentosum. Furthermore, the hairless mouse has been used to prove that UVA is in fact a complete carcinogen, through its ability to initiate and promote squamous cell carcinomas in that species. Mechanisms Animal studies indicate that UV is a complete skin carcinogen. UV is an initiator by virtue of its mutagenic capability, a promoter due to its ability to alter gene expression and thus stimulate proliferation, and it drives tumor progression by a combination of its mutagenic and growth-promoting activities. The most effective wavelengths for UV-induced NMSC in the

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hairless mouse are those in the UVB range, with peak activity at 293 nm. This action spectrum implicates DNA as the primary chromophore for NMSC. UVA (340 nm) can induce NMSC in hairless mice, but it is 10,000-fold less effective than UVB. In general, changes in fluence rate or interval between doses do not alter the shape or slope of the NMSC incidence curve, but may affect the latent period. The p53 tumor suppressor gene provides significant protection against UV-induced NMSC. UV-induced DNA damage is a potent inducer of wild-type P53 protein. P53 induces cell cycle arrest in cells with damaged DNA, thus permitting repair of minor DNA damage and stimulating apoptosis of cells too badly damaged for effective repair. This eliminates genetically altered and potentially transformed cells from the skin. When p53 is mutationally inactivated or deleted, deleterious mutations accumulate in UV-exposed keratinocytes, leading to the development of NMSC. More than 90% of UV-induced NMSC in the human and in the hairless mouse have mutationally inactivated p53 genes. Mutations are concentrated in exons 5–8 of the p53 gene. Most are missense point mutations arising on the nontranscribed strand of DNA. Most p53 mutations in NMSC are hallmark UV mutations, C to T and CC to TT, at dipyrimidine sites, suggesting that UV is the proximate carcinogen for these tumors. Skin Neoplasms Chronic natural or experimental exposure to UV leads to the development of hyperplastic and neoplastic skin lesions in a variety of animal species. In many cases, hyperplastic lesions appear to serve as precursors for neoplastic lesions. Actinic keratoses in humans appear to give rise to squamous cell carcinoma. In the hairless mouse, foci of epidermal hyperplasia can evolve into sessile or pedunculated papillomas, and squamous cell carcinomas may arise in those papillomas (Figure 14.52). With continued UV exposure, squamous cell carcinomas in the hairless mouse progress to increasingly invasive and anaplastic tumors. The behavior of UV-induced squamous cell carcinomas in humans and experimental animals is similar. Although clearly malignant and locally invasive, these tumors rarely metastasize. Squamous cell carcinoma has been linked to natural

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FIGURE 14.52 Gross appearance of skin lesions induced by chronic UV exposure in the hairless mouse. A number of pedunculated papillomas and three ulcerated squamous cell carcinomas are visible. Lesions arise and evolve independently. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 2, Academic Press, 2002, Vol. 1, Fig. 34, p. 586, with permission.

sunlight exposure in the unpigmented skin of dogs, the pinna and nasal planum of white cats, the vulva of cattle and goats and the periorbital region of Paint Horses, Quarter Horses, Appaloosas and draught horses, as well as the penile/preputial tissues in Quarter Horses and Appaloosas. Keratoacanthomas are invaginated keratin-filled masses lined by thickened epithelium that develop in humans and in some experimental animals in response to UV. In humans, these tumors are self-limiting and undergo involution, while in experimental animals they appear to be capable of giving rise to squamous cell carcinomas. Basal cell tumors similar to those caused by sunlight in humans rarely arise in experimental animals; however, a murine model of basal cell carcinoma, the Patched knockout mouse, has been developed. The only human sarcoma clearly associated with sunlight is the atypical fibroxanthoma of the elderly. However, chronic UV exposure in haired mice and in Monodelphis domestica can induce dermal fibrosarcomas and hemangiosarcomas, and sunlight exposure has been proposed as a cause of hemangiosarcoma in the sparsely haired skin of dogs. Some UVinduced spindle cell tumors of the dermis in mice may represent anaplastic squamous cell carcinomas, and carcinosarcomas or “collision tumors” have been described. A number of

models for human melanoma have been or are being developed, but none is entirely satisfactory. Chronic UV exposure induces benign and malignant melanomas and the precursor lesion of melanocytic hyperplasia in Monodelphis domestica. Genetically altered mice that develop a high incidence of melanoma have also been created; however, these mice tend to succumb very rapidly to metastatic disease, making them difficult to work with. Furthermore, in both mice and Monodelphis, melanomas arise in the dermis and show little or no junctional activity, unlike the situation in humans. Melanomas associated with natural sunlight exposure in animals include perineal melanomas of gray horses, and melanomas of the ears in Angora goats. Ocular Neoplasia In humans, squamous cell carcinoma of the conjunctiva is rare. It occurs with increased frequency in the tropics and in xeroderma pigmentosum patients, establishing a link with solar UV. Early papillary lesions are often found in association with chronic inflammation and stromal collagen degeneration. Squamous cell carcinoma of the eye is a serious economic problem in some breeds of cattle. The disease in cattle is clearly related to cumulative solar exposure, with pigmentation, genetic background, and viral infection also playing a role in the disease. The neoplasms develop from plaques and papillomas to carcinoma in situ and, ultimately, invasive squamous cell carcinoma. Squamous cell carcinoma of the conjunctiva has been associated with sunlight exposure in dogs. In mice chronically exposed to UV, ocular squamous cell carcinomas develop less frequently than corneal fibrosarcomas and hemangioendotheliomas. Precursor lesions include epithelial hyperplasia, neovascularization, and fibroplasia. Virtually all Monodelphis domestica chronically exposed to UV develop corneal fibroplasia and neovascularization that evolve into mesenchymal tumors of the cornea. It has also been observed that intraocular melanoma in humans has a higher incidence in those with blue eyes and in those living in the southern versus northern United States, suggesting a possible link to UV exposure. For additional information on ultraviolet radiation see Brash (1997), Elmets et al. (2014),

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9. MECHANISMS OF HYPERTHERMIA-INDUCED INJURY

Gallagher and Lee (2006), Harber and Bickers (1989), Kim et al. (2018), Kligman et al. (1985), Kowalska et al. (2021), Mukhtar and Elmets (1996), Nikula et al. (1992), Ullrich and Byrne (2012).

PART III HYPERTHERMIA 8. Clinical Use of Hyperthermia Hyperthermia, alone or in combination with other treatment modalities such as ionizing radiation or chemotherapy, has been used for cancer therapy. In that instance, use of hyperthermia depends on the increased heat sensitivity of malignant cells compared to normal cells. While normal tissue responds to hyperthermia (42– 43
C) by increasing blood flow up to 10 times normal, many neoplasms, in the range of 41– 42
C, show an initial increase in blood flow followed by a decrease or stasis within 1–2 h. This difference in flow gives the normal tissues a greater cooling capacity than tumor tissue. Therefore, the temperature tends to rise more in tumor than normal tissue, causing selective tumor necrosis. Tumor cell survival is further compromised by the hypoxic, acidic, and relatively nutrient-poor microenvironment of tumors. Hyperthermia enhances cell killing by ionizing radiation both additively and synergistically. The “thermal enhancement ratio” is the ratio of radiation dose producing a given effect to the reduced radiation dose required to produce the same effect in combination with heat. Additionally, in vitro and in vivo studies have repeatedly shown an increased tumor response when appropriate drugs are administered at elevated temperatures. Proposed mechanisms for chemomodulation include improved drug delivery by alterations in microvascular blood flow or pharmacokinetic parameters, enhanced cytotoxicity, increased cellular drug content, and increased depth of surface penetration of drug following intracavitary instillation.

9. MECHANISMS OF HYPERTHERMIAINDUCED INJURY Cell death in response to hyperthermia occurs 1–2 days after exposure. The fraction of cells

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surviving hyperthermia is a function not only of the absolute temperature, but also of the duration of hyperthermia. Heat induces tissue injury directly. The severity of the injury is dependent upon the critical thermal maximum (the level and duration of core heating). In humans, the critical thermal maximum is a body temperature of 41.6–42
C for 45 min to 8 h. All cellular structures are destroyed at extreme body temperatures (49–50
C) and cellular necrosis occurs in less than 5 min. Based on in vitro, laboratory animal, and clinical studies, it appears that for each 1
C rise in tissue temperature above 42
C, the time required for an equivalent effect is halved. Vascular changes play an important role in hyperthermia effects. In normal tissue, below 40
C, heating causes active hyperemia and vasodilation, with a resultant increase in tissue oxygen tension. However, at temperatures above 42
C, the microcirculation to the heated tissue collapses and tissue oxygen tension decreases. At the same time, the cellular metabolic rate increases by 10%–15% for each 1
C increase in temperature. When the metabolic demands of the cells can no longer be met by the blood flow, heat injury begins. Histologically, it is characterized by pyknosis, karyorrhexis, and coagulation of cytoplasm. At the molecular level, heat alters weak molecular interactions, including hydrogen bonds, ionic interactions, and hydrophobic interactions, to induce conformational change in and destabilization of macromolecules. Several subcellular targets for heat injury have been suggested to include cellular membranes, intracellular proteins, DNA, and lysosomes. Membrane fluidity is increased due to changes in lipids, and denaturation of proteins alters the function of vital cellular components, including membrane and cytoskeletal proteins and essential enzymes. Hyperthermia increases Kþ efflux, increases Ca2þ and Hþ influx, uncouples oxidative phosphorylation, causes translational arrest, alters mitotic spindle function, inhibits DNA synthesis, and delays ligation of newly synthesized DNA. Because of its effects on the mitotic spindle, hyperthermia can cause polyploidy, chromosome aberrations, and death in S phase. Although dividing cells are particularly heat sensitive, hyperthermia-induced cell death does not require cell division. Heat radiosensitizes some tumors, presumably by enhancing

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nuclear protein binding, thus restricting DNA accessibility for repair enzymes and inhibiting repair of radiation-induced DNA damage. Studies in vitro and in vivo have shown that both neoplastic and normal cells develop resistance to repeated hyperthermic episodes. “Acute thermotolerance” is produced by a single prior sublethal heat exposure. It typically develops within several hours and decays over a few days. “Chronic thermotolerance” develops after prolonged continuous heating. Induction of heat shock proteins is believed to be responsible for thermotolerance. These highly conserved acidic proteins ordinarily serve as molecular chaperones that supervise the folding of cellular proteins. Under conditions of heat stress, they suppress irreversible protein unfolding, thus preventing protein aggregation. Chronic thermotolerance, on the other hand, can occur without increased cellular levels of heat shock proteins. “Acclimatization” is an organism’s response to multiple exposures to a warm environment over several days, and is characterized by systemic adaptations (lower core body temperature, reduced heart rate, decreased metabolic rate, and increased sweating) that increase heat dissipation.

10. RESPONSE TO INJURY INDUCED BY HYPERTHERMIA The responses of normal tissues to hyperthermia are discussed below. Hyperthermic damage to normal tissue occurs with heatstroke, high fever, lightning strike, and burns.

10.1. Reaction of Specific Organs and Tissue to Hyperthermia Alimentary System Investigations of hyperthermic damage to the mouse small intestine indicate that the mucosa is very thermosensitive. Nonproliferating villous enterocytes are more susceptible to thermal injury than crypt cells. After heating the intestine to 41.5
C for 1 h, histologic examination 2 h later revealed swollen villous enterocytes, loss of microvilli, extrusion of cells from the villous tips, a pleomorphic inflammatory infiltrate in the lamina propria, and collapse of the villous

stroma. Stromal damage, especially edema and microvascular injury, may contribute to the epithelial injury. The translocation of bacteria and endotoxin from the intestine into the systemic circulation has been reported to occur in cases of burn and smoke inhalation injury. Similarly, in cases of heatstroke, the redistribution of blood flow from the splanchnic circulation to the periphery often results in intestinal ischemia. An ischemic gut facilitates the absorption of bacterial endotoxins with subsequent activation of inflammatory mediators and, ultimately, multiple organ dysfunction. In both animals and humans, liver enzyme values increase with temperatures above 42
C. Electron microscopic studies have shown increased numbers of autophagic vacuoles and dilation of both the Golgi apparatus and endoplasmic reticulum following hyperthermic treatment in humans and rats. Liver damage is frequently a feature of heatstroke. Elevations of alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) occur within 24 h of heatstroke in humans and experimental models of heatstroke in dogs and rats. Histopathologic changes include dilation of central and portal veins, congestion of centrilobular sinusoids, degeneration or necrosis of centrilobular hepatocytes, and cholestasis (Figure 14.53). Ultrastructural studies show ballooning or flattening of hepatocellular microvilli, breaks in hepatocyte plasma membranes, vesiculation of the endoplasmic reticulum, detachment of ribosomes, and swelling of mitochondria. Musculoskeletal System The thermosensitivity of cartilage has been studied by measuring growth inhibition necrosis of the tails of infant mice or rats. Heating the tails at 43
C for 1 h induced stunting but not necrosis in about 10% of the animals, whereas heating at 44
C for 1 h produced necrosis in more than 50% of the animals. Clamping the blood supply caused the infant rat tail to become much more thermosensitive, equivalent to a three-fold increase in heating time or a temperature decrease of 1.5
C. The effects of 30 min’ exposure at 40–48
C have been examined in pig muscle. One day after treatment, minimal myocyte necrosis was present in muscle heated to 43
C, while up to

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FIGURE 14.53 Liver: Yorkshire pig. This photomicrograph demonstrates the hepatocellular changes 4 h postinhalation injury as part of an oak wood–smoke toxin inhalation study. The centrilobular to midzonal hepatocellular sinusoids are dissociated with individualization, hepatocellular degeneration, necrosis, and multifocal vascular congestion. H&E stain. Bar ¼ 200 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.53, p. 1495, with permission.

30% of the muscle fibers were necrotic in muscle heated to 44–46
C. In areas of muscle heated above 46
C, edema, numerous inflammatory cells, hemorrhage, and coagulation necrosis of more than 30% of the muscle fibers were seen. One month after treatment, there was myofiber regeneration and focal fibrosis in muscle heated to 46–47
C. In tissue heated above 47
C, there was severe fibrosis and abscessation. Hyaline degeneration and necrosis of skeletal muscles (rhabdomyolysis) have been observed in people following heatstroke. Episodes of malignant hyperthermia (MH) are triggered by a variety of factors, including certain anesthetics and stress. The primary histologic finding in MH – an inherited disorder in humans, pigs, and some breeds of dogs – is severe acute rhabdomyolysis occurring in response to prolonged myofiber contraction following a sudden increase in myoplasmic calcium concentration due to a defect in the ryanodine receptor (RYR1) – a calcium-release channel located in the sarcoplasmic reticulum. Additionally, muscle rigidity, tachycardia,

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dyspnea, metabolic acidosis, and lifethreatening hyperthermia occur. Malignant hyperthermia-like episodes have also been reported in horses triggered by inhalant anesthetic agents or by injection of succinylcholine. An inherited defect in the RYR1 has been documented in Quarter Horses and may also be triggered by exercise, illness, breeding, concurrent myopathy, or other stress. Horses with the GYS1 mutation-positive form of polysaccharide storage myopathy and MH defect are much more difficult to treat. In animals not recently affected by hyperthermic episodes, myofibers may be normal or there may be a few degenerate fibers. Myofibers are separated by edema fluid in pigs dying acutely. Degenerative changes vary from segmental hypercontraction to coagulative necrosis. The most common histologic lesion seen in skeletal muscle is hypercontraction. Changes in myofibers are widespread and typically classified as multifocal monophasic; however, polyphasic injury may be observed in swine with recent nonfatal episodes of MH. Underlying chronic myopathic changes may also be observed. The cause of MH in domestic swine, including Pietrain, Yorkshire, Poland China, Duroc, and Landrace breeds, is a single point mutation in RYR1 at locus HAL-1843. A similar syndrome of MH appears to occur in Vietnamese pot-bellied pigs. A gene defect in HAL-1843 has been detected in some affected Vietnamese pot-bellied pigs while not detected in others suggesting more than one gene defect may lead to MH as in humans. The muscles most commonly affected in pigs include those of the back, loin, thigh, and shoulder. There are sporadic reports of MH-like episodes in various dog breeds. Exercise-induced hyperthermia has been observed in English Springer Spaniels and Labrador Retrievers. Ingestion of hops can also trigger MH-like episodes in susceptible breeds – most commonly in Greyhounds. Other susceptible canine breeds include Pointers, Saint Bernards, Bichon Frises, Golden Retrievers, and Border Collies. Chronic myopathic changes include internalization of nuclei, increased fiber size variation, and fiber hypertrophy in MHsusceptible dogs. Histologic lesions in dogs dying due to hyperthermia are similar to those reported in other MH-susceptible species. Malignant hyperthermia-like episodes have also been reported in cats with underlying myopathies

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such as X-linked muscular dystrophy, in which stress of restraint or anesthesia can trigger episodes of fatal hyperthermia. Nervous System Localized heating of brain tissue in rats and cats using interstitial microwave antennae or ultrasound, respectively, have shown similar results. Edema formation in both white and gray matter can occur in tissues held at 42
C for 50–70 min. Neuronal cell lysis occurs at temperatures greater than 43
C. Disruption of myelin tracts in the white matter is evident at brain temperatures of 43.0–43.5
C. The brain in victims dying of heatstroke is grossly swollen. The entire brain can swell within 24 h, depending on the extent and severity of the edema. In severe cases, edema may result in herniation of the brain. Histologic changes include degeneration and necrosis of neurons in the cerebral cortex, basal ganglia, and Purkinje cell layer. The neurologic dysfunction seen in cases of heatstroke is attributed to metabolic disturbances, cerebral edema and ischemia, metabolic encephalopathy, and possibly hypernatremic cerebral damage. The CNS, particularly the cerebellum, is quite susceptible to heat injury. A case of central pontine myelinolysis was reported in a heatstroke patient during the 1995 Chicago heat wave. Additionally, progressive cerebellar atrophy has been documented in survivors up to 10 weeks posthyperthermic insult. In three patients who had heatstrokes during the heat waves in France in 2003, the predominant histologic finding was severe diffuse loss of Purkinje cells. Degeneration of the Purkinje cell axons was evidenced by myelin pallor in the white matter of the folia and of the hilum of the dentate nuclei. Eye The avascular lens dissipates heat poorly and can reach high temperatures. Microwave exposures can produce cataracts in rabbits, but the same exposures produce facial burns but no cataracts in monkeys. In one study, hyperthermia was induced in rabbit corneas using ultrasound with the power set at 100% in continuous mode. Rabbit corneas held at 50
C for 10 s showed initial stromal damage with collagen disorganization, mild stromal edema, and initial signs of keratocyte damage consisting of nuclear

degeneration and partial destruction of Bowman’s membrane. Half of the corneas held at 60
C for 10 s were examined at time 0 and the other half after 1 week. At time 0, massive corneal damage with epithelial cell edema, collagen disorganization, severe stromal edema, intrastromal vacuole formation, plump keratocyte nuclei, and endothelial cell detachment was observed, as well as a severely impaired nerve plexus. At 1-week follow-up, corneas showed persistent stromal and endothelial cell edema with an increase in activated keratocytes and mitotic features in the stroma and epithelial layer. Cardiovascular System Whole-body hyperthermia at 41
C for 20 min can cause cardiac damage in rats. Lesions reported include edema, vacuolization, hyperemia, and subepicardial and subendocardial cell necrosis, inflammation, and interstitial fibrosis. There have been several reports of disseminated intravascular coagulopathy during whole-body hyperthermia. It is not known if consumption of coagulation factors occurs directly due to heating of the blood, or in response to substances released from heatdamaged tumor or normal tissue (endothelial cell injury). Hemorrhage due to fibrinolysis, hypofibrinogenemia, and thrombocytopenia secondary to intravascular clotting is also frequently seen in people or experimental animals dying of heatstroke. Focal necrosis of myocardial fibers is seen in heatstroke. Lesions in the myocardium in cases of malignant hyperthermia include granular degeneration of myocytes, contraction band necrosis, and myocytolysis. Urinary System Mouse kidney exposed to local hyperthermia in the range of 41–45
C for 35 min has foci of subcapsular tubular necrosis with a neutrophilic response and minimal calcification 1 week after exposure. After 4 weeks, the zone of necrosis and calcium deposits remains, but the neutrophils are absent. The kidneys are quite resistant to damage during systemic hyperthermia treatment when electrolyte and fluid balance are maintained. This resistance to damage is probably due to high renal blood flow. Patients suffering from heatstroke as a result of physical

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exertion and insufficient fluid intake may exhibit anuria or oliguria and elevated blood urea nitrogen. Proteinuria, renal casts, and tubular degeneration are sometimes seen. Acute renal failure is a potential complication in cases of severe heatstroke. The presence of rhabdomyolysis makes acute renal failure more likely to develop. Male Reproductive System One of the more sensitive organs to mild hyperthermia is the testis. In most mammals, even the thermal environment of the abdominal cavity will inhibit normal testicular development. Heating rat testes to 43
C for 15 min results in damage to transitional and late pachytene stages; spermatogonia are more heatresistant. As heating is extended to 30 min, the damage involves all spermatocyte and early spermatid stages. Sertoli cells undergo vacuolar degeneration secondary to germinal epithelial damage. Leydig cells are not affected by this hyperthermic treatment. After about 3 weeks, rat testes will recover from this mild damage. In rabbits, the minimal scrotal temperature at which testicular damage occurs is 40
C. At that temperature, more than 100 h of treatment is required for seminiferous tubule degeneration to occur. In a canine model for transurethral balloon laser hyperthermia (TUBAL-H) of the prostate gland, 11 normal and hyperplastic prostate glands were heated to between 40
C and 45
C for 30 min. Immediately following treatment, shedding of epithelial cells was observed histologically with no coagulative necrosis of tissue. After 8 weeks, changes consistent with atrophy were observed in the epithelial cells of the inner portion of the prostate gland. Immunohistochemical analysis revealed that the epithelial cells in the inner portion of the prostate gland expressed apoptosis related antigen (Lewis- Y) immediately after treatment and up to 4 weeks later. In another study, localized microwave hyperthermia was applied to the prostate gland in dogs using a new watercooled skirt-type antenna, operating at 915
MHz, as part of a new hyperthermia apparatus being developed for the treatment of the prostate gland in humans. The prostate gland of 20 male dogs was repeatedly heated to between 40
C and 47
C, under general anesthesia, for variable lengths of time up to 10 h.

923

All treatments by hyperthermia of the prostate gland resulted in the infiltration of mononuclear cells into the interstitium and polymorphonuclear cells into the glandular components. Permanent damage to prostatic tissue was dependent on time of exposure and temperature. Integumentary System Local application of heat can result in cutaneous burns. A temperature of 70
C or higher for several seconds in humans causes complete transepidermal necrosis, although a temperature of 50
C for periods of 10 min may not cause serious damage. Cutaneous burns are divided into three categories: the first-degree burn, in which damage is limited to the outer layer of the epidermis without significant dermal damage, other than erythema and mild edema; the second-degree burn, in which the epidermis is necrotic with vesicle and bulla formation, but with dermal sparing (Figure 14.54); and, finally, the third-degree burn, in which both the dermis and epidermis are damaged and the skin surface

FIGURE 14.54 Haired skin: Yorkshire pig. Seconddegree burn 48 h postburn injury with heated metal application. This section demonstrates the separation of the epidermis from the dermis (arrows) with bullae and vesicle formation (asterisk). The epidermis is fused with nuclear pyknosis and cytoplasmic coagulation. The underlying dermis is spared. H&E stain. Bar ¼ 50 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.55, p. 1498, with permission.

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14. RADIATION AND OTHER PHYSICAL AGENTS

FIGURE 14.55 Haired skin: Yorkshire pig. Thirddegree burn 48 h postburn injury with heated metal application. This section demonstrates the severe damage to both the epidermis (arrows) and the dermis with loss of architecture and coagulation of all tissues (asterisk). H&E stain. Bar ¼ 100 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, ed 3, Academic Press, 2013, Figure 44.56, p. 1498, with permission.

is charred or coagulated (Figure 14.55). Early microscopic evidence of mild thermal injury is nuclear and cytoplasmic swelling. More severe injury, as with a second-degree burn, results in rupture of nuclear membranes, pyknosis, and granular or homogeneously coagulated keratinocyte cytoplasm. In mild injury, dermal capillaries are dilated and there is interstitial edema. In severe injury, there may be coagulation of blood vessels and little evidence of exudation, except at the margins of the burn. The dermal collagen loses its fibrillar structure and becomes a homogenous gel. In pigs, subcutaneous fat heated to 40–48
C for 30 min develops a neutrophilic inflammatory infiltrate by 24 h after treatment. In subcutaneous tissues heated to 45
C, the inflammation resolves without residual injury. One month after treatment, subcutaneous tissues heated to 46–47
C contain giant cells and prominent fibroplasias. In sites heated above 47
C, chronic panniculitis is accompanied by foci of necrosis and abscesses. With the increased use of IEDs in combat environments, there has been a concomitant increase in the prevalence of blast trauma. Mechanisms of

injury from blast trauma are divided into four categories: primary, secondary, tertiary, and quaternary. Burns (flash-, partial-, and fullthickness) are included in the quaternary category. Flash burns occur as a result of the thermal component of the detonation on exposed skin. Secondary fires can cause additional burns and/or smoke inhalation injury. In addition to burns, victims of “dirty bombs” may also be exposed to radiation, toxic gases, other chemicals, and biological pathogens. For additional information on hyperthermia see Bazille et al. (2005), Born (2005), Bruchim et al. (2006), Bruchim et al. (2009), Cooper and Valentine (2016), Enkhbaatar and Traber (2004), Gibson et al. (2021), Hubbard et al. (1998), Kumar et al. (2020), Pargo and Gurtner (2016), Valentine (2017), Yeo (2004).

Disclaimer E. D. Lombardini and M. E. Pacheco-Thompson are Colonels in the US Army. The opinions or assertions herein are those of the authors and do not necessarily reflect the view of the Department of the Army or the Department of Defense.

Acknowledgments We would like to recognize and thank Drs Stephen A. Benjamin, Barbara E. Powers, Fletcher F. Hahn, Donna F. Kusewitt, and Mark A. Melanson for their extraordinary effort on previous editions of this chapter. Furthermore, we greatly appreciate Dr Scot J. Estep for all of his assistance and for providing materials as a subject matter expert in hyperthermia pathology. Finally, our gratitude to the myriad scientists at the Armed Forces Radiobiology Research Institute (AFRRI) for their guidance and support.

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Index ‘Note: Page numbers followed by “f” indicate figures “t” indicate tables and "b" indicate boxes..’

A A disintegrin and metalloprotease (ADAM), 577e578 A-B toxin, 647e648 Abalone, 372e373 Abamectin, 746e747 Absorption, distribution, metabolism, elimination (ADME), 212, 436 Acceptable daily intake (ADI), 71, 166, 738 Acceptable operator exposure level (AOEL), 738 Acetamiprid, 746 Acetylcholine (Ach), 590, 743 receptors, 742e743 Acetylcholinesterase (AChE), 593, 742, 825 Acetylcoenzyme A (acetyl-CoA), 590 Acetylsalicylic acid (ASA), 58, 189 Acid-sensing ion channels (ASICs), 591 Acrodynia, 712 Activated clotting time (ACT), 596e597 Activated partial thromboplastin time (aPTT), 596e597 Activation function-1 (AF-1), 434e435 Active Pharmaceutical Ingredient (API), 189, 200 influencing factors on concentration of APIs in plant, 193 Acute bovine liver disease (ABLD), 497e498 Acute hemorrhagic disease, 539e540 Acute kidney injury (AKI), 600e601 acute renal failure, 691e692 Acute lymphocytic leukemia (ALL), 870e871 Acute myeloid leukemia (AML), 870e871 Acute radiation syndrome (ARS), 859e860 and combined injury, 859e860 Acute reference dose (ARfD), 464, 738 Acute respiratory distress syndrome (ARDS), 637, 663e664, 685, 844e845 Ad libitum-fed rodents (AL-fed rodents), 111 Adequate intakes (AIs), 141 Adhesins, 641 Additives, natural color, 37 Adipocytes, 107e110 Adipose tissue, 107e110, 113 Adrenocorticotropic hormone (ACTH), 881e882 Adult T cell leukemia (ATL), 870e871 Adulterants, 194e196, 195t Adulteration Act, 76e77 Aetokthonos hydrillicola, 368e369

Aetokthonotoxin (AETX), 368e370 Aflatoxin B1, 401e405 Aflatoxins, 396, 401e411, 495, 736 biodistribution, metabolism, and excretion, 405 diagnosis, treatment, and control, 411 human risk and disease, 408e411 manifestations of toxicity in animals, 406e409 mechanism of action, 405e406 source/occurrence, 401 species susceptibility, 401e405 toxicity, 408 toxicology, 401e406 toxin, 401 Agave, 527e528 Agave lecheguilla, 494e495 Agouti gene, 777 Agriculture and Agri-Food Canada (AAFC), 84 Agrochemicals, 727e728 alphachloralose, 756 ARs, 751e753 cholecalciferol, 753e754 corn cob, 757 fungicides, 736e741 herbicides, 728e736 insecticides, 741e748 rodenticides, 748e758 AIN 93 diets, 118e119 Air pollution, 13 Alanine (ALT), 601 Alanine aminotransferase (ALT), 601, 731e732, 920 Alexandrium sp., 314 A. ostenfeldii, 318 A. peruvianum, 318 Alfalfa (Medicago sativa), 204 Algal compounds in food, 43e45 cyanotoxins, 44 domoic acid and amnesic shellfish poisoning, 44e45 marine algal toxins, 44 phycotoxins in food, 43 Algal toxins. See Phycotoxins Alimentary system, 872e877, 920 esophagus, 874e875 general reaction to ionizing radiation injury, 872 liver, 875e877 salivary tissue, 872e873 small intestine, 873e874 Alimentary toxic aleukia (ATA), 420, 431e432

929

Alkaline phosphatase (ALP), 492e493, 601e602, 731e732 Alkoxyalkyl mercury, 713 Allergy-like food poisoning, 53 Allethrin, 745 Aloe barbadensis, 236 Aloe vera (Aloe barbadensis), 204, 223e237, 234f gel, 236e237 latex, 235e236 toxicity findings for herbal remedies, 224te233t whole leaf extract, 234e235 Aloe-emodin, 236 Alpha-emitting radionuclides, 891 Alphachloralose, 756 clinical signs and pathology, 756 development and use, 756 human risk, 756 risk to environment/other species, 756 toxicology, 756 Alphatoxin, 642 Alpine blue borage (Echium vulgare), 492 Alsike clover (Trifolium hybridum), 498 Alternaria sp., 45, 197e199, 466 source/occurrence, 466 toxicology, 466e467 human exposure and disease, 468 manifestations of toxicity in animals, 467e468 mechanism of action, 467 toxins, 466e468 biodistribution, metabolism, and excretion, 467 chemical structure of, 466f Alternariol, 467 Aluminum (Al), 160 Aluminum phosphide, 755 absorption, distribution, excretion, and metabolism, 755 aluminum phosphide/zinc phosphide, 754e756 clinical signs, 755 development and use, 754 human risk, 755e756 other species, 755 toxicology, 754e755 Alzheimer type II cells, 349 Alzheimer’s disease (AD), 247, 257, 365, 367, 820 Amaranthus spp., 529e530 A. caudatus, 529e530

930 Amazonian giant leaf frog (Phyllomedusa bicolour), 553e554 Amber weed, 537e538 Amino acids (AAs), 60, 114e116, 511e512, 642e643 deficiency, 114e115 excess, 115e116 roles in neurotransmission, 61te63t 3-amino-9-methoxy-2,6,8-trimethyl-10phenyl-4,6-decadienoic acid (ADDA), 354 g-aminobutyric acid (GABA), 60e64, 732 2-aminoethanesulfonic acid, 115 6-aminoquinolyl-Nhydroxysuccinimidylcarbamate (AQC), 367e368 Amnesic shellfish poisoning (ASP), 44e45 Amphidinium genera, 330 Amyloid-beta (b-amyloid), 820 Amygdalin, 49 Amyotrophic lateral sclerosis (ALS), 365e367 Amyotrophic lateral sclerosiseparkinsonism dementia complex (ALS/PDC), 367 Anabaena flos-aquae, 355 Anaphylaxis, 57, 582, 602 Anatoxin-a(S), 44 Anatoxins (ANTXs), 355e358 clinical signs and pathology, 356e357 diagnosis, treatment, and control, 358 human exposure and disease, 357 source/occurrence, 355e356 anatoxin-a, 355f toxicology, 356 Androgen receptors (ARs), 772 Angiotensin-converting enzyme (ACE), 579 ACE-2, 859 Anguina funesta, 508e509 Aniline dyes, 6e7 Animal Feed Regulatory Program Standards (AFRPS), 88 Animal Feed Safety System (AFSS), 87 Animal toxins, 547e548. See also Zootoxins acquisition of food, 549f bacterial toxins as zootoxins, 660 clinical presentations and pathologic manifestations of zootoxin-mediated diseases, 595e604 deliberate administration, 557e559 diagnosis and treatment of zootoxinmediated diseases, 604e610 envenomation, 554e557 poisoning, 551e554 regulatory guidance regarding zootoxins, 610e615 sources of exposure, 550e559 strawberry poison dart frog, 550f toxins vs. venoms, 549 zootoxin classification, 562e595 Annual ryegrass toxicity, 508e509 liver of sheep, 509f structures of corynetoxins, 508f Anthocyanins, 49 Anthrax inhalation, 636e637

INDEX

Anthocyanins, 49 Antibodyedrug conjugates (ADCs), 638, 639f, 670 Antibody-toxin conjugates, 638, 639f Anticoagulant rodenticides (ARs), 749e753 clinical signs and pathology, 751e753 development and use, 749e750 metabolism, 751 rodenticides and chemical classes, 750t second generation of anticoagulants (SGARs), 750 toxicity data from assessment reports, 752t toxicology, 751 warfarin, chlorophacione, brodifacoum, and difethialone, 751t Anti-drug antibodies (ADA), 671 Antimicrobial resistance (AMR), 73e74 Antimony (Sb) toxicity, 680e683 diagnosis and treatment, 683 manifestations of toxicosis, 682e683 sources and exposure, 680 spots, 682 toxicology, 680e682 Antimony trioxide, 682 Antioxidant-responsive-element (ARE), 241 Antitoxins, 559, 637e638 Antivenin. See Antivenom Antivenom (antivenin), 559, 608 adverse reactions to, 608e609 curative therapies and complications, 608e609 adverse reactions to antivenom, 608e609 antivenom therapy, 608 practices for developing antivenom products, 614e615 therapy, 608 Apamin, 589f Aphanizomenon sp., 314 Apitherapy, 558e559 Apitoxin, 560te561t Aplastic anemia-prone (AA-prone), 871e872 Aplysiatoxins, 361e365 clinical signs and pathology, 363 diagnosis, treatment, and control, 364e365 human exposure and disease, 363e364 vesicles and papules on the abdomen, 363f source/occurrence, 361e362 toxicology, 362e363 Aquatic hypoxia, 308e309 Aquatic snails (Pomacea maculata), 370 Arachidonic acid (AA), 118e119, 582 Arachnidism, 616 Arginine, 114e115 deficiency, 115 excess, 116 Aristolochia sp. A. clematitis, 416, 490 A. fangchi, 490 Aristolochic acids (AAs), 216 Arndt-Schulz rule, 193 Arnica (Arnica montana), 201 Aromatic amines, 6e7 Arrowgrass (Triglochin spp.), 533e535

Arsanilic acid, 686e687 Arsenic (As), 680, 683e688 arsine, 687 chemical warfare considerations, 688 diagnosis and treatment, 687e688 inorganic arsenic, 683e688 organic arsenic, 686e687 toxicity, 684e685 Arsenic, inorganic, 683e688 manifestations of toxicosis, 685e686 sources and exposure, 683e684 toxicology, 684 Arsenobetaine, 687 Arsine, 687e688 Arthrinium spp., 511e512 Arthrogryposis, 521e523 Arthropods, 598e600 Arthrospira spp., 356e357 Aryl hydrocarbon (Ah), 10e11 Aryl hydrocarbon receptor (AhR), 435 Asbestos, 10 Asbestos Ban and Phase Out Rule, 10 Ascorbic acid. See Vitamin C Asian tiger snake (Rhabdophis tigrinus), 568 Aspartate (Asp), 60 Aspartate transaminase (AST), 601, 920 Aspergillus spp., 45, 116e117, 470, 511e512 A. carbonarius, 411 A. flavus, 184, 401, 495 A. niger, 411 A. ochraceous, 411 A. parasiticus, 401, 495 strains, 411 toxins, 468e471 cyclopiazonic acid, 468e470 sterigmatocystin, 470e471 tremorgenic mycotoxins, 471e472 Asthma, 329e330 Astragalus spp., 394, 511e512 A. bisulcatus, 519e521 A. lentiginosus, 500 A. praelongus, 519e521 A. pubentissimus, 500 Atlantic croaker (Micropogonias undulatus), 308e309 Atlantic horseshoe crab (Limulus polyphemus), 651 Atlantic mackerel (Scomber scombrus), 314e315 Atracotoxin, 556 Atrazine, 733e734 Atrioventricular block (AV block), 514e515 Atropa belladonna, 189, 505e506 Attention deficit hyperactive disorder (ADHD), 58 Australia, herbal remedies in, 276 Australian coastal taipan (Oxyuranus scutellatus), 593 Australian Quarantine and Inspection Service (AQIS), 86, 89 Australian Register for Therapeutic Goods (ARTG), 276 Autonomic nervous system (ANS), 457e458 Autonomic storm, 602, 604

931

INDEX

Auxin, chlorophenoxy herbicides as antiauxins, 729 Avian vacuolar myelinopathy (AVM), 368 Azadinium dexteroporum, 338 Azaspiracid shellfish poisoning (AZP), 44 Azaspiracids toxins (AZAs toxins), 44, 335e339 azaspiracid-1, 335f clinical signs and pathology, 336e338, 337f diagnosis, treatment, and control, 338e339 human exposure and disease, 338 source/occurrence, 336 sudan IIIestained section of liver, 338f toxicology, 336

B Bacillus thuringiensis kurstaki toxin (Btk toxin), 441 Bacterial exotoxins, 633 Bacterial toxins, 45e46, 635e636, 657e658. See also Animal toxins bacterial toxins as animal toxins, 552e553 clinical presentations and pathologic manifestations of, 657e665 diagnosis of bacterial toxinemediated diseases, 665e667 endotoxins, 651e657 exotoxins, 633e651 regulatory guidance regarding bacterial toxins, 668e671 systemic effects, 664 treatment of bacterial toxinemediated diseases, 665e668 Bald eagles (Haliaeetus leucocephalus), 368 Balkan endemic nephropathy (BEN), 416 Bassia spp., 527e528 Batrachotoxin, 584e589 Bcl2-associated X protein (BAX), 340 Beauvericin, 472e473 Bee sting toxins, 582 Belladonna (Herbae pulvis standardisatus), 205e206 Benchmark dose lower confidence limit (BMDL), 410 Benzamide, 747 Benzidines, 6e7 Benzopyrene, 6e7 Berberine, 258 Berylliosis, 690e691 Beryllium (Be) toxicity, 680, 688e691 chronic, 689e690 diagnosis and treatment, 690e691 disease, 688 hypersensitivity, 690e691 manifestations of toxicosis, 688e690 development of beryllium granuloma, 689f rickets in rats, 690 sources and exposure, 688 toxicology, 688 Beryllium granuloma, 690 Beryllium lymphocyte proliferation test (BeLPT), 690e691 Bioburden, 640 Bioconcentration, 562

Biogenic amines, 589e590 Biokinetic models, 699e700 Biologics Control Act, 668e669 Biosensors, 361 Bioterrorism, 635 Biotic stress, 184 Biotin, 137 Bipyridyl herbicides, 731 Birdsfoot trefoil (Lotus spp.), 533e535 Birdsville disease, 511e512 Bishop’s weed (Ammi majus), 538, 912 Bismuth toxicity, 691e692 diagnosis and treatment, 692 manifestations of toxicosis, 691e692 sources and exposure, 691 toxicology, 691 toxicosis, 692 Bisphenol A (BPA), 42, 767e768 Bitter cassava (Manihot utilissima), 46 Black Cohosh (Cimicifuga racemose), 204 Black mamba (Dendroaspis polylepis), 579 Black widow spiders (Latrodectus mactans), 568 Bladder cancer, 243 Blood group antigen-binding adhesin (BabA), 641 Blood poisoning, 630 Blood urea nitrogen (BUN), 415, 525, 606e607, 893 Blood vessels, 660 and blood components, 595e598 damage, 580 Bloodebrain barrier (BBB), 366, 709, 802 Blue capped ifrit (Ifrita kowaldi), 553 Blue herons (Ardea herodias), 326e327 Blue mussels (Mytilus edulis), 44 Blue-green algae, 306e307 Blue-ringed octopus (Hapalochlaena maculosa), 562 BMAA. See b-methylamino-L-alanine Bone morphogenetic proteins (BMPs), 158e159 Bordetella bronchiseptica, 633 BOTOX (onabotulinumtoxinA), 638e640 Botrys spp., 197e199 Botulinum neurotoxin (BoNT), 649e650 Botulism, 77, 634e635 Boxer crabs (Lybia spp.), 568 Brachiaria sp., 494e495 Bracken fern (Pteridium aquilinum), 539e541 acute hemorrhagic disease, 539e540 bracken staggers, 540e541 bright blindness, 540 enzootic hematuria, 539e540 human poisoning, 541 treatment and prevention, 541 Bradykinin-potentiating peptides (BPP), 579 Brahman-type cattle (Bos primigenus indicus), 459 Brassica sp., 47, 260e262, 264f green tea extract on rat liver, 261fe262f Brevenal, 329e330 Brevetoxins (BTXs), 44, 325e331 clinical signs, 326e327 diagnosis, treatment, and control, 329e330

gross and histologic findings, 327e328 West Indian manatee, 328f human exposure and disease, 328e329 source/occurrence, 325e326 structures of, 325f toxicology, 326 Bright blindness, 540 British anti-Lewisite (BAL), 687e688 British Industrial Biological Research Association (BIBRA), 3e4 Bromethalin, 757 clinical signs, 757 development and use, 757 other species, 757 toxicology, 757 Brown recluse spider (Loxosceles reclusa), 598e600 Buckwheat (Fagopyrum esculentum), 912 Buckyball, 799 Bufadienolides, 595 Buffleheads (Bucephala albeola), 368 Bufotenine, 554 Bufo toad (Incilius alvarius), 551e552 b-bungarotoxin (b-BTX), 590e591 Bureau of Food and Drugs (BFAD), 278 Burrowing asps (Atractaspis spp.), 579 Butenolide, 474e475 Butylated hydroxyanisole (BHA), 112 Butylated hydroxytoluene (BHT), 112 Bystander toxicity, 638

C Cadmium toxicosis, 695 C-reactive protein (CRP), 666e667 C14-demethylase inhibitors, 737e740 Cadmium (Cd) toxicity, 680, 692e700 diagnosis and treatment, 699e700 manifestations of toxicosis, 695e699 syndromes and lesions, 696te697t nephropathy, 695e697 sources and exposure, 692e693 susceptibility to cadmium-induced injury, 695t toxicity, 694 toxicology, 693e695 toxicosis, 698e700 Caffeine, 240, 242 Calbindin, 124e125 Calcinogenic glycoside-containing plants, 530e531 aorta from cow with enzootic calcinosis, 531f country, plant, and common names, 531t cow with enzootic calcinosis, 531f labdane acids, 532f Calcitonin geneerelated peptide (CGRP), 579 Calcitrol. See Vitamin D Calcium (Ca), 527e528, 703 Ca2+ channels, 592 Calcium, dietary, 111e112 deficiency, 142e143 excess, 143 parathyroid hormone, 142e143 Calcium oxalate monohydrate (COM), 527e528 Calcium therapy, oral, 529

932 California sea lions (Zalophus californianus), 321 Caloric excess, 106e110 Caloric restriction (CR), 110e111 Calystegia sepium, 505e506 Calystegines, 505e506 Camellia sinensis, 204 Campsis grandiflora, 209e211 Cancer, bioassays, 262 Candida spp., 116e117 C. albicans, 916 Cane toad (Rhinella marinus [formerly Bufo marinus]), 551e552 Cannabidiol (CBD), 50, 67, 193 Cannabinoid binding receptors (CBs), 238 CB1, 238 CB2, 238 Cannabis, 50, 67e69, 192, 237e238, 237f intoxication, 238 Cantharidin, 552, 598e600 Capillary leak syndrome, 579 Captopril, 610e611 Carbamates, 730, 742e744 Carbetamide, 730 toxicology, clinical signs, and pathology, 730 Carbohydrates, dietary, 116e117 carbohydrate-containing adjuvants, 652e653 carbohydrate-insulin model, 107 deficiency, 116 excess, 116e117 Carbon nanofibers (CNFs), 803e804 Carbon nanotubes (CNTs), 803e804, 807e808, 813e814 Carbon-based materials, 801 Carbon-based nanoparticles (NPs), 800 Carboxy-terminal cross-linked telopeptide of type 1 collagen (CTX-1), 115 Carcinogenicity, 236, 820 carcinogenicity data, 237 carcinogenicity studies, 3e4 carcinogenicity testing, methods, 23e25 testing in animal species, history of, 4e6 Carcinogens, 7, 241 Cardiac glycoside, 552 Cardioactive glycoside-containing plants, 514e515, 514t Cardiomyopathy, 474 Cardiotoxic animal toxins, 577 circulatory disturbances, 578e580 hemostasis abnormalities, 580e581 metalloproteinases, 577e578 neurotransmission derangement, 583e595 vascular permeability enhancement, 579 vascular tone modulation, 579 vascular wall damage, 580 Cardiotoxins, 577, 649 b-carotene, 121 Cascara sagrada (Rhamnus purshiana), 201 Cassava (Manihot esculenta C.), 533e535 Cassia spp., 518 C. fasciculata, 518 C. lindheimeriana, 518 C. nictitans, 518

INDEX

C. roemericana, 518 C. senna, 203e204 Castanospermine, 506 Castor bean plant (Ricinus communis), 541e542 Castrix, 137 Catechins, 259 Cathelicidin antimicrobial peptide (CAMP), 126 Caulophyllum thalictroides, 204 Caveolin-independent endocytosis (CIE), 818 CB2 agonist cannabidiol (CBD), 238 CeC chemokine receptor type 5 (CCR5), 644 Celecoxib, 246e247 Celiac disease (CD), 51, 53e54 Cell division and growth as target auxin mimics, 729 inhibition of, 729e730 microtubule organization inhibitors, 730 Cell phones, 17 2G cellular networks, 17 3G cellular networks, 17 4G cellular networks, 17 Centaurea spp., 510e511 C. repens, 510e511 C. solstitialis (yellow star thistle), 510e511 Center for Food Safety and Applied Nutrition (CFSAN), 82 Center for Veterinary Medicine (CVM), 87, 691e692 Centers for Disease Control and Prevention (CDC), 264 Centrosome, 811e812 Cephalosporium spp., 419 Cerium-144, 849 Cesium-137, 849 Cestrum diurnum, 530e531 Chamomile (Chamomilla recutita), 201, 238e239, 239f oils, 239 Channel blockers, 69 saxitoxin, 69 tetrodotoxin, 69 Checkpoint kinase 1 (CHEK1), 340e341 Chelation therapy, 704, 715 Chelonitoxism, 552e553 Chemical alternatives assessment (CAA), 29 Chemical Institute of Industrial Toxicology (CIIT), 7e8 Chenopodium spp., 527e528 Chernobyl, 847e848, 883, 905 disaster, 840 nuclear plant, 849 Cherries (Prunus spp.), 533e535 Chewing disease, 510e511 Chili peppers (Capsicum frutescens), 505e506 China Food and Drug Administration (CFDA), 85 China Food Safety Law (CFSL), 85 Chinese cobra (Naja atra), 594 Chinese medicine, 558e559 Chlamydomonas spp., 306 Chloralose, 756 Chlorantraniliprole, 747

Chlorella spp., 306 1-chloro-3-ethylamino-5-isopropyl-amino2,4,6-triazine, 733e734 Chlorine, 15 Chlorofluorocarbons (CFCs), 14 4-(4-chloroo-tolyloxy) butyric acid (MCBA), 729 Chlorophacinone, 751 Chlorophenoxy herbicides, 729 toxicology, clinical signs, and pathology, 729 Chloropropham, 730 Chlorotoxin from scorpion, 614 Cholecalciferol, 753e754. See also Vitamin D clinical signs and pathology, 753e754 development and use, 753 human risk, 754 medical data, 754 toxicology, 753 Cholera toxin (CTX), 641, 647e648 Cholesterol and dietary fiber, 126 Cholesterol-dependent cytolysins (CDCs), 649 Choline, dietary, 140e142 deficiency, 141e142 excess, 142 Choline acetyltransferase (ChAT), 590 Cholinergic signs, 742e743 Chondria armata, 44, 320 Chondrodendron tomentosum, 189 Chromalaena spp., 492 Chromium (Cr), dietary, 149e150 deficiency, 149e150 excess, 150 Chromium toxicity, 149e150, 700e702 diagnosis and treatment, 702 manifestations of toxicosis, 701e702 sources and exposure, 700 toxicology, 701 Chromosome damage, 855 Chronic interstitial nephropathy (CIN), 416e417 Chronic lymphocytic leukemia (CLL), 870e871 Chronic myeloid leukemia (CML), 870e871 Chronic obstructive pulmonary disease (COPD), 329e330, 663e664 Ciguatera fish poisoning (CFP), 44, 330 Ciguatoxins (CTXs), 44, 330e333, 584e589 clinical signs and pathology, 331 diagnosis, treatment, and control, 332e333 human exposure and disease, 331e332 maitotoxins (MTXs), 331 source/occurrence, 330 structure of CTX-1B, 330f toxicology, 331 Cimicifuga racemosa, 204 Cinnamon (Cinnamomi cortex), 203 Citrinin, 411e412 Clathrin-independent endocytosis (CIE), 818 Clathrin-mediated endocytosis, 817e818 Claviceps spp., 455e456 C. africana, 457 C. fusiformis, 457

INDEX

C. paspali, 455e456, 463 C. purpurea, 455e457, 463 Cleft palate, 524 Clenbuterol, 75 Climate change, 308 Clostridioides difficile, 633 Clostridium perfringens enterotoxin (CPE), 633e634 Clostridium sp., 194, 583e584 C. botulinum, 633e634 C. perfringens, 117 C. tetani, 633 Clothianidin, 746 Clupeotoxism, 44 Clustered regularly interspaced short palindromic repeat-Cas9 genome editing (CRISPR-Cas9 genome editing), 91 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), 24, 779 screening, 779e780 Clupeotoxism, 44 Coagulotoxic zootoxins, 580 Coagulotoxins, 570e571, 595 mechanisms, 580e581 Cobalamin, 139e140 deficiency, 140 excess, 140 Cobalt, 150e151 deficiency, 150 excess, 150e151 Cocaine, 518e519, 664 Cocklebur (Xanthium strumarium), 499e500 Cocoa (Theobroma cacao), 245e248, 245f procyanidins, 246e247 Coffee, 239e245, 240f animal studies, 244e245 consumption, 241, 243 drinking, 243 human health, 241e243 pharmacokinetics, 240e241 Codex Alimentarius Commission (CAC), 71, 79e80 Codex Committee on Food Additives (CCFA), 71 Color Additive Amendment of 1960, 79 Colorado River toad. See Sonoran Desert toad Coloring agents, 37 COMMD1, 153 Commiphora mukul, 204 Committee on the Validation of Alternative Methods (ICCVAM), 26e27 Common dolphins (Delphinus sp.), 321 Common kraits (Bungarus caeruleus), 594 Common loons (Gavia immer), 326e327 Common Mechanism Group (CMG), 733e734 Common vampire bat (Desmodus rotundus), 579 Compartment syndrome, 605 Complementarity-determining regions (CDRs), 643e644

Complementary Medicines (CM), 187b Complete blood count (CBC), 606e607, 666e667 Complex-Trait Consortium (CTC), 24e25 Compound annual growth rate (CAGR), 190 Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), 16e17 Conazoles, 737e740 Cone snails, 573 Coneflowers, 248 Conotoxins, 562 a-conotoxin, 585te588t d-conotoxin, 585te588t k-conotoxin, 585te588t m-conotoxin, 585te588t u-conotoxin, 558e559, 585te588t Constitutive androstane receptor (CAR), 435, 740e741 Consumer Affairs Agency (CAA), 89 Consumer Packaging and Labelling Act (CPLA), 84 Consumer Protection Act, 80 Consumer Right Protection Law (CRPL), 85 Contaminants, 13, 39e40, 166, 194 Contaminants in Food Chain (CONTAM), 13, 39e40, 166, 194, 454 Contract research organization (CRO), 6 Control of Drugs and Cosmetics Regulations Act, 278 Conventional pharmaceuticals, 187b, 196, 197t Convolvulus arvensis, 505e506 Coolia genera, 330 Coots (Fulica americana), 368 Copper, dietary, 111e112, 151e153 deficiency, 278 excess, 152e153 oxidative stress, 152e153 Copper (Cu) toxicity, 823e824 Coral (Palythoa spp)., 313t, 370e371 Cormorants (Phalacrocorax auritus), 326e327 Corn cob, rodenticide, 757 clinical signs and pathology, 757 development and use, 757 human risk, 757 risk to other species, 757 toxicology, 757 Coronary heart disease (CHD), 241 Corticotrophin-releasing hormone (CRH), 882 Corynebacterium diphtheria, 637e638 Coumarin, 750 COVID-19 pandemic (coronavirus disease 2019 pandemic), 190, 633 Crinotoxin, 557 CRISPR-associated proteins (Cas), 24 Creosote bush (Larrea tridentata), 203e204 Crooked calf syndrome, 524 Crotalaria spp., 492 Crotalus atrox toxoid, 609e610 Crotoxin, 593e594

933 Culmorin, 475e476 Curly dock (Rumex spp.), 528e529 Culmorin, 475e476 Cyanobacteria, 306 Cyanobacterial genera, 356 Cyanobacterial supplement, 346 Cyanogenic glycosides, 46e47 Cyanogenic plants, 533e535 Cyanoglossum spp., 492 Cyanohydrin, 46 Cyanotoxins, 44, 309, 365. See individual toxins Cycad seeds (Cycas revoluta T.), 533e535 Cycle-inhibiting factors (CIFs), 641 Cyclic adenosine monophosphate (cAMP), 592e593 Cyclic imine (CI), 317e319 clinical signs and pathology, 318e319 diagnosis, treatment, and control, 319 gymnodimine structures, 317f human exposure and disease, 319 pinnatoxin A structures, 317f prorocentrolide structures, 318f spirolides AeD and two desmethyl analogues structures, 317f source/occurrence, 317e318 toxicology, 318 Cyclomodulins, 641 Cyclooxygenase 2 (COX2), 656e657, 820 Cyclopiazonic acid, 468e470 chemical structure of, 469f human exposure and disease, 469e470 manifestations of toxicity in animals, 469 source/occurrence, 468 toxicology, toxicokinetics and mechanism of action, 468e469 Cylindrocarpon spp., 419 Cylindrospermopsin (CYN), 339e345 clinical signs and pathology, 341e344 diagnosis, treatment, and control, 344e345 human exposure and disease, 344 source/occurrence, 339e340 structure of, 339f toxicology, 340e341 Cylindrospermopsis spp., 314e315 C. raciborskii, 339 Cylindrospermum spp., 356 Cymopterus spp., 538 Cynomolgus monkey (Macaca fascicularis), 321 Cynosurus echinatus, 497e498 Cyromazine, 747 Cysteine, 115, 909 Cystic fibrosis (CF), 329e330, 663e664 Cytochromes P450 (CYP), 59, 124e125, 204, 216, 240, 260, 274, 405, 470, 816 enzymes, 340 Cytochromes P450 3A (CYP3A), 869 Cytokine release syndrome (CRS), 644 cytokine storm, 422e423 Cytolysins, 640e641 Cytotoxins, 571

934

INDEX

D Damage-associated molecular patterns (DAMPs), 581e582 Damp building related illness (DBRI), 432 Dandelion (Taraxacum officinale), 204 Danthron, 236 Dark Agouti rats (DA rats), 414e415 Datura spp., 505e506 D. metel, 209e211 Debinding protein (DBP), 753 Dead zone, 308 Death camas (Zigadenus spp.), 513e514 Death domains (DDs), 655e656 1,2-dehydropyrrolizidine alkaloids, 490e491 Delaney clause, 79 Delphinium spp., 509e510 D. andersonii, 509e510 D. barbeyi, 509e510 D. bicolor, 509e510 D. geyerii, 509e510 D. glaucescens, 509e510 D. glaucum, 509e510 D. nuttallianum, 509e510 D. occidentale, 509e510 Dendrimers, 801 Dendrotoxin, 591e592 Dendroaspis natriuretic peptide (DNP), 579 Deoxynivalenol (DON), 398, 427e428, 432e433 Department of Agriculture, Forestry and Fisheries (DAFF), 87, 90 Department of Environmental Affairs (DEA), 90 Department of Health (DoH), 87, 90 Department of Trade (DTI), 87 Department of Trade and Industry (DTI), 90 Depleted uranium (DU), 896 Dermatitis, 220e223. See also Organ toxicity allergic contact dermatitis, 220 photosensitization dermatitis, 220e223 primary irritant dermatitis, 220 Dermatitis herpetiformis (DH), 53 Dermonecrotic toxin (DNT), 633 13-desmethyl spirolide, 319 Desmethylbromethalin (DMB), 757 Desmodus rotundus salivary plasminogen activator (DSPA), 580 Developmental origins of health and disease (DOHaD), 776e777 Dew poisoning, 498 Di(2-ethylhexyl) phthalate (DEHP), 774 Diacetoxyscirpenol (DAS), 419 human risk and disease, 431e432 manifestations of toxicity in animals, 426e429, 427t poultry, 429 ruminants, 429 swine, 428e429 Diafenthiuron, 747 Diamide insecticides, 747 Diaporthe toxica, 496e497 Diarrheic shellfish poisoning (DSP), 44, 333 Dichlorodiphenyldichloroethylene (DDE), 773 Dichlorodiphenyltrichloroethane (DDT), 15, 744

4-(2,4-dichlorophenoxy) butyric acid (2,4DB), 729 2,4-dichlorophenoxyacetic acid (2,4-D), 729 Dicoumarol, 131 Dietary contaminants, 166e168 analyses, 166 mycotoxins, heavy metals, phytoestrogens, and other contaminants, 167e168 pesticides, 166e167 Dietary Supplement Health and Education Act (DSHEA), 279 Dietary supplements, 39 Diethylstilbestrol (DES), 777 Diets, 105e106 Digitalis, 514e515 Digitalis spp. D. lanata, 189 D. purpurea, 184 Dihydropterin (BH2), 138 1,25-dihydroxy Vitamin D. See Vitamin D Diisononyl phthalate, 774 Dilative cardiomyopathy, 115 Dimercaprol, 687e688 Dimercaptopropane sulfonate (DMPS), 687e688 Dimercaptosuccinic acid (DMSA), 687e688, 700 Dimethylsulfoxide (DMSO), 148 Dinitrophenols, 728 Dinoflagellates Gonyaulax spp., 314, 318e319, 372e373 Lingulodinium spp., 372e373 Ostreopsis spp., 330, 370e371 Prorocentrum spp., 318, 330, 333 Protoceratium spp., 372e373 Ptychodiscus spp., 325e326 Pyrodinium spp., 314 Vulcanodinium rugosum, 318 Dinophysis spp., 333 Dinophysistoxins (DTXs), 333e335 clinical signs and pathology, 334 diagnosis, treatment, and control, 335 human exposure and disease, 334e335 source/occurrence, 333 toxicology, 333e334 okadaic acid, 333f Dinotefuran, 746 Dioxin-like compounds, 10e11 Dioxins, 10e11 Dipeptidyl peptidase-4 (DPP4), 579 Diquat, 730e732 poisoning, 732 Discoid lupus erythematous (DLE), 913 Disinfection by-products (DBPs), 15 Disintegrin, 577e578 Disseminated intravascular coagulation (DIC), 596e597, 657 Distiller’s grains (DGS), 149 Diuron, 733 DMI (demethylation inhibitors) fungicides, 737e740 DNA-dependent RNA polymerase, 164e165 Dolichospermum spp., 314, 316, 355, 360e361 D. circinale, 315

Domestic Animal Infectious Diseases Control Act, 89 Domoic acid (DA), 35, 44e45, 66, 319e325. See also Okadaic acid (OA) clinical signs and pathology, 321e324 acute domoic acid toxicosis, 323f California sea lion, 323f domoic acid cardiomyopathy, 324f domoic acid toxicosis, 322fe323f diagnosis, treatment, and control, 325 human exposure and disease, 324 occurrence and species susceptibility, 319e321 toxicology, 321 structures of DA, kainic acid, and glutamic acid, 320f Dopamine, 331 Drechslera biseptata, 497e498 Dried distillers’ grains with solubles (DDGS), 440 Dronabinol, 192 Drug and Cosmetic Act, 277 Ducks, ring-necked (Aythya collaris), 368 Dungeness crabs (Metacarcinus magister), 320 Dutchman’s breeches (Thamnosma texana and Thamnosma montana), 538 Dysbiosis, 54e55

E Easter lily (Lilium longiflorum), 526 Eastern diamondback rattlesnake (Crotalus adamanteus), 555e556 Eastern tiger snake (Notechis scutatus), 590e591 Echinacea spp., 201, 248, 248f Echium spp., 492 Edema factor (EF), 636e637, 647e648 Egg Products Inspection Act (EPIA), 82e83 Eggplant (Solanum melongena), 505e506 Elderberry (Sambucus spp.), 533e535 Electro-Hyper-Sensitivity (EHS), 18 Electroencephalography (EEG), 324, 775 Electromagnetic Field (EMF), 845 Electromagnetic radiation (EMR), 845 Elemental mercury toxicosis, 711 chronic, 711 Elemental sulfur, 148 Emerging mycotoxins, 396 Emodin, 236 Emulgent. See Food emulsifier Emulsifiers, 38 Emulsions, 38 Endocannabinoids, 68 Endocrine disruption (ED), 736e737. See also Endocrine disruptors environmental contaminants and emerging endocrine disruptors, 773e776 mechanisms of, 773 Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), 771 Endocrine disruptors, 766e775 bioinformatics and integrative and functional enrichment omics approaches, 788e789

INDEX

CRISPR screening, 779e780 emerging models in EDC research, 778e780 environmental chemicals, 766e773 epigenetic effects of, 776e778 examples of disruption of endocrine pathways, 773e776 general anesthetics as, 775e776 glyphosate, 774e775 history of endocrine disruptor research, 766e768 machine learning and EDCS, 789e793 mechanisms of endocrine disruption, 773 omics technologies to evaluate endocrine disruption, 780e788 phthalates, 774 from reactive to proactive endocrine disruptor analysis, 778 regulatory approaches to, 771e773 routes of exposure to, 769e771 ToxCast bioanalytical plots, 770f types of chemicals with endocrinedisrupting activity, 768 zebrafish model, 778e779 Endocrine Guideline Optimization (ERGO), 772e773 Endocrine modifiers, 69e70 estrogenic mycotoxins, 70 goitrogens, 69 phytoestrogens, 69e70 Endocrine-disrupting chemicals (EDCs), 435, 440, 766, 767f Endocytosis, 817 Endotoxemia, 651 Endotoxemia, metabolic, 665 Endotoxin units (EUs), 651, 666 Endotoxins, 630, 651e657 bacterial endotoxin, 654f contaminating therapeutic products, 652e653 endotoxin limits, 652e653 endotoxin-mediated cell signaling, 655e656 infection, 651 ingestion, 651e652 inhalation, 652 lipid A, 655f monitoring, 652 pathogenesis of endotoxin-induced immune dysfunction, 656e657 sources of exposure, 651e653 structure and functional attributes of, 653e655 testing, 652 toxicology, 653e657 Enhancer of zeste homolog 2 (EZH2), 777 Enniatins, 472e473 Enolpyruvyl shikimate phosphate synthase glyphosate, 735 inhibition of, 735 Enterobacter, 194 Enterococcus, 194 Enterohemorrhagic Escherichia coli (EHEC), 658 Enterotoxemia, 648

Enterotoxigenic Escherichia coli (ETEC), 633, 658 Enterotoxins, 648 Envenomation envenoming, 549e550 process, 549e550 with zootoxins, 554e557 bites, 555e556 stings, 556e557 Environmental carcinogenicity testing current considerations for, 25e27 new directions for, 27e29 Environmental contaminants, 10e13, 40e41 dioxins and dioxin-like compounds, 11t Environmental pollutants air pollutants, 13e15 examples of, 8e22 general environmental contaminants, 10e13 ground and soil contamination, 16e17 microplastics and nanoplastics, 20e22 radiofrequency radiation, 17e20 water pollutants, 15e16 workplace exposure, 9e10 Environmental Protection Agency (EPA), 801 Environmental toxicity testing current considerations for, 25e27 new directions for, 27e29 Environmental toxicologic pathology current considerations for environmental toxicity and carcinogenicity testing, 25e27 alternative testing strategies, 26e27 human relevancy, 25e26 mechanism of action vs. mode of action, 25 examples of environmental pollutants, 8e22 history of carcinogenic testing in animal species, 4e6 methods of toxicity and carcinogenicity testing, 23e25 fish models, 23e24 transgenic mouse models, 24e25 new directions for environmental toxicity and carcinogenicity testing, 27e29 safe and sustainable alternatives, 28e29 principles of evaluations for carcinogenic potential, 6e8 role of lifestyle and environment on human health, 22e23 Enzootic hematuria, 539e540 Enzyme immunoassays (EIAs), 606 Enzyme-linked immunosorbent assays (ELISAs), 310, 606, 666 Ephedra (Ephedra sinica), 248e250, 249f treatment-related cardiotoxic lesions, 250f Epicatechin, 259 Epicatechin-3-gallate, 259 Epichloe spp., 456e457 Epigallocatechin, 259 Epigallocatechin-3-gallate (EGCG), 259 Epinephrine, 458e459 Epithelial growth factor (EGF), 910

935 Eprinomectin, 746e747 Epsilon toxin (ETX), 637, 644, 648, 662 Equine leukoencephalomalacia (ELEM), 442 Ergot alkaloids, 455e465 chemical structure of selected ergot alkaloids and dopamine, 456f diagnosis, treatment and prevention, 465 human risk and disease, 463e464 manifestations of toxicity in animals, 460e463 pharmaceutical use, 464e465 poultry, comb gangrene, 461 source/occurrence, 455e457 Claviceps purpurea sclerotium on cereal grain, 455f Claviceps spp., 455e456 Epichloe spp., 456e457 toxicology, 457e460 biodistribution, metabolism, and excretion, 457e458 mechanism of action, 458e459 species susceptibility, 459e460 toxins, 457 Ergovaline, 506e508, 507f Erythroxylum coca (coca), 518e519 Escherichia coli, 197e199, 237 Estimated daily intake (EDI), 71 Estrogen, androgen, and thyroid (EAT), 771 Estrogen receptor 1 (ESR1), 769 Estrogen receptor 2 (ESR2), 769 Estrogenic mycotoxins, 70 Estrogenic receptors (ERs), 434e435 17a-ethinylestradiol (EE2), 769 Ethylene glycol, 6e7 Ethylenediaminetetraacetic acid (EDTA), 700 Ethylenethiourea (ETU), 736e737 Eukaryotic elongation factor 2 (eEF2), 648 European adder (Vipera aspis), 583 European Chemical Agency (ECHA), 748 European Commission (EC), 84, 86e87 European corn borer (Ostrinia nubilalis), 441 European Food Safety Authority (EFSA), 84, 86e87, 401, 728e729 European Food safety Authority Panel on Contaminants (EFSA CONTAM), 466 European Generation of Novel, Integrated and Internationally Harmonized Approaches for Testing Metabolism Disrupting Chemicals (GOLIATH), 772 European hedgehog (Erinaceus europaeus), 568 European Union (EU), 84, 417 European Union General Food Law (EUGFL), 84 Evasins, 641 Evidence-based medicine, 190 Excitatory amino acids (EAAs), 60, 64 Exotoxins, 630, 633e651 classification by function, 640e642 bacterial colonization factors, 641e642 cytolysins, 640e641 classification by mechanism of action, 642e648

936

INDEX

Exotoxins (Continued) pores by membrane-damaging exotoxins, 645f type I exotoxinsesuperantigens hijacking immune response, 642e644 type II exotoxins-membrane-damaging toxins, 644e647 type III exotoxinseintracellular effector enzymes, 647e648 classification by target organ spectrum, 648e651 exposure, 633e634 key bacterial toxin attributes, 632t sources of exposure, 633e640 infection, 633 ingestion, 633e636 inhalation, 636e637 therapeutic products, 637e640 toxicology, 640e651 Exposome, 786e787 Exposomics, 786e788 Extracellular signaleregulated kinase (ERK), 644e646 Extremely low frequencies (ELFs), 845 Extrinsic pathway, 580

F Facial eczema, 497 Fagopyrism, 538 Fagopyrum spp., 538 F. esculentum, 538 F. tataricum, 538 Fair Packaging and Labeling Act of 1966, 80 Fat necrosis, 461e462, 462f Fatty acidebinding protein 4 (FAB4), 340 Fatty acids (FAs), 107e110 deficiency, 119 excess, 119 Fatty acyl transferase (FAT), 128e129 Federal Communications Commission (FCC), 17 Federal Food, Drug, and Cosmetic Act (FD&C Act), 81e82 Federal Meat Inspection Act (FMIA), 77, 82e83 Feed safety, 34 Fenoxycarb, 747e748 Fermentable Oligosaccharides, Disaccharides, and Monosaccharides and Polyols (FODMAPs), 57, 59 Fescue foot, 460e461 Feverfew (Tanacetum parthenium), 201 Fiber, 117e118 Fiber, dietary, 117e118 Fibrin degradation products (FDPs), 596e597 Fidelity Level (FL), 201 Field Emission Scanning Electron Microscope (FESEM), 808 Fipronil, 746 Fire ants (Solenopsis spp.), 553e554 Fireweed (Senecio madagascariensis), 492 First-generation anticoagulant rodenticides (FGARs), 750 Fish kill, 314e317

Fish models, 23e24 Flavin adenine dinucleotide (FAD), 134 Flavin mononucleotide (FMN), 134 Flavonoids, 247 Flavor enhancers, 37e38 Flavorings, 37 Flavorings, natural, 37e38 Flavorings, synthetic, 37 substances, 38 Florida snail kite (Rostrhamus sociabilis), 370 Flos carthami, 203 Flubendiamide, 747 Fluorescence in situ hybridization (FISH), 667 Fluorine, 111e112 Fluorine (F), dietary, 111e112, 153e156 deficiency, 154 excess, 154e156 Fluoride toxicity, 155 Fluorosis, dental, 155 Fluorosis, osseous, 155 Fluoxetine, 771 Flying foxes (Pteropus mariannus mariannus), 367 Folates. See Folic acid Folic acid, 137e139 deficiency, 138e139 excess, 139 Follicle-stimulating hormone (FSH), 426, 881 Food Additive Petitions (FAP), 87 Food additives, 60, 71e72 acceptable daily intake (ADI), 71 Food Additives Amendment, 78 Food allergies, 51e52, 58 Food and Agriculture Organisation of the United Nations (FAO), 51 Food and Agriculture OrganizationeWorld Health Organization (FAOeWHO), 416 Food and Drug Act and Regulations (FDA&R), 84 Food and Drug-Related Crime Investigation Bureau (FDRCIB), 85e86 Food and Drugs Act (1920), 76e77 Food and Veterinary Office (FVO), 88e89 Food coloring, 37, 58, 71e72 Food contact substances (FCSs), 41e43 Food contaminants, 72e76 functional foods, 75e76 residues, 73e75 Food Directorate (FD), 84 Food effects, diet and fasting, 110 Food emulsifier, 38 Food intolerance, 53, 57 Food Labeling Act, 89 Food packaging, 41e43 Food poisoning, 633e634 Food processing, 454 contaminants, 41e43 Food processingeinduced chemicals (FPICs), 42 Food regulation, 76e90 Australia and New Zealand, 86 Brazil, 86

Canada, 84 China, 85 Europe, 84e85 feed for animal consumption, 87e90 Japan, 85 regulation and approval of foods for human consumption, 81e87 South Africa, 87 United Kingdom, 86e87 United States, 81e84, 87e88 Food residues, 396 Food safety, 34 assessment, 70e76 challenges and future developments in, 90e92 risk/safety assessment in food, 70e71 Food Safety Act, 87 Food Safety and Inspection Service (FSIS), 82e83 Food Safety Basic Act (FSBA), 85, 89 Food Safety Law, 85 Food Safety Modernization Act (FSMA), 80, 668 Food Safety Order of 1991, 87 Food Safety Programme (FSP), 89 Food Sanitation Act (FSAct), 85, 89 Food Standards Agency (FSA), 87 Food Standards Australia New Zealand Act (FSANZ), 86 Food Standards Scotland (FSS), 86e87 Foods, 34e37, 105 adverse reactions to food constituents, 51e58 bacterial toxins, 668 chemicals foreign to food, 36t chemicals intentionally added to food, 37e39 compounds with toxic properties naturally present in foods, 46e47 contamination, 39e46 genetically modified, 48e49 hominid development to modern human, 34f mechanism of action of clinical disorders related to food, 59e70 novel foods, 47e51 safety assessment of food, 70e76 toxicology, 34 Foodborne illness, 76, 633e634 Formaldehyde, 14 Fortified foods, 38 Fructose, toxicity, 135 Fructus aurantii, 203 Fugu, 552e553, 635e636 Fukushima, 847e848 Fukuyoa spp., 331 Fullerenes, 799 Fumonisin B1 (FB1), 45, 445e446 Fumonisins (FB), 45, 440e455, 736 chemical structure of, 441f diagnosis, treatment, and prevention, 454 human risk and disease, 452e454 manifestations of toxicity in animals, 446e452 regulations and guidances, 454e455

937

INDEX

source/occurrence/exposure, 440e441 toxicology and MOA, 441e446 biodistribution, metabolism, and excretion, 443 fumonisin-induced species-specific target organ toxicity, 442t mechanism of action (MOA), 443e446 species susceptibility, 442e443 toxins, 441e442 Functional deficits, 615 Functional foods, 38, 75e76 Fungicide Resistance Action Committee (FRAC), 737 Fungicides, 736e741. See also Herbicides; Insecticides; Rodenticides strobilurins or quinol oxidation site of complex III inhibitor fungicides, 741 triazole-containing azole fungicides/DMIfungicides/C14-demethylase inhibitors, 737e740 mefentrifluconazole, 739e740 prothioconazole, 738e739 succinate dehydrogenase inhibitors (SDHI), 740e741 tebuconazole, 738 Funnel web spiders, 555e556 Furocoumarins, 538 Fusaproliferins, 475e476 Fusaric acid, 475e476 Fusarium spp., 45, 419e420, 434 F. culmorum, 420 F. graminearum, 420 F. oxysporum, 197e199 F. poae, 394e396 F. sporotrichioides, 394e396 F. sulphureum, 197e199 F. verticillioides, 451e453 toxins, 472e476. See also Trichothecenes beauvericin and enniatins, 472e473 diagnosis, treatment, and control, 476 human exposure and disease, 473 manifestations of toxicity in animals, 473 source/occurrence, 472 toxicology, toxicokinetics and mechanism of action, 472e473

G G-protein coupled receptors (GPCRs), 592e593 GABA type A receptor (GABAAR), 775 Gambierdiscus spp., 331 G. toxicus, 330 Gamma aminobutyric acid (GABA), 331, 472 Gamma-glutamyl transferase (GGT), 492e493 Gangrenous syndrome, 460e461, 461f Garlic (Allium sativum L.), 201, 204, 217e218, 250e252, 251f Gelsolinase, 578 General Administration of China (GACC), 85 General Administration of Customs (GAC), 89

General anesthetics (GAs), 775 as endocrine disruptors, 775e776 General Food Law, 87 Generally Recognized As Safe (GRAS), 49, 78, 87, 223e234, 251, 257, 266 Genetically modified organisms (GMOs), 48, 76, 441 foods, 48e49 plants, 76 Giant cells (GCs), 788e789 Gila monsters (Heloderma suspectum), 555 Ginger (Zingiber officinale), 204, 256e257, 256f Ginkgo (Ginkgo biloba), 204, 252e256, 252f Ginkgo biloba extract (GBE), 252e253 on nasal tissue, 255f on rat liver, 253f on thyroid gland, 254f Ginseng (Panax ginseng), 201, 204, 257e258, 257f Ginseng (Panax notoginseng), 209e211 Global system for mobiles (GSMs), 17e18 Glomerular filtration rate (GFR), 415 Glucoseregulated protein 78 (GRP78), 704e705 Glucosinolates, 47 Glufosinate, 732e733 toxicology, clinical signs, and pathology, 732e733 Glutamate (Glu), 60e64 excitotoxicity, 60 receptors, 60e64, 64f g-glutamyl transpeptidase (transferase) (GGT), 450, 580e581, 641 Glutathione (GSH), 340e341 interaction with selenium, 162e163 interaction with vitamin E, 128 Glutathione peroxidase (GSHPx), 435e436 interaction with selenium, 162e163 interaction with vitamin E, 128 Glutathione reductase, 128 interaction with selenium, 162e163 interaction with vitamin E, 128 Glutathione-S-transferase p (GST-P), 450 Glutathione-S-transferases (GSTs), 340e341 Gluten, 53e57 Gluten-free diet (GFD), 54 Gluten-related disorders (GRDs), 53e57 ataxia, 53 neuropathy, 53 Glycine (Gly), 60 Glycosides, 46 Glycosylphosphatidylinositol (GPI), 644 Glyphosate, 735, 774e775 toxicology, clinical signs, and pathology, 735 Goat weed, 537e538 Goitrogens, 69 Gold nanoparticulates, 823 Golden poison dart frog (Phyllobatesv terribilis), 568 Goldenseal (Hydrastis Canadensis), 258e259, 258f

Good Agricultural and Collection Practices (GACP), 199e200 Good Agricultural Practices (GAPs), 194, 200 Good Laboratory Practice (GLP), 194, 611e614, 670 Good Manufacturing Practices (GMP), 194, 199e200, 275, 277 Good Supply Practice (GSP), 194 Grain Inspection, Packers, and Stockyards Administration (GIPSA), 411 Gram-negative bacteria (GNB), 651 Granulocyte colony-stimulating factor (GCSF), 858e859 Granulocytopenia, 865e866 Grapes (Vitis spp.), 526e527 Grass carp (Ctenopharydon idella), 370 Great horned owl (Bubo virginianus), 368 Green bottle fly (Lucilla sericata), 611 Green mamba snake (Dendroaspis angusticeps), 579 Green sea turtles (Chelonia mydas), 363e364 Green tea (Camellia sinensis), 259e260, 259f Green treefrogs (Hyla cinerea), 370 Green turtle (Chelonia mydas), 327 Groundsel (Senecio sp.), 268e270 Group A Streptococcus (GAS), 633 Guanine nucleotide (CpG), 776 Guanitoxin (GNT), 44, 358e361 clinical signs and pathology, 359e360 lacrimation with chromodacryorrhea, 360f diagnosis and treatment, 361 human exposure and disease, 360e361 source/occurrence, 358e359 spherical vegetative cells, 359f structure of, 359f toxicology, 359 Guggul (Commiphora mukul), 204 Guillain-Barre´ syndrome (GBS), 583 Gulf War Illness (GWI), 66e67 Gut microbiota and adverse reactions to food, 59e60 Gymnodimines, 318e319 Gymnodinium, 314

H Hallucinogens, 67 Halogenated acetic acids, 15 Halogeton (Halogeton glomeratus), 527e528 Harbor seals (Phoca vitulina), 321 Harmful algal blooms (HABs), 306e308 aquatic hypoxia, 308e309 Hawaiian monk seals (Neomonachus schauinslandi), 330 Hawksbill turtle (Eretmochelys imbricata), 327 Hazard Analysis and Critical Control Points (HACCP), 57e58, 79 Hazard Analysis of Risk-Based Preventative Controls (HARPC), 80 Head blight, 419e420 Health and Human Services (HHS), 683 Health-based guidance values, 443e445 Health Canada (HC), 84, 88

938 Health Products and Food Branch (HPFB), 84, 88 Health Promotion Act, 89 Health Promotion Law (HPL), 85 Heatstroke, 844 Heavy metals, 167e168, 680, 682 Helenium amarum, 490 Helicobacter pylori, 633 Heliotrope (Heliotropium europaeum), 492 Hematopoietic system, 425, 865e872 Hemlocks (Tsuga spp), 512e513 a-hemolysin (HlyA), 641, 648e649 Hemolysins, 640e641, 648e649 Hemolytic uremic syndrome (HUS), 660 Hemostasis, abnormalities, 580e581 coagulotoxic mechanisms, 580e581 coagulotoxic zootoxins, 580 Hemostatic disruption, 596e597 Hemotoxins, 570, 595 Hepatic veno-occlusive disease (HVOD), 269, 492 Hepatoma Type A, 7 Hepatoma Type B, 7 Hepatosis dietetica, 130 Hepatotoxic plants, 490e500 PA-containing plants, 490e494 plants containing fungal hepatotoxins, 495e498 saponin-containing plants, 494e495 Hepatotoxicity, 218, 694 Herb-herb interaction, 204e209, 205te207t Herbal remedies, 190, 276e279 acceptability of, 196e197 active pharmaceutical, 192e197 adulterants, 194e196 apothecary to pharmacy, 189e190 comparison between properties of, 196, 197t contaminants, 194 direct toxicity, 209 herbal remedies and prescription medicines, 208te209t dose and response, 193e194 drug interactions, 204 efficacy and effectiveness, 200e201 effectiveness experimental evidence, 201 randomized clinical trials using herbal remedies, 201 traditional knowledge of efficacy, 201 efficacy experimental evidence, 201 randomized clinical trials using herbal remedies, 201 traditional knowledge of efficacy, 201 evidence for herbal remedy efficacy, 190e192 empirical evidenceetraditional knowledge, 190e192 experimental evidenceecontrolled, 192 herb-herb interaction, 204e209 hypersensitivityeidiopathic allergic reactions, 211 indirect toxicity, 209e211

INDEX

selected herbal remedies, 210t influencing factors on concentration of APIs in plant, 193 interactions, 204 herbedrug interaction, 204 international regulatory, 275e279 herbal remedy, 280te284t select list of countries and regulatory requirements, 276e279 pharmacodynamics, 218 properties of herbal remedies and conventional drugs, 196 quality, 193, 199e200 active pharmaceutical ingredient, 200 efficacy and safety, 197e199 manufacturing processes and controls, 200 quality control, 200 randomized clinical trials using, 201 safety, 203e211 adverse reactions, 203e204 safety, side effects and toxicity, 203 toxicologic pathology of, 223e275 aloe vera, 223e237 cannabis, 237e238 chamomile, 238e239 cocoa, 245e248 coffee, 239e245 echinacea, 248 ephedra, 248e250 garlic, 250e252 ginger, 256e257 Ginkgo biloba, 252e256 ginseng, 257e258 goldenseal, 258e259 green tea, 259e260 I3CeBrassica sp. glucosinolates, 260e262 Kava kava, 262e265 milk thistle, 266e267 mint, 267e268 rattlepods, yellow weed, and groundsel, 268e270 saw palmetto, 270e271 senna, 271 St. John’s Wort, 271e272 tobacco, 272e274 turmeric oleoresin, 274e275 toxicokinetics, 212e216 toxicology of, 211e223 genotoxicity and carcinogenesis, 211e212 herbal pharmacodynamics, 218 herbal toxicokinetics, 212e216 lethality, 211, 212t microbiome, 216e218 organ toxicity, 218e223 use and regulations Australia, 276 Canada, 276 China, 276e277 European Union, 277 India, 277 Japan, 277e278 Korea, 278 Malaysia, 278 Philippines, 278

United States of America, 191t, 278e279 Herbicide Resistance Action Committee (HRAC), 728e729 Herbicides, 728e736. See also Fungicides; Insecticides; Rodenticides activation of reactive oxygen species, 730e735 classified in terms of mode of action, 728t inhibition of cell division and growth, 729e730 d-hexatoxins (HXTX), 556 Hiroshima, 847 Hirudo seu whitmania, 203 Histamine, 52 fish poisoning, 57 Histidine, 552e553 Hodgkin’s disease, 896e897 Holocyclotoxin, 585te588t Homogentisate (HG), 734e735 Hoodia gordonii, 204 Hooded pitohui (Pitohui dichrous), 553 Hormesis, 193 Hormones, 766 Horse chestnut (Aesculus hippocastanum), 201 Horsefly (Tabanus spp.), 203 House mouse (Mus musculus), 756 House mouse (Mus musculus domesticus), 750 House musk shrew (Suncus murinus), 635 Household technologies and nonionizing radiation, impact of, 845e846 3ß-Hsd gene, 308e309 HT-2 toxin, 419 Human Equivalent Dose (HED), 242 Human exposure, 453 Human health radiation, 845e846 role of lifestyle and environment on, 22e23 Human relevancy, 25e26 Human renal tubular epithelial cells (HK-2), 255 Humpback whales (Megaptera novaeangliae), 314e315 Hydrated sodium calcium aluminosilicate (HSCAS), 411 Hydrilla verticillata, 368e370 Hydrogen cyanide (HCN), 46 Hydrogen sulfide (H2S), 149 Hydroxyanthraquinones (Has), 236 Hydroxyphenylpyruvate dioxygenase inhibitors, 734e735 4-hydroxyphenylpyruvate dioxygenase (HPPD), 734e735 5-hydroxytryptamine (5-HT), 423, 458e459, 554, 589e590 Hymenoptera, 557 Hyoscyamus spp., 505e506 Hypericin, 271, 537e538 Hypericism, 537e538 Hypericum perforatum, 204, 537e538 Hypersensitivity, 211 Hypervitaminosis A, 123 Heterocyclic amines (HCAs), 92

INDEX

4-hydroxyphenylpyruvate dioxygenase (HPPD), 734e735 Hyperthermia, 843e845, 919 clinical use of, 919 mechanisms of hyperthermia-induced injury, 919e920 reaction of specific organs and tissue to, 920e924 alimentary system, 920 cardiovascular system, 922 eye, 922 integumentary system, 923e924 male reproductive system, 923 musculoskeletal system, 920e922 nervous system, 922 urinary system, 922e923 response to injury induced by, 920e924 Hypothalamicepituitaryegonadal axis (HPG axis), 734

I Idiopathic allergic reactions, 211 IgA glomerulonephritis (DON), 430 Imidacloprid, 746 Immunogenicity, 671 Immunomodulating drugs, 691 Immunotoxins safety assessment, 670e671 regulatory guidance for, 670e671 toxic effects associated with, 671 Imperial Chemical Industries (ICI), 730e731 Improvised explosive devices (IEDs), 844 Indian river lagoon (IRL), 311 Indigenous traditional medicine, 187b Indochinese spitting cobra (Naja siamensis), 581 Indole acetic acid (IAA), 729 Indole-3-carbinol (I3C), 260e262 Indospicine, 511e512 Inducible nitric oxide synthase (iNOS), 656e657 Infant Formula Act, 80 Inflammation induction, 581e583 adaptive immune responses to zootoxins, 583 innate immune responses to zootoxins, 582e583 Inflammatory bowel disorders (IBDs), 59 Informant Agreement on Remedies (IAR), 201 Informant Consensus Factor, 201 Inhibitor cysteine knot (ICK), 584 Inositol triphosphate (IP3), 592e593 Insect growth p0580 regulators (IGRs), 747e748 Insecticides, 741e748. See also Fungicides; Herbicide; Rodenticides new insecticides, 746e748 cyromazine, 747 diafenthiuron, 747 diamide insecticides, 747 fenoxycarb, 747e748 lufenuron, 748

macrocyclic lactone endectocides, 746e747 neonicotinoids, 746 phenylpyrazoles, 746 pymetrozine, 748 spiropidion, 748 organochlorines, 744e745 organophosphates and carbamates, 742e744 clinical signs and pathology, 743e744 human risk, 744 toxicology, 742e743 pyrethrins and pyrethroids, 745e746 Integrated Risk Information System (IRIS), 41 Intercellular adhesion molecule 1 (ICAM-1), 906 Interferon gamma (IFN-g), 644, 853 Interleukin-1 receptor-like type 1 (IL1RL1), 583 Interleukins (IL), 630, 638 IL-6, 577e578 Internal emitters, 848e851, 848t International Agency for Research on Cancer (IARC), 5e6, 18, 25e26, 37, 237, 416, 683, 714, 820, 845 International Cooperation on Alternative Test Methods (ICATM), 26e27 International Council For Harmonisation (ICH), 611e614, 670 International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), 3e4, 7 International Nuclear Event Scale (INES), 840, 851 International Organization for Standardization (ISO), 611e614, 797 International system of units (SI units), 847 Intracisternal A particle (IAP), 777 Intrinsic factor (IF), 139e140 Invasins, 641e642 Investigational New Drug (IND), 614, 670e671 In vitro diagnostic directive (IVDD), 640e641 Iodine, dietary, 156e158 deficiency, 157e158 excess, 158 goiter, 157e158 thyroid hormone synthesis, 156e158 Iodine-131 therapy, 156e158, 849, 881 Ion channel acid-sensing, 591 ligand-gated, 591 transient receptor potential vanilloid 1 (TRPV1), 592 voltage-gated, 552 Ionizing radiation, 841e843, 861. See also Ultraviolet radiation (UV radiation) acute radiation syndromes and combined injury, 859e860 alimentary system, 872e877 cardiovascular system, 896e899 cell and tissue radiosensitivity to, 856e857

939 endocrine system, 880e882 external radiation, 841e842 fetal effects, 903 general tissue and organ effects of, 857e858 haired skin, 857f hematopoietic and lymphoid systems, 865e872 bone marrow, 866f general reaction to ionizing radiation injury, 865e868 lymphoid tissues, 868e870 radiation leukemogenesis, 870e872 integumentary system, 905e908 interaction of ionizing radiation with biological materials, 853e854 internally deposited radionuclides, 842e843 ionizing radiation, 846e853 carcinogenesis, 860e862 ionizing radiationeinduced cell death, 856 mechanisms of, 853e862 molecular mediators, 858e859 musculoskeletal system, 892e896 nature and action of, 846e853 Chernobyl and Fukushima, 847e848 external radiation and internal emitters, 847e851 pathology associated with, 851e853 radiation biophysics, 847 nervous system, 877e880 reproductive tract, 903e905 ovary, 904e905 testes, 903e904 respiratory system, 885e892 pleural surface, 866f response to injury induced by, 862e908 special senses, 882e885 ear, 883 eye, 883e885 general reaction to ionizing radiation injury, 882e883 subcellular and cellular effects of, 854e856 urinary system, 899e903 vascular and connective tissue effects of, 862e865 heart, coronary artery, 864f heart and lungs, 863f phases of radiation injury, 863t small-caliber artery, 864f Ionotropic glutamate receptors (iGluRs), 60e64, 65f Ipomoea spp., 501, 505e506 I. lonchophylla, 505e506 I. meulleri, 505e506 Iron, 112 deficiency, 682, 703 excess, 158e160 Iron (Fe), dietary, 158e160 Irradiation, 894 Irritable bowel syndrome (IBS), 57, 59 Isobaric tagging for relative and absolute quantitation (iTRAQ), 781 Isoforms, 816 Isopropylamine (IPA), 735

940

INDEX

Isorhamnetin, 252, 255e256 Isothiocyanates, 47 Ivermectin, 746e747

J Japan Agricultural Standards Law (JASL), 85 Japan Existing Chemical Database (JECDB), 3e4 Japanese Agricultural Standards (JAS), 85 Japanese Agricultural Standards Act (JAS Act), 89 Japanese medaka (Oryzias latipes), 338 Jararaca (Bothrops jararaca), 610e611 Joint FAO/WHO Expert Committee on Food Additives (JECFA), 43, 70e71, 77e78, 416 Joint Food Standards Treaty, 86 Jun N-terminal kinase (JNK), 655e656

K K-ras gene, 892 proto-oncogene, 892 Kabirimine, 318 Kadethrin, 745 Kaempferol, 252, 255e256 Kamboˆ vaccine, 553e554 Karenia, 325e326 K. selliformes, 318 KashineBeck disease, 432 KashineBeck osteoarthritis (T-2 toxin), 430 Kava (Piper methysticum), 203e204 Kava kava (Piper methysticum), 201, 262e265, 266f centrilobular hepatocellular inflammation and fatty change, 263f Kelch-like ECH-associated protein 1 (Keap1), 241 Kemp’s ridley turtle (Lepidochelys kempii), 327 Kentucky-31 (KY-31) tall fescue, 455 Keratinocyte-derived chemokine (KC), 858e859 Keshan disease, 130, 474 Kidney toxicity, 450, 469, 600e601, 899e902 Killdeer (Charadrius vociferus), 368 King cobra (Ophiophagus hannah), 568 Klamath weed, 537e538. See also St. John’s Wort Kochia weed (Kochia scoparia), 499e500 Komodo dragons (Varanus komodoensis), 555 Korea, herbal remedies in, 278 Krimpsiekte, 595 Kyoto Encyclopedia of Genes and Genomes (KEGG), 783e786

L Lactate dehydrogenase (LDH), 517, 731e732, 920 Lactose intolerance, 57 Lance-leaf sage (Salvia reflexa), 499e500 Lantana spp., 498e499 L. camara, 498e499 Large-volume parenterals (LVPs), 669

Larval zebrafish (Danio rerio), 369 Latrotoxins (LTX), 593 Law on Farm Product Quality and Safety (LFPQS), 85 Law on the Inspection of Import and Export Commodities (LIIEC), 85 LC-quadrupole/time-of-flight MS in tandem mode (LC-QTOF-MS/MS), 354 Lead (Pb), 680, 702e709 acute exposure, 705 chronic exposure, 705e706 diagnosis and treatment, 708e709 effects on heme synthesis, 704f human exposure and disease, 706e708 lead-induced anemia, 708t lead-induced nephrosis, 707f lead lines, 707 manifestations of toxicosis in animals, 705e706 palsy, 707e708 sources and exposure, 702e703 toxicology, 703e705 Leaky gut, 54e57 Least weasel (Mustela nivalis), 755 Leatherback turtle (Dermochelys coriacea), 327 Leptin, 107e110 Lesser scaup (Aythya affinis), 368 Lethal factor (LF), 636e637, 647e648 Leucine, 115 Leukocidins, 640e641, 649 Lifestyle, 22e23 Lily (Lilium spp.), 526e527 cat poisoned with, 526f Limits of detection (LODs), 433 Limulus amebocyte lysate (LAL) assay, 651, 666, 669 Linear energy transfer (LET), 847 Linear no threshold model (LNT model), 852 Linuron, 733 Lipidomics, 781e783 Lipid A, 655f Lipids, 118e119, 781e782 deficiency, 119 excess, 119 lipid-soluble vitamins, 119 Lipids, dietary, 118e119 deficiency, 111 excess, 111e112 gastrointestinal function, 117e118 Lipofuscin, 130 Lipomatosis, 461e462 Lipopolysaccharides (LPS), 44, 244, 416, 651. See also Endotoxin Lipopolysaccharides-binding protein (LBP), 655 Listeria spp., 641e642 Listeriolysin O (LLO), 641 Liver, 7, 601e602, 739e740, 875e877 cancer, 452e453 in laboratory animals, 408e409 injury, 448 microphysiological systems, 24 toxicity

hepatoblastomas, 259 hepatocellular adenomas, 259 hepatocellular hypertrophy, 259 hepatocellular necrosis, 698 Locoweed (Astralagus and Oxytropis spp.) intoxication, 500e505. See also Swainsonine Lodgepole pine (Pinus contorta), 532e533 Loggerhead turtle (Caretta caretta), 327 Lolium spp. L. arundinaceum, 455 L. perenne, 497 Loop mediated amplification (LAMP), 666 Lufenuron, 748 Lung toxicity, 602, 663e664, 886e889 cancers, 702, 890e891 Lupines (Lupinus spp.), 513, 521e523 calf with crooked calf disease, 522f Lupinosis, 496e497 liver from sheep with, 497f Lupinus spp., 521e523 L. formosus, 521e523 L. sulphureus, 521e523 Luteinizing hormone (LH), 426, 734 Lymphocyte functioneassociated antigen 1 (LFA-1), 641 Lymphocyte necrosis, 426 Lyngbyatoxins, 361e365 Lysergic acid diethylamide (LSD), 457e458, 554 Lysine, 114 Lysophosphotidylcholine (LPC), 582e583

M Macaques (Macaca fascicularis), 366 Macrocyclic lactones, 746e747 endectocides, 746e747 Macrocyclic trichothecenes process, 429e430 SG-induced atrophy, 431f toxins, 420 Macronutrients, 111e166 amino acids, 114e116 carbohydrates, 116e117 fiber, 117e118 lipids, 118e119 proteins, 113e114 Magnesium, dietary, 144e146 deficiency, 145 excess, 146 Magnetic resonance imaging (MRI), 41 Maitotoxins (MTXs), 331 Major histocompatibility complex (MHC) class II, 643 Malayan krait (Bungarus candidus), 592 Malignant bone tumors, 894 Malignant hyperthermia (MH), 921e922 Mallard duck (Anas platyrhynchos), 368 Malondialdehyde (MDA), 435e436 Manatee. See Brevitoxins; West Indian manatees Mancozeb, 736e737 Manganese (Mn), dietary, 112, 160e161 deficiency, 160e161 excess, 161

INDEX

Manganese-dependent superoxide dismutase (Mn-SOD), 160 Mannheimia haemolytica, 633 Many-banded krait (Bungarus multicinctus), 590e591 Margin of exposure (MOE), 410 Marijuana (Cannabis sativa), 189, 192 Marine algal toxins, 44. See also Phycotoxins Marjolin “ulcer”, 609 Mass spectrometry (MS), 310, 433, 781e782 Matrix Gla protein (MGP), 130e131 Matrix metalloproteinases (MMPs), 164e165, 879 Maximum residue limits (MRLs), 39, 45, 73 international regulatory documents, 74t Maximum tolerated dose (MTD), 249, 279, 730 Mechanism of action (MOA), 25, 58, 728 Mectins, 746e747 Medaka (Oryzias latipes), 23e24 Median lethal dose (MLD), 731e732 Medicated feed, 38e39 Mediterranean monk seals (Monachus monachus), 314 Mefentrifluconazole, 739e740 Membrane attack complexes (MACs), 582 Membrane fluidity, 919e920 Membrane-bound form (mCD14), 655 Membrane-damaging toxins (MDTs), 642 Menaquinone (MK), 130e131 MK-4, 131 MK-7, 131 Mentha piperita, 204 Mercury (Hg), 709e715 diagnosis and treatment, 714e715 elemental, 711 inorganic, 711e712 intoxication, 714e715 ions, 709 organic, 712e714 sources and exposure, 709 toxicology, 709 toxicologic effects, 710te711t Mercury, inorganic, 711e712 Mesenchymal stem cells (MSCs), 866e867 Metabolism-disrupting chemicals (MDCs), 782e783 Metabolizable energy, 106 Metabolomics, 781e783 Metabotropic glutamate receptors (mGluRs), 60e64 Metal phosphides, 754e756 Metalloproteinases, 577e578 Metallothionein, 693 Metals. See also individual metals antimony, 680e683 arsenic, 683e688 beryllium, 688e691 bismuth, 691e692 cadmium, 692e700 chromium, 700e702 lead, 702e709 mercury, 709e715 plutonium, 715e716

thallium, 716e719 uranium, 719e721 Methemoglobin, 535e536 Methicillin-resistant S. aureus (MRSA), 658 Methionine, 114 and choline, 141 and cobalamin, 139e140 excess, 115 and folate, 138 and sulfur, 148e149 5-methoxy-N,N-dimethyltryptamine (5MeO-DMT), 554, 589e590 Methoxychlor, 772 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), 354 2-methyl-4-chlorophe noxyacetic acid (MCPA), 729 b-methylamino-L-alanine (BMAA), 309, 365e368 analytical methods for detection and quantification, 367e368 animal studies, 366 human exposure and disease, 367 mechanism of action, 366e367 sources/occurrences/exposures, 365 toxicology, 365 7,8-methylenedioxylycoctonineb (MDL), 509e510 Methyllycaconitine (MLA), 509e510 Methylmercury, 713 Methylsulfonylmethane (MSM), 148 Microbiome, 59, 216e218, 629e630, 783e786 and cytochrome P450, 59 and food additives, 60 and immunity, 59e60 Microbiota, 783 Microcystins (MCs), 345e355. See also Phycotoxins Microcystis, 346 M. aeruginosa, 345e346 M. viridis, 345e346 M. wesenbergii, 345e346, 352e353 Micronutrients, 111e166 minerals, 142e166 vitamins, 119e142 Microplastics, 20e22 MicroRNAs (miRNAs), 781 Microseira wollei, 361 Microtubules, 813 interactions, 813 organization inhibitors, 730 carbamates, 730 Microwaves (MV), 845 exposures, 922 Milk sickness, 515e516. See also White snakeroot Milk thistle (Silybum marianum), 201, 266e267, 267f hepatoblastoma, 266f hepatocellular carcinoma, 267f photomicrographs of lesions, 265f Millimeter waves (MMWs), 17 Minamata disease, 712e713. See also Methylmercury

941 Minerals, 142e166 calcium, 142e143 magnesium, 144e146 major minerals, 142e149 phosphorus, 143e144 sodium/potassium, 146e148 sulfur, 148e149 trace minerals, 149e166 Minimal erythemal dose (MED), 911 Ministry of Agriculture (MAPA), 86 Ministry of Agriculture, Forestry, and Fisheries (MAFF), 85, 89 Ministry of Agriculture and Rural Affairs (MARA), 85, 89 Ministry of Commerce (MOFCOM), 85 Ministry of Environment (MMA), 86 Ministry of Health (MS), 86 Ministry of Health, Labour and Welfare (MHLW), 85, 89 Ministry of Public Security, 85 Mint (Mentha sp.), 267e268, 268f Mistletoe (Viscum album), 200, 204 Mitogen-activated protein kinases (MAPKs), 242, 340e341, 422, 644e646, 655e656 Mitotic spindle interactions, 811e818 additional cytopathologic interactions, 815e818 centrosomal interactions, 812e813 chromosomal interactions, 813e815 microtubule interactions, 813 Modern Crop Protection Compounds, 737 Modulins, 642 Molecular initiating events (MIEs), 780 Molybdenum (Mo), dietary, 112, 161e162 deficiency, 161 excess, 161e162 Moniliformin, 474. See also Mycotoxins Monkey-faced disease, 523e524 Monkey, rhesus macaque (Macaca mulatta), 690, 699, 706 Monoamine oxidase (MAO), 271 Monocyte activation test (MAT), 652, 669 Monodelphis, 918 M. domestica, 918 Monofluorophosphate (MFP), 154e155 Monomeri flavanol, 246 Monosodium glutamate (MSG), 37, 65e66, 112 Monterey cypress (Cupressus macrocarpa), 532e533 Moorea spp., 314 M. producens, 361 M. wollei, 315 Moschus sp. (Asiatic musk deer), 203 Mountain wood tick (Dermacentor sp.), 590e591 Mouse lymphoma assay (MLA), 234 Mucor spp., 116e117 Multi-walled carbon nanotubes (MWCNTs), 803, 810e811 MultiOmics pathway resolution, 789 Muscarinic ACh receptors (mAChRs), 592e593 Muscarinic toxins (MTs), 592e593

942

INDEX

Mushrooms, 67 Mussel, green-lipped (Perna canaliculus), 204 Mussels (Mytilus spp.) M. edulis, 335 M. galloprovincialis, 338 Mycobacterium tuberculosis, 916 Mycotoxins, 45, 167e168, 394e397, 736 aflatoxins, 401e411 emerging mycotoxins, 465e476 Alternaria toxins, 466e468 Aspergillus and Penicillium toxins, 468e471 ergot alkaloids, 455e465 fumonisins, 440e455 mycotoxin-related liver diseases and syndromes, 497e498 ochratoxins, 411e417 partial listing of, 395t patulin, 417e419 target organ toxicity, 398te400t trichothecene mycotoxins, 419e433 zearalenone, 433e440 Myeloid differentiation factor 88 (MyD88), 582e583, 655e656 Myeloid differentiation factor-2 (MD-2), 655e656 Myeloid leukemia (ML), 871e872 Mylabris spp., 203 Myotoxic plants, 514e521. See also Teratogenic plants cardioactive glycoside-containing plants, 514e515 Cassia or Senna spp., 518 Erythroxylum coca, 518e519 rayless goldenrod and white snakeroot, 515e517 seleniferous plants, 519e521 Thermopsis spp., 517 Myotoxins, 649

N N-(methylsuccinimido) anthranoyllycoctonine (MSAL), 509e510 N-acetylglucosamine (NAG), 653, 700 N-acetylglucosamine-1-phosphate transferase (GPT), 508e509 N-methyl-D-aspartate (NMDA), 321 N-methyl-D-aspartate receptor (NMDAR), 824e825 N-nitroso compounds (NOCs), 37 N-oxides, 490e491 Na+ (sodium) channels, 591 Nabilone, 192 Nagasaki, 840 Nano-enabled products, 801 Nanomaterials (NMs), 50e51, 798 Nanomedicine, 797e798, 801 Nanoparticles, 798 Nanoparticulates (NP), 798 centrosomal interactions, 812e813 current and future nanotechnology applications, 801e803 development of nanotechnology, 800e801 enhanced toxicity of, 804e805

enhanced toxicity of nanoscale particulates, 804e805 experimental toxicologic pathology of, 804e828 cytopathology, 809e818 target organ and tissue toxicity, 818e827 visualizing NPs in tissue, 805e809 future trends in nanopathology and nanotoxicology, 828e829 historical perspective, 798e800 human exposures, 803e804 human relevance of experimental studies in animals, 827e828 nanopathology, future trends in, 828e829 neurotoxicity, 820e825 quantum chemistry, 805 size, 805 solubility, 804 surface area, 804 target organ and tissue toxicity, 818e827 cardiovascular pathology, 825e826 lymphatic pathology, 826e827 neurotoxicity/neuropathology of NPs, 820e825 pulmonary pathology, 819e820 Nanoplastics, 20e22 Nanotechnology, 50e51, 798 applications, 801e803 development of, 800e801 Nanotoxicology, 828e829 Narthecium ossifragum, 494e495 National Academies of Sciences (NAS), 112 National Aeronautics and Space Administration (NASA), 79 National Agency of Sanitary Surveillance (ANVISA), 86 National Ambient Air Quality Standard (NAAQS), 14 National Cancer Act (1971), 5 National Cancer Institute (NCI), 3e5, 23 bioassay program, 6 National Food Authority (NFA), 86 National Health Commission (NHC), 85 National Institute for Occupational Safety and Health (NIOSH), 3e4 National Institute of Environmental Health Sciences (NIEHS), 3e4, 6, 437 National Institutes of Health (NIH), 3e5, 771e772 National Nanotechnology Initiative (NNI), 797 National Research Council (NRC), 106 National Toxicology Program (NTP), 3e4, 6, 18e19, 111e112, 192, 437, 449e450 Natural Health Product Directorate (NHPD), 276 Natural Health Products Regulations under Food and Drugs Act, 276 Natural products, 200e201 Natural toxins as food contaminants, 43e46 algal compounds in food, 43e45 bacterial toxins, 45e46 mycotoxins, 45 Natural-ingredient diets, 106

Naturally occurring radioactive materials (NORMs), 841 Naval Medical Research Institute (NMRI), 738 Necrotizing cardiomyopathy, 130 Necrotizing fasciitis (“flesh-eating bacterial disease”), 658 Necrotoxins, 571, 602e603 Nematocysts, 557 Nematode (Caenorhabditis elegans), 369 Neonicotinoids, 746 Neosaxitoxin, 314e317, 314f Neotyphodium spp. N. coenophialum, 506e508 N. lolii, 506e508 Nephropathy, mycotoxin, 414 Nephrotoxic plants, 524e531. See also Toxic plants Amaranthus spp., 529e530 calcinogenic glycoside-containing plants, 530e531 lily and grapes, 526e527 oaks, 525e526 oxalate-containing plants, 527e529 Nephrotoxicity, herbal remedies, 218 Nerve conduction velocity (NCV), 594 Nerve growth factor (NGF), 569 Netpen liver disease in salmon, 313t, 345e355 Neural tube defects (NTDs), 445e446 Neuromuscular junction (NMJ), 584 and peripheral synapses, 603e604 Neuromuscular syndrome, 707e708 Neuronal nitric oxide synthase (nNOS), 824e825 Neurotoxic esterase (NTE), 743 Neurotoxic plants, 500e514. See also Teratogenic plants Centaurea spp., 510e511 death camas, 513e514 hemlocks, 512e513 larkspur, 509e510 lupines, 513 nitro-toxins, 511e512 plant-induced storage diseases, 500e506 ryegrass toxicity, 506e509 Neurotoxic shellfish poisoning (NSP), 44, 326 Neurotoxic zootoxins, 584, 585te588t, 593 Neurotoxicity, of herbal remedies, 220 of nanoparticles, 820e825 Neurotoxins, 571e573, 584, 649e651 formic acid, 572f mellitin, 572f a-neurotoxins, 594 Neurotransmission, 60e69 cannabinoids, 67e69 domoic acid, 66 excitatory amino acids, 60 excitotoxicity of glutamate, 60 glutamate, GABA, and glutamate receptors, 60e64 monosodium glutamate, 65e66 serotonin, 67

943

INDEX

Neurotransmitter availability, 589 release, 591 Neutrophil extracellular traps (NETs), 641, 657 New Zealand Food Safety Authority (NZFSA), 86, 89 NF-E2-related factor-2 (Nrf2), 241 Niacin, 135e136 deficiency, 135e136 excess, 136 Nicotinamide adenine dinucleotide (NAD), 135 Nicotinamide adenine dinucleotide phosphate (NADPH), 135, 731 Nicotinic acetylcholine (nACh), 509e510 Nicotinic acetylcholine receptors (nAChRs), 521e523, 592 Nicotinic acid, 135e136 Nierembergia veitchii, 530e531 Nitrates, 37 nitrate-accumulating plants, 535e536, 536t Nitric oxide (NO), 114e115, 247 Nitriles, 47 Nitrites, 37 3-nitro-4-hydroxyphenylarsonic acid, 686e687 4-nitro-phenylarsonic acid, 686 Nitro-toxins, 511e512 Nitropropionic acid (NPA), 511e512 Nitrosamines, 6e7 Nixtamalization process, 453 NLRP3 (Nod-like receptor family pyrin domain containing 3), 582e583, 644e646 No Observable Adverse Effect Level (NOAEL), 112e113, 166, 242, 253, 344e345, 350, 425 Nod-like receptor pyrin domain containing 3 (NLRP3), 644e646 Nodularia spumigena, 345e346 Nodularins (NDs), 345e355 clinical signs and pathology, 348e351 H and E-stained section of liver, 350f immunohistochemically stained liver, 351f liver from male mice, 352f cyclic heptapeptide and pentapeptide cyanotoxins, microcystin-LR structures, 346f diagnosis, treatment, and control, 354e355 source/occurrence, 345e347 Microcystis spp., 346f toxicology, 347e348 Nodularin-R (ND-R), 345e346 Nonalcoholic fatty liver disease (NAFLD), 107, 660 Nonalcoholic steatohepatitis (NASH), 107, 351 Nonallergic food hypersensitivity and intolerance, 58 Nonceliac gluten/wheat sensitivity (NCGS), 53, 57 Noncoding RNA (ncRNA), 776 Nonmelanoma skin cancer (NMSC), 916

Nonsteroidal anti-inflammatory drugs (NSAIDs), 608, 910 Nordberg-Kjellstro¨m model, 699e700 Northern fur seals (Callorhinus ursinus), 321 Norway rats (Rattus norvegicus), 754 Nostoc spp., 345e355, 365e368 Novel carbohydrates, 50 Novel emulsifiers, 49 Novel foods, 47e51. See also Foods colors, 49 and examples, 48t GM foods, 48e49 Novel oils, 50 Novel preservatives, 49 Novel proteins, 49e50 Novel sweeteners, 49 Noxiustoxin, 585te588t NTP Interagency Center for Evaluation of Alternative Toxicological Methods (NICEATM), 26e27 Nuclear factor kappa B (NF-kB), 582e583, 655e656 Nuclear medicine, 841 Nuclear weapons testing, 842 Nucleolin, 818 Nucleus tractus solitarus (NTS), 423 Nutrients, 307e308 general references for nutrient requirements, vitamins and minerals, 108te109t overdose, 112 Nutrition Labeling and Education Act (NLEA), 80 NX-2, 420e421

O O-antigen, 653 Oaks (Quercus spp.), 525e526 Obesity, 106e110 Occupational exposures, 842 Occupational Safety and Health Administration (OSHA), 9 Ochratoxins, 411e417 biodistribution, metabolism, and excretion, 412e413 diagnosis, treatment, and prevention, 417 human risk and disease, 416e417 manifestations of toxicity in animals, 414e416 mechanism of action, 413e414 ochratoxin A, B, and C, 411e412, 414 ochratoxin A-hydroquinone, 413 source/occurrence, 411e412 ochratoxin A and phenylalanine, 411f toxicology, 412e414 species susceptibility, 412 Ocular neoplasia, 918e919 Odoribacter spp., 786 Okadaic acid (OA), 44, 333e335. See also Domoic acid (DA) clinical signs and pathology, 334 diagnosis, treatment, and control, 335 human exposure and disease, 334e335 source/occurrence, 333 toxicology, 333e334

Oleander (Nerium oleander), 514t Olestra, 50 Olfactory sensory neurons, 821e822 Oligodendrocyte, 879 Oligodendrocyte type 2 astrocyte (O-2A), 877 Omics, 772 bioinformatics and integrative and functional enrichment, 788e789 technologies to evaluate endocrine disruption, 780e788 exposomics, 786e788 lipidomics and metabolomics, 781e783 microbiome, 783e786 transcriptomics and proteomics, 780e781 Operation Enduring Freedom (OEF), 844 Operation Iraqi Freedom (OIF), 844 Opisthonos, 469 Opossum (Didelphis virginiana), 615 Oral allergy syndrome (OAS), 51 Organic anion transporter proteins (OATs), 412e413 Organic anion transporting polypeptides (OATPs), 347e348, 412e413 Organic arsenic, 683, 686e687. See also Arsenic manifestations of toxicosis, 686e687 sources and exposure, 686 toxicology, 686 Organic chloride pesticides (OCPs), 92 Organic mercury, 712e714. See also Mercury mercury toxicosis, 714f Organisation for Economic Cooperation and Development (OECD), 771, 797 Organochlorines, 744e745 clinical signs and pathology, 745 development and use, 744 human risk, 745 toxicology, 744 Organomercurials, 712e713 Organophosphate-induced delayed neuropathy (OPIDN), 742e744 Organophosphates (OP), 742e744 Osteoarthropathy, 692 Osteomalacia, 127 Osteoporosis, 697e698 Osteoradionecrosis, 893 Ostreopsis, 370e371 Otter. See Southern sea otters Over the counter (OTC), 277 Oxalate-containing plants, 527e529 kidney from sheep poisoned with halogeton, 529f Oxalis spp., 527e528 Oxazolidine-2-thiones, 47 Oxytropis spp., 394, 501

P P-glycoprotein (Pgp), 204 p-ureidobenzenearsonic acid, 686 p53 gene, 856, 876, 892 Palm Island mystery disease, 339 Palytoxicosis, 44 Palytoxins (PLTXs), 44, 370e372, 371f Panicum spp., 494e495

944 Panton Valentine leukocidin (PVL), 649 Paralytic shellfish poisoning (PSP), 44, 314 Paralytic shellfish toxins (PSTs), 314 Paraquat, 730e732 Parathyroid hormone (PTH), 142 Parkinson’s disease, 247, 365, 367 Particular nutritional uses (PARNUTSs), 51 Particulates not otherwise regulated (PNOR), 803 Paspalum spp., 463 Pasteurella multocida, 633 Paterson’s curse (Echium plantagineum), 492 Pathogen-associated molecular patterns (PAMPs), 582e583, 642e643 Pathology quality assurance, 6 Pathology Working Group (PWG), review process, 6 Pattern recognition receptors (PRRs), 655 Patulin, 417e419 chemical structure of, 417f diagnosis, treatment, and control, 419 human exposure and disease, 419 manifestations of toxicity in animals, 418e419 source/occurrence, 417 toxicology, 417e418 biodistribution, metabolism, and excretion, 418 mechanism of action, 418 toxin, 417e418 Pelargonium sidoides, 204 Pellagra, 135 Penicillium sp., 45, 511e512 P. chrysogenum, 184 P. crustosum, 472 P. cyclopium, 468 P. verrucosum, 411 toxins, 468e471 cyclopiazonic acid, 468e470 fusarium toxins, 472e476 sterigmatocystin, 470e471 tremorgenic mycotoxins, 471e472 Per-and polyfluoroalkyl substances (PFASs), 41 Perchlorate, 42 Perennial ryegrass staggers, 506e508 Perfluorinated substances (PFAS), 11e13 Perfluorocarbons (PFCs), 42, 92 Perfringolysin (PFO), 649 Permethrin, 745 Permissible exposure limit (PEL), 803 Peroxisome proliferatoreactivated receptors (PPARs), 774 Persistent chemical pesticides, 15 Persistent organic pollutants (POPs), 35 Pertussis toxin (Ptx), 647e648 Pesticides, 166e167, 744 Petroleum oils, 728 Pharmacodynamics/pharmacokinetics (PK/PD), 189e190 Pharmacy apothecary to pharmacy, 189e190 biologically active secondary metabolites in herbal remedies, 185te187t World Health Organization, 187b

INDEX

nomenclature for select herbal remedies, 188te189t Phenol-soluble modulin (PSM), 642 Phenothrin, 745 Phenylalanine, 116 Phenylarsonics, 686 Phenylpyrazoles, 746 Phomopsis sp., 197e199, 419 Phosphatase types 1 (PP1), 334 Phosphatase types 2A (PP2A), 334 Phosphatidyl ethanolamine Nmethyltransferase (PEMT), 141 Phospholipase A2 (PLA2), 576e577 zootoxic mechanisms of, 577f Phospholipase C, 164e165 Phospholipase D, 578 Phosphonic acids, 732e733 glufosinate, 732e733 Phosphorus, dietary, 143e144 deficiency, 143e144 excess, 144 Photoallergy, 910 Photosensitization diseases, 538e539, 912 Photosensitization, hepatogenous, 538 Photosensitizers, 912 Photosensitizing plants, 536e539 drugs and other toxicants, 538 fagopyrism, 538 furocoumarins, 538 hepatogenous photosensitization, 538 hypericism, 537e538 photosensitization sequelae, 538e539 primary photosensitization plant, 537 skin from horse with secondary photosensitization, 537f Phototoxicity, 912 Phthalates, 42 Phycology, 306 Phycotoxins, 368e373 anatoxins (ANTXs), 355e358 AZAs, 335e339 BTXs, 325e330 CI, 317e319 CTXs, 330e333 cylindrospermopsins, 339e345 DA, 319e325 diagnostic expertise and instrumentation, 310e311 in food, 43 guanitoxin, 358e361 HAB, 306e308 important marine and freshwater toxins, 309e310 b-methylaminoalanine, BMAA, 365e368 microcystins and nodularins, 345e355 okadaic acid and dinophysistoxins, 333e335 PLTXs, 370e372 saxitoxin and neosaxitoxin, 314f STX, 314e317 vacuolar myelinopathy and aetokthonotoxin, 368e370 aetokthonotoxin structure, 369f YTXs, 372e373 Phylloerythrin, 538

Physalis spp., 505e506 Physiological homeostasis, 766 Phytoestrogens, 69e70, 167e168 Pigtailed macaque (Macaca nemestrina), 418 Pigweed, red (Amaranthus retroflexus), 529e530 Pine needles, 532e533 Pinnatoxins, 317e319, 317f Pitohui, hooded (Pitohui dichrous), 553 Pithomyces chartarum, 497 Planktothrix, 314 Plants, 184. See also Poisonous plants containing fungal hepatotoxins, 495e498 alsike clover, 498 cocklebur and other potent hepatotoxic plants, 499e500 Lantana spp., 498e499 lupinosis, 496e497 plant/mycotoxin-related liver diseases and syndromes, 497e498 sporidesmin, 497 sporidesmin intoxication, 497f structures of phomopsins, sporidesmin, and cytochalasins, 496f influencing factors on concentration of APIs in, 193 myotoxic plants, 514e521 nephrotoxic plants, 524e531 neurotoxic plants, 500e514 oxalates, 527e528 plant-associated toxins, 490 plant-induced storage diseases, 500e506 swainsonine or locoweed intoxication, 500e505 saponins, 494e495 teratogenic plants, 521e524 Platy fish (Xiphophorus sp.), 916e917 Plutonium (Pu), 715e716, 849 diagnosis and treatment, 716 manifestations of toxicosis, 716 sources and exposure, 715 toxicology, 715e716 Plutonium-239, 849 Pneumolysin (PLY), 649 Poison Act, 278 Poison dart frogs (Dendrobates spp.), 553 Poison hemlock (Conium maculatum), 512, 521e524 Poisonous plants, 490, 532e542. See also Plants hepatotoxic plants, 490e500 myotoxic plants, 514e521 nephrotoxic plants, 524e531 neurotoxic plants, 500e514 teratogenic plants, 521e524 Poisons, 569 arrow frogs, 553 Pollen-food allergy syndrome, 51 Poly ADP-ribose polymerase activation (PARP activation), 844e845 Poly(L-glutamic acid) polymer (PGA), 614 Polyaromatic hydrocarbons (PAHs), 274 Polybrominated diphenyl ethers (PBDEs), 35

INDEX

Polychlorinated biphenyls (PCBs), 10e11, 35, 768 Polychlorinated dibenzo-p-dioxins (PCDDs), 10e11 Polychlorinated dibenzofurans (PCDFs), 10e11 Polycyclic aromatic hydrocarbons (PAHs), 6e7, 42e43 Polyethyl enimine (PEI), 818 Polyketides, 417e418 Polymeric flavanol, 246 Polyphenols, 245e247 Polysaccharides, 569 Ponderosa pine (Pinus ponderosa), 532e533 Poppy, opium (Papaver somniferum), 189 Porcine nephropathy, 415 Porcine pulmonary edema (PPE), 442, 448 Pore-forming toxins (PFTs), 644 Portimine, 318 Potassium (K+) channels, 591e592 Potassium (K), dietary, 111e112, 146e148 deficiency, 147 excess, 147e148 Potatoes (Solanum tuberosum), 505e506 Poultry, 416, 429 Poultry Products Inspection Act (PPIA), 82e83 PR/SET Domain 6 (Prdm6), 777 Prebiotics, 51 PrecticX, 50 Pregnane X receptor (PXR), 271, 435, 740e741 Preservatives, 37 Presynaptic receptor activity, 592 Primary photosensitization plant, 537 Probiotics, 38, 51 Procoagulant, 580 Progressive retinal degeneration, sheep, 540 Pronghorn antelope (Antilocapra americana), 497e498 Propazine, 733e734 Propylthiouracil (PTU), 736e737 Prorocentrolide, 318 Protective antigen (PA), 636e637, 647e648 Protein C (PROC), 340 Protein kinase A (PKA), 128e129 Protein kinase C (PKC), 235e236 Protein phosphatase 2a (PP2A), 128e129 Protein-calorie malnutrition (PCM), 110 Proteineprotein interactions (PPIs), 789 Proteins, 113e114, 194e196 deficiency, 113 excess, 113e114 protein-bound bismuth, 691 protein-energy malnutrition, 110 Proteomics, 780e781 Prothioconazole, 738e739 Prothrombin time (PT), 596e597, 666e667 Provisional mean (PM), 432 Pseudo-allergy, 552e553 Pseudo-nitzschia australis, 44, 321e322 Pseudomelanosis coli, 235 Pseudomonas exotoxin A (PE), 638 Pseudomonas sp., 194, 197e199 Pseudo-procoagulant, 571

Psilocin, 67 Psilocybin, 67 Pteriatoxins, 318 Puffer fish (Fugu rubripes), 635e636 Pulegone (Mentha sp.), 267e268 Pulmonary edema, 446e448 Pulmonary intravascular macrophages (PIMs), 664 Pure Food and Drug Act, 77, 80, 668e669 Pymetrozine, 748 Pyrethrins, 745e746 clinical signs and pathology, 746 development and use, 745 human risk, 746 pyrethrin 1, 745 toxicology, 745e746 Pyrethroids, 745e746 clinical signs and pathology, 746 development and use, 745 human risk, 746 toxicology, 745e746 Pyrethrum (Chrysanthemum cinerariaefolium), 745 Pyridiniums, 730e732 toxicology, clinical signs, and pathology, 731e732 Pyridoxine, 136e137 Pyridoxine-5’-phosphate (PLP), 136 Pyrogen, 652 Pyrrolizidine alkaloids (PAs), 268e269, 490e494 animal species susceptibility, 493e494 chemical structure and diversity, 490e491 pyrrolizidine alkaloids, 491f clinical signs and pathology, 492e493 human exposure and disease, 494 plant sources, 492 toxicology, 492

Q Quality assurance (QA), 6 Quillaja saponaria, 494e495 Quercetin, 240, 252, 255e256 Quicksilver, 711 Quinol oxidation site of Complex III inhibitor (QoI), 741

R Rabbit pyrogen test (RPT), 652, 669 Radiation hyperthermia, 919 impact of household technologies and nonionizing radiation, 845e846 injury, 841 ionizing radiation, 846e853 leukemogenesis, 870e872 mechanisms of hyperthermia-induced injury, 919e920 mechanisms of ionizing radiation injury, 853e862 mechanisms of ultraviolet radiation injury, 909e910 and physical agents, 840 response to injury induced by hyperthermia, 920e924

945 response to injury induced by ionizing radiation, 862e908 response to injury induced by ultraviolet radiation, 910e919 sources and occurrence, 841e845 hyperthermia, 843e845 ionizing radiation, 841e843 UV radiation, 843 therapy, 842 toxicity, 220, 241, 442, 734, 745 toxicology, 840 ultraviolet radiation, 908e909 Radiation, target organ toxicity bone, 892e895 neoplasms, 894 sarcomas, 894 cardiovascular (CV), 241 coronary arteries, 897e898 heart, 898e899 pericardium, 898 ear, 883 endocrine system, 880e882 abdominal viscera, 882f adrenal gland, 882 pancreas, 882 parathyroid gland, 881e882 thyroid gland, 880e881 eye, 883e885, 913e914 hyperthermia, 922 lens, 883e884 other ocular structures, 885 retina, 884e885 nervous system, 877e880, 922 brain, 874e875 general reaction to ionizing radiation injury, 877e878 peripheral/cranial nerves, 878 spinal cord, 878e879 oocytes, 904 pancreas, 882 parathyroid gland, 881e882 pulmonary fibrosis, 888 pulmonary neoplasia, 888 respiratory system, 885e892 general reaction to ionizing radiation injury, 885e886 lung, 886e889 neoplasia, 889e892 upper respiratory tract, 886 skin, neoplasms, 917e918 urinary system, 899e903, 922e923 kidney, 900e902 ureter, 902 urinary bladder, 902e903 vascular tissue effects of ionizing radiation, 862e865 Radiofrequency electromagnetic fields (RFEMFs), 17 Radiofrequency radiation (RFR), 17e20, 19te20t, 845 Radiomimetic effect, 426, 427t Radium, 849 Radium jaw, 842 Radix sp., 203 R. aconiti, 203

946 Radix sp. (Continued) R. euphorbiae, 203 R. phytolaccae, 203 Radon, 850 Ragwort (Senecio jacobaea), 492 Rainbow trout (Oncorhynchus mykiss), 23e24 Ramazzini Institute (RI), 3e4, 19e20 Randomized controlled trials (RCTs), 192 Rattlepods (Crotalaria sp.), 268e270, 269f reported intraperitoneal LD50 values, 269t Rayless goldenrod (Haplopappus heterophyllus), 515e517 goat poisoned with, 517f Reactive oxygen species (ROS), 236, 240, 242, 413, 446, 728e729, 731, 825 activation of, 730e735 hydroxyphenylpyruvate dioxygenase inhibitors, 734e735 inhibition of cellular metabolism, 735e736 phosphonic acids, 732e733 pyridiniums, 730e732 thiocarbamates, 735e736 triazines, 733e734 ureas and thioureas, 733 Recombinant bovine somatotropin (rBST), 50, 75 Recombinant factor C (rFC), 652, 669 Recommended Daily Allowance (RDA), 112 Red beard sponge (Microciona prolifera), 553 Red clover (Trifolium pretense), 204 Red tides, 306e307, 554 Reference dose (RfD), 45 Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), 771 Relative biological effect (RBE), 854 Residues, 73e75 Resmethrin, 745 Resorcylic acid lactones (RALs), 70 Restricted Use Pesticide (RUP), 730e731 Retinoid X receptors (RXRs), 121 Retinol, 121 Rhizoma sp. R. rhei, 203 R. sparganii, 203 R. zedoariae, 203 R. zingiberis, 203 Rhizopus sp., 116e117 Rhuem sp., 527e528 Riboflavin, 134e135 deficiency, 134e135 excess, 135 Ribosomal protein L6 (RPL6), 340 Ribotoxic stress response, 422 Ricin, 542 Ricinus spp., 541e542 Rickets, 126e127 Riddelliine, 268e270 Risk/safety assessment in food, 70e71 Riskebenefit assessment (RBA), 70 RNA sequencing (RNA-seq), 779e780 Rocky Mountain wood tick (Dermacentor andersoni), 590e591 Rodenticides, 748e758. See also Fungicides; Herbicide; Insecticides alphachloralose, 756 ARs, 749e753

INDEX

bromethalin, 757 cholecalciferol, 753e754 corn cob, 757 inorganic compounds, 754e756 rodenticides and chemical classes, 749t strychnine, 758 Roentgen (R), 847 Roman chamomile (Chamaemelum nobile), 238e239 Rumex sp., 527e528 Ryanodine receptor type 1 (RYR1), 844, 921e922 Ryegrass toxicity, 506e509 annual ryegrass toxicity, 508e509 perennial ryegrass staggers, 506e508

S Sale of Drug Act, 278 Salmonella sp., 197e199, 633, 641e642 S. typhimurium, 80, 266, 413, 446 Saponin-containing plants, 494e495 goat fed switchgrass, 495f Sarafotoxins, 579 Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), 468e469 Sarcobatus sp., 527e528 Saw Palmetto (Serenoa repens), 270e271, 270f Saxitoxin (STX), 44, 69, 314e317 clinical signs and pathology, 315 diagnosis, treatment, and control, 316e317 human exposure and disease, 315e316 source/occurrence, 314e315 toxicology, 315 Schulz’ law, 193 Scombroid poisoning, 57 Scopolia, 505e506 Scorpion mouse (Onychomys torridus), 591 Scorpion neurotoxins, 558e559 Scorpions, 557 Scombrotoxin, 585te588t Seafood monitoring programs, 316 Sea sponge (Halichondria spp.), 333 H. okadai, 333 Seafood poisoning, 44 Secondary bile acids, 59 Secondary hyperparathyroidism, 127 Second generation of anticoagulant rodenticides (SGARs), 750 Seleniferous plants, 519e521 cow with chronic selenosis, 520f sheep acutely poisoned with, 520f Selenium (Se), dietary, 113, 162e163 deficiency, 162e163 excess, 163 Selenium toxicity, 519e521 chronic, 519e521 Semen spp. S. crotonis, 203 S. persicae (Taoren, peach kernel), 203 S. pharbitidis, 203 Senecio sp., 492 S. riddellii, 490 Senna (Senna alexandrina), 271, 271f, 518 Sepsis, 664 Sepsis shock syndrome, 664 Septicemia, 630 Serenoa repens, saw palmetto, 204

Serotonin, 67, 458e459 Service berry (Amelanchier alnifolia), 533e535 Sesbania sp., 494e495 Setaria sp., 527e528 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 126 Shiga toxin (Stx), 641, 647e648, 658 Shigella spp., 194, 633, 641e642 Shellfish poisoning, 44e45 Short chain fatty acids (SCFAs), 59 Short-chain polyunsaturated FAs (SCPUFAs), 118e119 Shrikethrush (Colluricincla spp.), 553 Sick building syndrome, 420, 428, 430 Silicate tetrahedron (SiO4), 10 Silymarin, 266 Simazine, 733e734 Single guide RNA (sgRNA), 779 Single-cell RNA-sequencing (scRNA-seq), 780e781 Single-walled carbon nanotubes (SWCNTs), 799e801, 803 Sinusoidal obstruction syndrome (SOS), 269, 492 Slow loris venom, 549 Small molecule drugs (SMDs), 669 Snake oil, 548f Snake venoms, 600 SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) complex, 591 Sodium, dietary, 146e148 deficiency, 147 excess, 147e148 Sodium-iodide symporter (NIS), 42, 741 Sodium saccharin, 112 Soil contamination, 16e17 Solanum, 505e506 S. dimidiatum, 506 S. kwebense, 506 S. malacoxylon, 530e531 S. torvum, 530e531 S. verbascifolim, 530e531 Soluble fiber, 117e118 Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), 591 Sonoran desert toad (Incilius alvarius), 551e552 Sorbitol dehydrogenase (SDH), 492e493 South American opossum (Monodelphis domestica), 916e917 South American rattlesnake (Crotalus durissus), 593 Southern sea otters (Enhydra lutris), 321 Spanish fly (blister beetle extract), 551 Sphinganine (Sa), 441 Sphinganine:sphingosine ratio (Sa:So ratio), 445 Sphingomyelin, 141 Sphingomyelinase D, 578 Sphingosine (So), 441 Sphingosine-1-phosphate (S1P), 445, 782 Spirea ulmaria, 189 Spiropidion, 748 Spirulina sp. See Arthrospira

INDEX

Sporidesmin, 497 Sprague Dawley rats (SD rats), 443, 845e846 Spring parsley (Cymopterus watsonii), 538, 912 St. John’s Wort (Hypericum perforatum), 271e272, 272f, 537e538, 912 Stabilizers, 38 Stachybotryotoxicosis, 432 Stachybotrys chartarum, 420 Stanleya pinnata, 519e521 Staphylococcal enterotoxin B (SEB), 637 Staphylococcal enterotoxin-like proteins (SEls), 648 Staphylococcal enterotoxins (SEs), 635, 648 Staphylococcus aureus, 633 State Administration for Market Regulation (SAMAR), 85, 89 Sterigmatocystin, 470e471 human exposure and disease, 471 manifestations of toxicity in animals, 470 source/occurrence, 470 toxicology, toxicokinetics, and mechanism of action, 470 chemical structure of, 470f Sterol biosynthesis inhibitors (SBIs), 737 Stevia sp., 49 S. rebaudiana, 204 Stings, 556e557 Stomach cancer, 702 Strategic Programs on Endocrine Disruptors (SPEED), 771 Strobilurins, 741 Strobilurus tenacellus, 741 Strontium, 895 Strychnine, 758 clinical signs, 758 risk to other species, 758 toxicology, 758 Stylocheilus longicauda, 361 Succinate dehydrogenase inhibitor fungicides (SDHI fungicides), 740e741 Sucrose, 117 toxic response in rat, 117 Sulfate (SOe2 4 ), 148 Sulfite (SOe2 3 ), 148 Sulfur, dietary, 148e149 deficiency, 149 excess, 148e149 inorganic, 148 Sulfuric acid, 728 Sunburn cells, 911 Superantigens (SAgs), 642, 665 Superfund legislation, 16e17 Superoxide dismutase (SOD), 435e436, 859 Supplements. See Dietary supplements Surfactants, 646e647 surfactant/emulsion adjuvants, 649 Susceptible population exposure, 57 Swainsona galegifolia, 500 Swainsonine clinical disease, 501 alkaloids of several plant-origin glycosidase inhibitors, 502t deer fed locoweed, 503f, 505f

in human health and medicine, 504e505 calystegines, 505e506 castanospermine, 506 or locoweed intoxication, 500e505 pathology, 503e504 photomicrograph of cerebellum, 503f poisoning damages, 504 reproductive effects, 504 toxin and toxicity, 501 Sweet clover (Melilotus spp.) and mold, 131 Sweet potato (Ipomoea batatas), 505e506 Sweet vernal grass (Anthoxanthum odoratum) and mold, 131 Swimmer’s itch, 313t, 361 Swine, 405 fumonisins manifestations of toxicity in animals, 448e449 lung from case of fumonisin toxicoses, 449f T-2 toxin and diacetoxyscirpenol, 428e429 zearalenone, 438e439 Swordtail fish (Xiphophorus sp.), 916e917 Symbioimine, 318 Symphytum, 492 Systemic lupus erythematous (SLE), 913

T T cell receptors (TCRs), 643 T-2 toxin, 419 chemical warfare considerations for, 433 human risk and disease, 431e432 manifestations of toxicity in animals, 426e429, 427t macrocyclic trichothecenes, 429e430 poultry, 429 ruminants, 429 swine, 428e429 T-2 mycotoxin on pig intestine, 428f Tachypleus amebocyte lysate (TAL), 651, 666 Tagatose, 49 Tall fescue (Lolium arundinaceum), 456 Tannins, 525 Tarantula, Peruvian green velvet (Thrixopelma pruriens), 594 Taurine, 115 Tebuconazole, 738 Technologically enhanced naturally occurring radioactive material (TENORM), 841 Tentoxin, 467 Tenuazonic acid, 467e468 Tenulin, 490 Teratogenic plants, 521e524. See also Myotoxic plants; Neurotoxic plants; Photosensitizing plants lupine, 521e523 poison hemlock, 524 Veratrum californicum, 523e524 Terpenoids, 190 Tetanospasmin, 633 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 10e11 Tetrahydrocannabinol (THC), 50, 192 delta-9 tetrahydrocannabinol (dTHC), 237

947 Tetrahydropterin (BH4), 138 Tetramethrin, 745 Tetrodotoxin (TTX), 69, 309, 635e636 molecular structure of, 635f Thallium (Tl), 716e719 diagnosis and treatment, 719 manifestations of toxicosis, 717e718 sources and exposure, 716e717 toxicology, 717 Thermal enhancement ratio, 919 Thermopsis spp., 517 skeletal muscle, 517f Thermotolerance, 920 Thevetia spp. T. peruviana, 514e515 T. rhombifolia, 517 Thiacloprid, 746 Thiamethoxam, 746 Thiamine, 133e134 Thickeners, 38 Thiocarbamates, 735e736 Thiocyanates, 47, 157 Thioredoxin reductase (TXNRD1), 340e341 Thioureas, 733 human risk, 733 toxicology, clinical signs, and pathology, 733 Thorium, 850 Thorotrast, 850 Threshold for saturation of renal clearance (TSRC), 729 Thyroid-stimulating hormone (TSH), 156, 741, 880e881 Thyroperoxidase (TPO), 741 Ticks, 563te567t, 583e584, 585te588t, 592 Tipton weed, 537e538. See also St John’s wort Tissue transglutaminase 2 (tTG), 54 Titanium dioxide (TiO2), 823e824 Toad licking, 554 Tobacco (Nicotiana tabacum), 189, 272e274, 273f plants, 693 smoke, 273 smoking, 273 a-tocopherol transport protein (a TTP), 130 Tolerable daily intake (TDI), 40, 419, 431e432, 454 Tolerable weekly intakes (TWIs), 40 Toll/interleukin-1 receptor (TIR), 655e656 Toll-like receptor (TLR) TLR4, 655e656 Topomerase II (Topo II), 236 Tox21, 27 Toxemia, 630 Toxic equivalence factor (TEF), 10e11 Toxic equivalent, 10e11 Toxic plants, 532e542. See also Poisonous plants bracken fern, 539e541 cyanogenic plants, 533e535 nitrate-accumulating plants, 535e536 photosensitizing plants, 536e539 pine needles, 532e533 Ricinus spp., 541e542

948 Toxic shock syndrome (TSS), 664 Toxic Substances Control Act (TSCA), 35, 801 Toxigenesis, 630 Toxinology, 550 Toxins, 548e549 Toxoid, 559, 638 Toxungen, 549e550 Trace minerals, 149e166 chromium, 149e150 cobalt, 150e151 copper, 151e153 fluorine, 153e156 iodine, 156e158 iron, 158e160 manganese, 160e161 molybdenum, 161e162 selenium, 162e163 zinc, 164e166 Traditional and complementary medicine (TCM), 187b Traditional knowledge, 190e192 of efficacy, 201 Traditional medicine (TM), 187b, 190 Transforming growth factor-b (TGF-b), 858e859 Transforming growth factor-beactivated kinase 1 (TAK1), 655e656 Transgenic mouse models, 24e25 Transglutaminase 2 (TG2), 53 Transient receptor potential vanilloid 1 (TRPV1), 592 Transitional and complementary medicine (TCM), 190 Transurethral balloon laser hyperthermia (TUBAL-H), 923 Traumatic brain injury (TBI), 253 Tremorgenic mycotoxins, 471e472 human exposure and disease, 472 manifestations of toxicity in animals, 472 source/occurrence, 471 toxicology, toxicokinetics, and mechanism of action, 471e472 structure of penitrem A, 471f Triazines, 733e734 toxicology, clinical signs, and pathology, 734 Triazole-containing azole fungicides, 737e740 Tribulus terrestris, 494e495 Tricarboxylic acid (TCA), 133 Trichoderma sp., 419 Trichodesma sp., 492 Trichothecene mycotoxins, 419e433 diagnosis, treatment, and prevention, 433 chemical warfare considerations for T-2 toxin, 433 general, 433 livestock, 433 human risk and disease, 430e433 deoxynivalenol, 430e431 macrocyclic trichothecenes, 432e433

INDEX

T-2 toxin and diacetoxyscirpenol, 431e432 manifestations of toxicity in animals, 425e430 deoxynivalenol, 425e426 general, 425 T-2 toxin and diacetoxyscirpenol, 426e429 source/occurrence, 419e420 toxicology, 420e425 biodistribution, metabolism, and excretion, 421e422 mechanism of action, 422e423 partial listing of trichothecene toxins and comparative toxicity, 424t toxicity and species susceptibility, 423e425 toxins, 420e421 trichothecenes and mycotoxins, 421f Trichothecium sp., 419 Triglycerides (TGs), 107e110 Trimethylamine-N-oxide (TMAO), 141 Trisetum flavescens, 530e531 Trivalent Cr, 149e150. See also Chromium Tropical Ataxic Neuropathy, 47 Tryptophan, 114, 909 metabolites, 59 Tumor necrosis factor alpha (TNF-a), 445, 577e578, 642, 879e880 Tumor necrosis factor receptoreassociated factor 6 (TRAF6), 655e656 Tupistra chinensis, 209e211 Turkey X disease, 401. See also Aflatoxin Turmeric Oleoresin (Curcuma longa), 274e275, 274f Type 2 diabetes mellitus (T2DDM), 106 Tyrosine, 115, 909

U U.S. Environmental Protection Agency (EPA), 3e4, 26, 82e83, 728e729, 801 U.S. Food and Drug Administration (FDA), 3e4, 18e19, 82, 592 UK Food Standards Agency (FSA), 86e87 Ultraviolet radiation (UV radiation), 307e308, 843, 908e909. See also Ionizing radiation carcinogenesis, 916e919 animal models, 916e917 epidemiologic evidence, 916 mechanisms, 917 ocular neoplasia, 918e919 skin neoplasms, 917e918 mechanisms of ultraviolet radiation injury, 909e910 nature and action of, 908e909 response to injury induced by, 910e919 eye, 913e914 immune system, 914e916, 915t integument, 910e913 ultraviolet radiation carcinogenesis, 916e919 Ultraviolet-B radiation (UVB radiation), 124e125 Uncertainty factor (UF), 740

Undifilum oxytropis, 394 United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), 852 United States Department of Agriculture (USDA), 82 United States Pharmacopoeia (USP), 198te199t Uranium (U), 719e721 diagnosis and treatment, 721 manifestation of toxicosis, 720e721 sources and exposure, 719 toxicology, 719e720 Ureas, 733 human risk, 733 toxicology, clinical signs, and pathology, 733 Uridine diphosphate glucuronyltransferases (UGTs), 435 Urinary system, 899e903, 922e923 kidney, 900e902 ureter, 902 urinary bladder, 902e903

V Vacina do sapo, 553e554 Vacuolar myelinopathy (VM), 368e370 Valerian (Valeriana officinalis), 201 Vascular cell adhesion molecule (VCAM), 906 VCAM-1, 656e657 Vascular endothelial growth factor (VEGF), 501, 579, 877 Vascular leak syndrome (VLS), 671 Vascular tissue effects of ionizing radiation, 862e865 Vasculotoxins, 580, 595 Veno-occlusive disease (VOD), 269, 492 Venomics, 558 Venoms, 557e559, 569 aggression and defense, 558 mixture, 549e550 therapeutic applications, 558e559 zootoxins, 583 Venomous animals, 555, 557 Veratrum californicum, 523e524 monkey-faced lamb disease, 523f Verticimonosporium, 419 Vetch (Vacia sativa), 533e535 Veterinary Drug Directorate (VDD), 88 Vibrio cholera, 633 Viral hepatitis (HBV), 409 Vitamins, 121e124 deficiency, 122e123 A, 122e123 B, 133e134 C, 132e133 D, 125e127 E, 129e130 K, 131 excess, 123e124 A, 123e124 B, 134 C, 133 D, 127e128

INDEX

E, 130 K, 131e132 Vitamin B, 133e140 Vitamin B1. See Thiamine Vitamin B2. See Riboflavin Vitamin B3. See Niacin Vitamin B6. See Pyridoxine Vitamin B7. See Biotin Vitamin B12. See Cobalamin Vitamin C, 106, 119e121, 132e133 deficiency, 132e133 excess, 133 Vitamin D, 106, 124e128. See also Cholecalciferol deficiency, 125e127 excess, 127e128 Vitamin D receptor (VDR), 124e125 Vitamin E, 128e130 deficiency, 129e130 excess, 130 Vitamin K, 130e132 deficiency, 131 excess, 131e132 Vitamin K deficiency bleeding (VKDB), 131 Vitamin K1, 130e131 Vitamin K2, 130e131 Vitellogenin 1 (vtg1), 779 Vomitoxin, 425e426

W Wallerian degeneration, 686e687 Warfarin, 753 Water buffalo (Bubalus bubalis), 461 Water disinfection by-products, 8 Water fleas (Ceriodaphnia spp.), 369 Water hemlock (Cicuta spp.), 512e513 Water pollutants, 15e16 Water-soluble vitamins, 119e121 West Indian manatees (Trichechus manatus), 326 Western diamondback rattlesnake (Crotalus atrox), 609e610 Wheat-associated allergy (WA), 53, 57 White adipose tissue (WAT), 107e110 White clover (Trifolium repens), 533e535 White snakeroot (Ageratina altissima L.), 515e517 Whitetail deer (Odocoileus virginianus texanus), 497e498

Wholesome Poultry Products Act (WPPA), 84 Whole leaf aloe vera. See Aloe vera (Aloe barbadensis) Wild tree tobacco (Nicotiana glauca), 521e523 Wimmera ryegrass (Lolium rigidum), 508e509 Withdrawal periods, 73 Workplace exposure, 9e10 World Health Organization (WHO), 314, 416, 562, 615, 845

X X-ray-associated tumors, 892 Xenobiotics, 630 metabolism, 816 Xeroderma pigmentosum, 916 Xocolatl, 245

Y Yellow Burrweed (Amsinckia spp.), 268e270 Yellow star thistle (Centaurea solstitialis), 511f Yersinia spp., 633 Y. enterocolitica, 641e642 Y. pseudotuberculosis, 641e642 Yessotoxins (YTXs), 372e373 Yttrium, 889

Z Zearalenol, 436 Zearalenone, 70, 396, 433e440 chemical structures of, 435f diagnosis, treatment, and prevention, 440 human risk and disease, 439e440 manifestations of toxicity in animals, 436e439 source/occurrence, 433e434 toxicology, 434e436 biodistribution, metabolism, and excretion, 436 mechanism of action, 434e436 species susceptibility, 436 toxins, 434 Zebrafish (Danio rerio), 23e24, 308e309, 778e779 model, 778e779

949 Zeranol, 70 Ziconotide, 558e559 Zinc (Zn), 112, 164e166, 703 deficiency, 165e166 excess, 166 Zootoxicosis, 552e553 Zootoxins, 548e549, 600e601 adaptive immune responses to zootoxins, 583 classification, 562e595 clinical presentations and pathologic manifestations of, 595e604 diagnosis and treatment of, 604e610 diagnosis, 605e607 field tests, 605e606 history and physical examination, 605 molecular procedures, 606 by function, 569e573 coagulotoxins, 570e571 necrotoxins, 571 neurotoxins, 571e573 relationship between enzymatic and nonenzymatic zootoxins, 570f innate immune responses to zootoxins, 582e583 by mechanism of action, 573e595 cell and tissue destruction, 576e578 selected zootoxin mechanisms of actions, 574te575t shapes, sizes, and mechanisms of zootoxin, 573f pathology procedures, 606e607 poisoning with dermal or mucosal contact, 553e554 ingestion, 551e553 poison-filled glands, 551f inhalation, 554 potency, 549 prophylactic measures, 609e610 regulatory guidance regarding zootoxins, 610e615 practices for developing antivenom products, 614e615 practices for developing zootoxin-based medical products, 611e614 sources and major indications of medicinal zootoxins, 610e611 sources and major indications of, 610e611 structure of short 3FTx, 569f