Biomarkers in Toxicology [2 ed.] 0128146559, 9780128146552

Table of contents :
Cover
Biomarkers in Toxicology
Copyright
Dedication
Contributors
Foreword
Part I: Toxicity Testing Models and Biomarkers
1. Introduction
References
2. Rodent Models for Toxicity Testing and Biomarkers
Introduction
The Rat
Use in Toxicological Research
Characteristics
Strain Differences
Normal Physiological Values
Study Designs
Routes of Test Article Administration
Oral Routes
Dietary Versus Gavage Methods
Dietary Method
Gavage Method
Test Article Preparation
Equipment
Technique
Neonatal Administration
Capsule
Water
Intravenous Route
Lateral Tail Vein
Bolus Injection
Tail Vein Infusions
Jugular Vein
Bolus Injection
Infusion
Saphenous, Lateral Marginal, and Metatarsal Veins
Dorsal Penis Vein
Sublingual Vein
Intraperitoneal Route
Intramuscular Route
Subcutaneous Route
Topical Route
Rectal Route
Intranasal Route
Inhalation Route
Intratracheal Administration
Regimen
End Point Measurement Techniques
Observations and Physical Examinations
Neurobehavioral Examination
Functional Observational Battery
Locomotor Activity
Cardiovascular Parameters
Electrocardiography
Recording Methods
Restraint
Position
Tethered
Leads
Telemetry
ECG Waveform
Heart Rate
Blood Pressure
Indirect Measurement
Tail Cuff Method
Hindpaw Method
Direct Measurement
Blood Collection Techniques
Retro-Orbital Plexus
Tail
Tail Clip
Venipuncture
Arterial Puncture
Cardiac Puncture
Abdominal Aorta and Vena Cava
Jugular Vein
Proximal Saphenous and Metatarsal Vein
Proximal Saphenous Vein
Metatarsal Vein
Sublingual Vein
Decapitation
Cannulation
Jugular Vein
Inferior Vena Cava
Abdominal Aorta
Subcutaneous Ports
Urine Collection
Necropsy
Summary
The Mouse
Use in Toxicological Research
Normal Physiological Values
Species Differences
Strain Differences
Study Designs
Acute Toxicity Studies
Short-Term Toxicity Studies
Chronic Toxicity Studies (26weeks–2years)
Carcinogenicity Studies (18–24months)
Teratology Studies
Segment II Teratology Studies
Genetic Toxicity Studies
Mouse Micronucleus Assay
Heritable Translocation Assay
Microbial Host-Mediated Assay
Special Studies
Mouse Ear Swelling Test
Dermal Carcinogenicity (Skin Painting) Study
Routes of Test Substance Administration
Oral Administration
Gavage
Description of Technique
Dietary Admixtures
Description of Technique
Drinking Water
Description of Technique
Intravenous Injection
Description of Technique
Intraperitoneal Injection
Description of Technique
Intramuscular Injection
Description of Technique
Subcutaneous Injection
Description of Technique
Intradermal Injection
Description of Technique
Topical Administration
Description of Technique
Inhalation
Chamber (Whole Body)
Head/Nose Exposure (Head Only/Nose Only)
End point Data Collection
Clinical Observations and Physical Examinations
Clinical Laboratory Evaluations
Postmortem Procedures
Summary
The Hamster
Species
Syrian Hamster
Chinese Hamster
European Hamster
Armenian Hamster
Turkish Hamster
Rumanian Hamster
Dzungarian Hamster
South African Hamster
Spontaneous Tumors
Animal Identification
Dosing Procedures
Oral Administration
Subcutaneous Administration
Intradermal Administration
Intramuscular Administration
Intraperitoneal Administration
Intravenous Administration
Blood Collection Techniques
Retro-orbital Method
Cardiac Puncture
Tail Clipping Method
Femoral Vein Method
Jugular Vein Method
Saphenous Vein
Urine Collection
Physical Parameters
Neonatal Body Weights
Body Weights and Weight Gains
Dentition
Life Spans
Sexual Maturity
Respiratory Rate and Oxygen Consumption
Blood Pressure
Heart Rate
ECG Patterns
Clinical Laboratory
Glucose
Lipids
Urea Nitrogen
Enzymes
Alkaline Phosphatase
Alanine Aminotransferase
Aspartate Aminotransferase
Creatine Kinase and Lactic Dehydrogenase
Thyroid Hormones
Reproductive Hormones
Adrenal Hormones
Proteins
Hematology Values
Erythrocytes
Leukocytes
Coagulation
Trypanosomes
Blood Gases and pH
Urine Values
Species Peculiarities
Strain-Related Considerations
Typical Study Protocols
Carcinogenicity Toxicity Testing
Inhalation and Intratracheal Studies
Teratology Studies
Toxicology Studies
Chinese Hamster Ovary Cell Chromosome Aberrations
Syrian Hamster Embryo Cell Transformation Assay
Models of Diseases
Cardiomyopathy
Dental Caries
Diabetes Mellitus
Leprosy
Muscular Dystrophy
Osteoarthritis and Degenerative Joint Disease
Pancreatic Cancer
Concluding Remarks and Future Directions
References
3. Göttingen Minipigs as Large Animal Model in Toxicology
Pigs and Minipigs in Translational Research
Legislation and Species Selection
Göttingen Minipigs as an Animal Model
Dosing Routes and Formulations
Age and Sex
Hematology
Blood Biochemistry
Organ Weight, Background Findings, and Comparative Aspects
Safety Pharmacology
Immune System
Concluding Remarks and Future Directions
References
4. Nonhuman Primates in Preclinical Research
Introduction
Clinical Pathology in NHPs
Preanalytical Variation
Hematology
Coagulation and Hemostasis
Clinical Biochemistry
Urinalysis
Background Microscopic Lesions in NHPs
Mononuclear Cell Infiltrates
Cardiovascular
Gastrointestinal and Hepatic
Renal
Skeletal Muscle
Concluding Remarks and Future Directions
References
5. Biomarkers of Toxicity in Zebrafish
Introduction
Zebrafish Background
Ecotoxicological Biomarkers of Toxicity
Examples of Biomarkers of Fish Toxicity
An Example of an Established Biomarker: Vitellogenin
Cautions
Concluding Remarks and Future Directions
Acknowledgments
References
6. Mechanistic Toxicology Biomarkers in Caenorhabditis elegans
Introduction
C. elegans and Biomarkers
Mechanistic Biomarkers
Oxidative Stress Markers
Mitochondrial Markers
Neurodegenerative Diseases and Gene–Environment Interactions
Alzheimer's Disease
Parkinson's Disease
Concerns Unique to C. elegans
Concluding Remarks and Future Directions
Acknowledgments
References
7. Potential of Small Animals in Toxicity Testing: Hope from Small World
Introduction
Toxicity Testing Using Small Animals
Drosophila melanogaster
Drosophila Model in the Prediction of Cellular Toxicity
Prediction of Developmental, Reproductive, and Behavioral Toxicity Using Drosophila melanogaster
Toxicomics Profiling Using Drosophila
Predication of Toxicity Using Eisenia fetida
Prediction of Toxicity Using Daphnia magna
Concluding Remarks and Future Directions
Acknowledgments
References
8. Alternative Animal Toxicity Testing and Biomarkers
INTRODUCTION
USE OF ALTERNATIVES TO ANIMAL TESTING AND BIOMARKERS
REASONS FOR DEVELOPING ALTERNATIVES TO ANIMAL TESTS AND BIOMARKERS
EXAMPLES OF ORGANIZATIONS' RESEARCHING AND FUNDING ALTERNATIVES TO ANIMAL TESTING AND BIOMARKER DEVELOPMENT
ACHIEVING 3RS WITH MINIMAL TO NO ANIMAL TESTING BY USING BIOMARKERS
In Silico Computer Simulation
In Vitro Cell Culture Techniques
Limitations of Alternative In Vitro Tests
APPLICATIONS OF IN VITRO TESTS AND BIOMARKERS
Genetic Toxicology Testing, In Vitro
Bacterial Mutation Assay (Ames Assay)
In Vitro Micronucleus and Chromosome Aberration Assays in Cultured Cells (Cytogenetic Assays)
Comet or Single-Cell Gel Electrophoresis Assay
Integrated Genotoxicity Testing Using Fewer Animals and Collecting Most Information
Unique Zebrafish Model
In Vitro Pharmacologic Activity Testing
Isolated Tissues
Receptor Binding Studies
In Vitro Drug Disposition Studies
Major Challenges of In Vitro Absorption, Distribution, Metabolism, and Elimination
Advances in Technologies
Systemic Toxicity Testing in Product Development
CONCLUDING REMARKS AND FUTURE DIRECTIONS
References
9. Adverse Outcome Pathways and Their Role in Revealing Biomarkers
Introduction
Adverse Outcome Pathways: Principles of Development and Assessment
Adverse Outcome Pathway Framework: Similarities and Differences With Biomarkers
Adverse Outcome Pathways as a Tool to Retrieve Biomarkers
Biomarkers Depicted from the Adverse Outcome Pathway–Knowledge Base
Regulatory Use of Adverse Outcome Pathways
Concluding Remarks and Future Directions
References
Part II: Systems Toxicity Biomarkers
10. Central Nervous System Toxicity Biomarkers
Introduction
Measuring Central Nervous System Dysfunctions
Biomarkers in Neurotoxicology
Biomarkers of Exposure
Biomarkers of Susceptibility
Biomarkers of Effects
Case Study 1: The Organophosphorus Insecticides
Toxicity and Neurotoxicity of Organophosphorus
Biomarkers of Exposure
Biomarkers of Susceptibility
Biomarkers of Effect
Case Study 2: Mild Traumatic Brain Injury
Concluding Remarks and Future Directions
Acknowledgments
References
11. Peripheral Nervous System Toxicity Biomarkers
Introduction
Types of Biomarkers of the Peripheral Nervous System
Peripheral Nervous System Biomarker Methodology
Neurological and Physical Examination
Protocol
Sample Requirements for Biomarker Testing
Biomedical Imaging and Informatics
Electrodiagnostic Tests
Blood Tests
Testing for Toxins
Molecular Diagnostics
Molecular, Cellular, and Histological Techniques
Toxicogenomics and Proteomics Approaches
Metabonomics
Biomarkers of Traumatic Brain Injury Affecting Peripheral Nervous System
Biomarkers of Nerve Tissue Injury in the Peripheral Nervous System
Biomarkers of Changes in Electrophysiology
Ion Channels as Biomarkers
Biomarkers of Axonal Injury
Biomarkers of Altered Axonal Transport
Biomarkers of Axonal Regeneration
Potential Biomarkers of Pain
Potential Biomarkers of Psychiatric Diseases, Neurodegenerative Diseases, and Insomnia
Potential Biomarkers of Motor Function, Emotional Behavior, Temperature Regulation, Sensory Perception, Locomotion, and Psy ...
Potential Biomarkers of Sleeping Disorders (Apnea, Insomnia, and Desynchronosis)
Biomarkers of Peripheral Nervous System Infection
MicroRNAs as Potential Biomarkers of Peripheral Nervous System Injury
Chemical-Induced Peripheral Neuropathy
Pharmacogenomic Biomarkers for Preventing Chemical-Induced Peripheral Neuropathy
Epigenetic Biomarkers of Axonal Regeneration
Biomarkers From Studies on Inherited Peripheral Neuropathies
Multiple System Atrophy–Related Biomarkers
Biomarkers of Demyelination Injuries
Biomarkers of Neuromuscular Junction Injuries
Biomarkers From Surrogate Tissue Analysis for Peripheral Nervous System Disorders
Biomarkers of Cholinergic System and Its Relevance to the Peripheral Nervous System
Peripheral Nervous System Biomarkers for Exposure to Organophosphates
Biomarkers of Organophosphorus-Ester Induced Delayed Neurotoxicity
Autonomic Nervous System
Autoimmune Autonomic Disorders
Autoantibodies as Biomarkers for Peripheral Nervous System Disorders
Nonimmune Nodopathies
Role of Peripheral Nervous System in Parkinson's Disease and Lewy Body Disease
Neural Stress System of Peripheral Nervous System
Biomarkers of Enteric Nervous System
Emerging Avenues of Peripheral Nervous System Biomarker Developments: Exosomes
Personalized Medicine
Concluding Remarks and Future Directions
References
12. Cardiovascular Toxicity Biomarkers
Introduction
Physiology of the Cardiovascular System
Basic Functions
Cardiac Cycle
Nervous System
Immune System
Neurohormonal System
Vascular System
Xenobiotics
Epigenetic Modifications
Mitochondrial Dysfunction
Proteomics
Cardiac Toxicity
Biomarker of Myocardial Ionic Dysfunction
Electrocardiogram
Metabolomics
Biomarkers of Wall Stretch
Natriuretic Peptide
Choline
Fatty Acid–Binding Protein
Biomarkers of Necrosis
Troponin
Creatine Kinase
Myoglobin
Biomarkers of Inflammation
C-Reactive Protein
Galectin-3
Myeloperoxidase
Tumor Necrosis Factor
Remodeling
ST2 and sST2
sST2 Soluble
Matrix Metalloproteinase-9
Growth Differentiation Factor 15
Neurohormonal Action
Fibrinogen
Copeptin
Vascular Biomarkers
Homocysteine
Lp-PLA2
Placental Growth Factor
Pregnancy-Associated Plasma Protein-A
Other Markers
Heart Rate Variability
Rho Kinase
Plasma Ceramides
Markers of Drug-Induced Toxicity
Cardiac Biomarkers of Illegal Drugs, Chemicals, and Terror Agents
Anthrax
Pharmaceuticals
Illegal Drugs
Environmental Chemicals
Weaponized Chemicals
Future Trends in Cardiovascular Biomarkers
MicroRNAs as Cardiac Biomarkers
Risk Prediction of Cardiovascular Events
Concluding Remarks and Future Directions
References
13. Respiratory Toxicity Biomarkers
Introduction
The Respiratory System
Causes of Lung Injury and Disease
Types of Lung Injuries and Pathologies
Acute Lung Injuries
Asthma
Bronchitis, Emphysema, and Chronic Obstructive Pulmonary Disease
Pulmonary Fibrosis and Granuloma
Pulmonary Hypertension
Pulmonary Phospholipidosis
Sampling Methods for Analysis of Biomarkers of Lung Toxicity
Bronchoalveolar Lavage
Sampling Sputum, Nasal Lavage, and Exhaled Breath
Blood and Urine Sampling
Types of Lung Toxicity Biomarkers
Exposure Biomarkers
Biochemical Biomarkers
Biomarkers in Exhaled Breath Condensate
Biomarkers in Bronchoalveolar Lavage Fluid, Nasal Lavage Fluid, and Sputum
Total Protein and Albumin
Activities of Lactate Dehydrogenase, γ-Glutamyltransferase, N-Acetylglucosaminidase, and Proteases/Antiproteinases
Oxidation By-Products and Antioxidants
Cytokines, Chemokines, and Growth Factors
Bronchoalveolar Lavage Fluid Cells as a Determinant of Lung Inflammation
Circulating Biomarkers of Lung Injury and Pathology
Surfactant, Other Lung-Specific Proteins, and Immunological Biomarkers
Collagen and Elastin Fragments
Cytokines, Metalloproteases, and Acute-Phase Proteins
Thrombosis Biomarkers
Circulating Progenitor Cells
Microparticles
miRNA
Biomarker Identification Using Proteomic and Metabolomic Profiling
Biomarker Identification Using Genomic Profiling of Nucleated Blood Cells
Urine Biomarkers of Lung Injury and Disease
Concluding Remarks and Future Directions
Acknowledgments
Disclaimer
References
14. Hepatic Toxicity Biomarkers
Introduction
Overview of Liver Physiology, Toxicity, and Pathology
Bioactivation and Detoxification
Protein Synthesis and Catabolism
Bilirubin Processing
Covalent Binding
Necrosis and Apoptosis
Inflammation (Immune) Hepatitis
Steatosis
Review of Existing Biomarkers of Liver Toxicity
Alanine Aminotransferase
Aspartate Aminotransferase
Alkaline Phosphatase
Total Bilirubin
Other Existing Hepatotoxicity Biomarkers
Review of Emerging Biomarkers
Genetic Biomarkers of Hepatotoxicity
Genomics Biomarkers of Hepatotoxicity
Proteomic Biomarkers of Hepatotoxicity
Metabolomics Biomarkers in Hepatotoxicity
MicroRNAs as Biomarkers of Hepatotoxicity
Biomarker Qualification and Validation
Concluding Remarks and Future Directions
References
15. Conventional and Emerging Renal Biomarkers
Introduction
Characteristics of Biomarkers
Traditional Biomarkers
Glomerular Filtration Rate
Proteinuria
Enzymuria
Newer Biomarkers
Cystatin C
Neutrophil Gelatinase–Associated Lipocalin
Kidney Injury Molecule-1
Interleukin 18
Liver-type Fatty Acid Binding Protein
Genomics, Proteomics, Metabolomics
Concluding Remarks and Future Directions
References
16. Gastrointestinal Toxicity Biomarkers
Introduction
NSAID-Induced Gastrointestinal Toxicity
Chemotherapy-induced Gastrointestinal Toxicity
Radiation-induced Gastrointestinal Toxicity
Inflammatory Bowel Diseases
Biomarkers for Gastrointestinal Damage
Blood Biomarkers
Citrulline
Diamine Oxidase
CD64
Gastrins
C-reactive Protein
Orally Administered Probes
Matrix Metalloproteinases
Pepsinogen I and II
Fecal Biomarkers
Calprotectin
Lactoferrin
Polymorphonuclear Neutrophil Elastase
Bile Acids
I-FABP and L-FABP
MicroRNA
Fecal S100A12
Other Novel Approaches
Concluding Remarks and Future Directions
Metabolome-based Biomarkers
References
17. Reproductive Toxicity Biomarkers
Introduction
Male Reproductive Biomarkers
Spermatogenesis
Synthesis of Steroids, Gonadotropins, and Peptide Hormones in Males
Male Puberty
Male Reproductive Aging
Female Reproductive Biomarkers
Folliculogenesis
Synthesis of Steroids, Gonadotropins, and Peptide Hormones in Females
Female Puberty
Uterus
Placenta
Female Reproductive Aging
Concluding Remarks and Future Directions
References
18. Biomarkers of Toxicity in Human Placenta
Introduction
Placental Development and Structure
Toxic and Hormonally Active Chemicals in Human Placenta
Placental Functions and Molecular Pathways Involved in Toxicity
Xenobiotic Metabolism and Its Regulation in Human Placenta
Placental Transporters
Placental Hormone Production
Molecular Stress Protein Pathways Involved in Toxicity of Human Placenta
Possibilities for Studying Pathways and Proteins Involved in Toxicity of Human Placenta
Potential Biomarkers of Exposure and Toxicity in Human Placenta
Toxic Compounds and Their Metabolites as Biomarkers
Placental DNA Adducts as Biomarkers
Xenobiotic-Metabolizing Enzymes, Placental Hormones, and Stress-Related Proteins as Biomarkers of Exposure and/or Toxicity ...
Epigenetic Changes in Human Placenta as Potential Biomarkers of Toxicity
Use of Placenta in Regulatory Toxicology—Considerations by Ecvam and Other Organizations
OECD Guidelines
Role of European Center for the Validation of Alternative Methods
Experimental Methods Concerning Placenta
Concluding Remarks and Future Directions
References
19. Early Biomarkers of Acute and Chronic Pancreatitis
Introduction
Anatomical, Physiological, and Metabolic Considerations for Pancreatic Injury
Acute Pancreatitis
Chronic Pancreatitis
Biomolecular Basis of Pancreatitis
Biomarkers of Acute and Chronic Pancreatitis
Abdominal Imaging Techniques
Functional Biomarkers of Endocrine and Exocrine Pancreatitis
Amylase
Isoamylase
Lipase
Carbohydrate-Deficient Transferrin
Activation By-Products of Pancreatic Trypsinogen and Carboxypeptidase B
Elastase and Phospholipase A2
Procalcitonin
C-Reactive Protein and Pancreatitis-Associated Protein
Cathepsins
Peptides
Inflammatory Cytokines and Chemokines
Biomarkers of Endocrine and Autoimmune Pancreatitis
Ethanol Metabolites and Conjugates
Differentially Altered Proteins, Metabolites, Small Molecules, and microRNA
Comparison of Biomarkers Between Acute and Chronic Pancreatitis
Biomarkers for Early Detection of Pancreatic Cancer
Breathe Analysis
Concluding Remarks and Future Directions
References
20. Skeletal Muscle Toxicity Biomarkers
Introduction
Muscle Types
Pesticides
Cholinesterase Inhibitors
Behavioral Effects
Cholinergic Effects
Noncholinergic Effects
Excitotoxicity and Oxidative/Nitrosative Stress
Creatine Kinase and Creatine Kinase Isoenzymes
Lactate Dehydrogenase and Lactate Dehydrogenase Isoenzymes
Histopathological Alterations
Strychnine
Paraquat
Metals
Therapeutic Drugs
Lipid-Lowering Drugs
Drugs Used Against AIDS
Antiinflammatory Drugs
Antitumor and Anticancer Drugs
Drugs of Abuse
Venoms and Zootoxins
Snake Toxins
Spider Venom
Botulinum Toxin
Myotoxic Plants
Concluding Remarks and Future Directions
References
21. Ocular Biomarkers in Diseases and Toxicities
Introduction
Ocular Biomarkers
Molecular Biomarkers in Ocular Surface Disease
Molecular Biomarkers in Keratoconus
Molecular Biomarkers in Glaucoma
Molecular Biomarkers in Retinal Disease
Molecular Biomarkers in Age-Related Macular Degeneration
Molecular Biomarkers in Ocular Oncology
Systemic Agents and Ocular Toxicity
Concluding Remarks and Future Directions
References
22. Biomarkers of Ototoxicity
Introduction
Structure and Function of the Normal Auditory System
Anatomic Structure of the Human Auditory System
Hearing Mechanisms
Overview on Ototoxicity
Classification of Chemicals Causing Ototoxicity
Pharmaceuticals
Antibiotics
Aminoglycosides
Macrolides
Salicylates and Nonsteroidal Antiinflammatory Drugs
Antineoplasic Drugs
Cisplatin
Vincristine
Loop Diuretics
Antimalarial Drugs
Phosphodiesterase Type 5 Inhibitors
Antiepileptic Drugs
Industrial Chemicals
Factors Modifying Chemical Ototoxicity
Underlying Mechanisms of Ototoxicity
Functional and Molecular Biomarkers for the Evaluation of Ototoxicity
Functional Assessment of the Hearing Damage
Pure-Tone Audiometric Testing
Distortion Product Otoacoustic Emission
Acoustic Immitance Test
Acoustic Brain Stem Response Testing
Molecular Biomarkers
Alternative Models
Concluding Remarks and Future Directions
References
23. Blood and Bone Marrow Toxicity Biomarkers
Introduction
Hematopoietic System
Bone Marrow
Hematopoiesis
Hematopoietic Cells
Erythrocytes
Platelets
Monocytes
Neutrophils
Eosinophils
Basophils
Lymphocytes
Soluble Mediators
Mechanisms of Hematotoxicity
Biomarkers of Hematotoxicity
Markers of Hematopoietic/Hematologic Toxicity
Complete Blood Count
Markers of Erythrocyte Toxicity
Markers of Platelet Toxicity
Markers of Bone Marrow Toxicity
Bone Marrow Evaluation
Progenitor Cell Colony Formation
Markers of Hemostatic Toxicity
Concluding Remarks and Future Directions
References
24. Immunotoxicity Biomarkers
Introduction
The Immune System
Lymphoid Organs and Tissues
Cells of the Immune System
B Cell Lymphocytes
T Cell Lymphocytes
Neutrophils
Macrophages
Natural Killer Cells
Mast Cells
Soluble Mediators of Immunity
Cytokines
Chemokines
Other Soluble Mediators
Mechanisms of Immunotoxicity
Biomarkers
Biomarkers of Immune Status
Cell Counts
Organ Weight
Histopathology
Antibody Assays
T-dependent Antibody Response
Delayed-type Hypersensitivity Response
Lymphocyte Phenotyping
Natural Killer Cell Activity
Phagocytosis and Chemotaxis
Proliferation Tests
Cytokine Profiling
Other Soluble Mediators
Biomarkers of Hypersensitivity
Local Lymph Node Assay
Human Repeat-Insult Patch Test
Immunoglobulin E Levels
Biomarkers of Autoimmunity
Concluding Remarks and Future Directions
References
Part III: Chemical Agents, Solvents and Gases Toxicity Biomarkers
25. Bisphenol A Biomarkers and Biomonitoring
INTRODUCTION
BISPHENOL A BIOMARKER ISSUES IN BIOMONITORING
EXPOSURE AND SELECTED OUTCOME STUDIES
LARGE GENERAL POPULATION BIOMONITORING PROGRAMS
National Health and Nutrition Examination Survey, United States
Canadian Health Measures Survey
Generation R Study, The Netherlands
Norwegian Mother and Child Cohort Study, Norway
Other General Population Exposure Studies in Various Countries
PREGNANT WOMEN, MOTHER–INFANT/CHILD PAIRS, INFANTS, CHILDREN
Pregnant Women
Mother–Infant/Child Pairs
Children
FOOD PACKAGING AND BISPHENOL A ALTERNATIVES
CONCLUDING REMARKS AND FUTURE DIRECTIONS
References
26. Insecticides
Introduction
Organophosphates and Carbamates
Mechanism of Toxicity
Biomarkers
Acute Toxicity
Intermediate Syndrome
Delayed Toxicity
Chronic Toxicity
Chlorinated Hydrocarbons
Mechanism of Toxicity
Biomarkers
Pyrethrins and Pyrethroids
Mechanism of Toxicity
Biomarkers
Amitraz
Mechanism of Toxicity
Biomarkers
Neonicotinoids
Mechanism of Toxicity
Biomarkers
Fipronil
Mechanism of Toxicity
Biomarkers
Ivermectin and Selamectin
Mechanism of Toxicity
Ivermectin
Selamectin
Biomarkers
Rotenone
Mechanism of Toxicity
Biomarkers
Concluding Remarks and Future Directions
References
27. Herbicides and Fungicides
INTRODUCTION
BACKGROUND
TOXICOKINETICS
MECHANISM OF ACTION
BIOMARKERS AND BIOMONITORING OF EXPOSURE
Herbicides
Phenoxy Acid Derivatives
Triazines and Triazoles
Phenylurea Herbicides
Protoporphyrinogen Oxidase–Inhibiting Herbicides
Substituted Anilines
Bipyridyl Derivatives
Amides and Acetamides
Imidazolinones
Triazolopyrimidine
Phosphonomethyl Amino Acids or Inhibitors of Aromatic Acid Biosynthesis
Others
Fungicides
Dialkyldithiocarbamates
Anilinopyrimidines
Chloroalkylthiodicarboximides (Phthalimides)
Halogenated Substituted Monocyclic Aromatics
CONCLUDING REMARKS AND FUTURE DIRECTIONS
References
28. Polychlorinated Biphenyls, Polybrominated Biphenyls, and Brominated Flame Retardants
Introduction
Polychlorinated Biphenyls
Physicochemical Properties and Environmental Levels
Human Health Effects and Modes of Action
Polybrominated Biphenyls
Physicochemical Properties
Human Health Effects and Modes of Action
Brominated Flame Retardants
Physicochemical Properties
Human Health Effects and Modes of Action
Thyroid Hormone Disruption as a Biomarker of Exposure and Effect
Perturbed Calcium Homeostasis and Kinase Signaling as Biomarkers of Effect
Induction of Cytochrome P450 Enzymes as a Biomarker of Exposure and Effect
Concluding Remarks and Future Directions
Acknowledgments
References
29. Polycyclic Aromatic Hydrocarbons
Introduction
Biomarkers of Exposure
Hydroxylated Metabolites
DNA Adducts
Protein Adducts
Other Biomarkers of Exposure
Biomarkers of Effect
Genetic Alterations
Biomarkers of Susceptibility
Concluding Remarks and Future Directions
Acknowledgments
References
30. Metals
Introduction
Classification of Biomarkers
Selection of an Ideal Biomarker: Benefits and Drawbacks
Biomonitoring of Exposure to Heavy Metals
Lead
Biomarkers of Exposure
Biomarkers of Effects
Biomarkers of Susceptibility
Arsenic
Biomarkers of Exposure
Biomarkers of Effects
Biomarkers of Susceptibility
Mercury
Biomarkers of Exposure
Limitations of Biomarkers of Exposure
Biomarkers of Effects
Biomarkers of Susceptibility
Cadmium
Biomarkers of Exposure
Biomarkers of Effects
Biomarkers of Susceptibility
Chromium
Biomarkers of Exposure
Biomarker of Effects
Biomarkers of Susceptibility
Thallium
Biomarkers of Exposure
Biomarkers of Effects
Biomarkers of Susceptibility
Manganese
Biomarkers of Exposure
Biomarkers of Effects
Biomarkers of Susceptibility
Concurrent Exposure of Heavy Metals
Current Concerns and Biological Relevance
Concluding Remarks and Future Directions
References
31. Melamine
Introduction and Historical Background
Toxicology of Melamine
Toxicity
Pharmacokinetics/Toxicokinetics
Mechanism of Action
Treatment
Biomarkers
Melamine+Cyanuric Acid Biomarkers
Melamine Only Biomarkers
Concluding Remarks and Future Directions
References
32. Biomarkers of Petroleum Products Toxicity
Introduction
Chemical Markers
Polycyclic Aromatic Hydrocarbons
Tissue and Body Fluid Levels of Petroleum Hydrocarbons
Biochemical Biomarkers
CYP Biomarkers
Biomarkers of Responses to Oxidate Stress
Genetic Biomarkers of Petroleum Exposure
Morphologic Biomarkers
Teratology
Biomarkers in Birds
Concluding Remarks and Future Directions
References
33. Biomarkers of Chemical Mixture Toxicity
Introduction
Potential Chemical Mixtures
Pesticides
Organochlorinated Compounds
Halogenated Flame Retardants
Polycyclic Aromatic Hydrocarbons
Metals
Biomonitoring for Assessing Human Exposure to Chemical Mixtures
Biological Specimens
Blood
Urine
New Matrices for Assessing Cumulative Exposure
Milk
Saliva
Hair
Meconium
Biomolecular Adducts
Risk Assessment of Combined Actions of Chemical Mixtures
Biomarkers of Target Organ Toxicity of Chemical Mixtures
Biomarkers of Liver Damage
Kidney
Nervous System
Lungs
Nonspecific Biomarkers of Toxic Response
Oxidative Stress
DNA Damage and Genotoxicity
Concluding Remarks and Future Directions
References
34. Biomarkers of Toxic Solvents and Gases
Introduction
Solvents
Gases
Biomarkers of Toxic Solvent and Gas Exposure
The Use of the Parent Compound as a Biomarker of Exposure
The Use of Metabolites of Toxic Solvents or Gases as Biomarkers of Exposure
The Use of Adducts as Biomarkers of Toxic Solvent and Gas Exposure
The Use of Novel Biomarkers of Toxic Solvent and Gas Exposure
Biomarkers of Effects of Toxic Solvent and Gas Exposure
Interpretation and Use of Biomonitoring Data
Concluding Remarks and Future Directions
References
Part IV: Biotoxins Biomarkers
35. Freshwater Cyanotoxins
Introduction
Hepatotoxins
Microcystins
Introduction
Chemistry
Toxic Effects
Cellular Transport
Mechanism of Action
Biomarkers
Cylindrospermopsin
Introduction
Chemistry
Toxic Effects
Mechanism of Action
Biomarkers
Neurotoxins
Anatoxin-a
Introduction
Chemistry
Toxic Effects
Mechanism of Action
Biomarkers
Anatoxin-a(s)
Introduction
Chemistry
Toxic Effects
Mechanism of Action
Biomarkers
Saxitoxins
Introduction
Chemistry
Toxic Effects
Mechanism of Action
Biomarkers
Concluding Remarks and Future Directions
References
36. Mycotoxins
Introduction
Aflatoxins
Background
Chemistry and Legal Regulation of Aflatoxins
Toxicokinetics of Aflatoxins
Aflatoxin Metabolism and Chemical Biomarkers of Aflatoxin Exposure
Histopathological Biomarkers of Exposure to Aflatoxins
Sterigmatocystin
Ochratoxins
Production and Occurrence
Toxicokinetics and Biotransformation of Ochratoxin A
Chemical Biomarkers of Ochratoxin A
Histopathologic Biomarkers of Exposure to Ochratoxins A
Fumonisins
Chemistry of Fumonisins
Toxicokinetics, Metabolism, and Chemical Biomarkers
Histopathological Biomarkers
Deoxynivalenol Trichothecene
Toxicokinetics, Biotransformation, and Biomarkers
Zearalenone
Background
Toxicokinetics, Biotransformation, and Biomarkers
Concluding Remarks and Future Directions
References
37. Poisonous Plants: Biomarkers for Diagnosis
INTRODUCTION
ASTRAGALUS AND OXYTROPIS SPECIES (LOCOWEEDS, NITROTOXIN SPP., AND SELENIUM ACCUMULATORS)
Locoweeds
Toxicology
Toxin
Conditions of Grazing
Biomarkers of Poisoning (AST, GGTP, White Cell Count, Cellular Vacuolation, Serum Swainsonine, α-Mannosidase)
Prevention of Poisoning and Management Recommendations
Nitro-Containing Astragalus (Milkvetches)
Toxicology
Biomarkers of Poisoning (Clinical, Pathology, History of Ingestion, Methemoglobin)
Prevention and Treatment
Seleniferous Astragalus
Toxicity
Biomarkers of Poisoning (Liver Biopsy Se, Whole Blood Se, Enzyme, Hair, Mane, Tail, Hoof Samples)
Prevention of Poisoning
LARKSPURS (DELPHINIUM SPP.)
Toxicology
Biomarkers of Poisoning (Blood Alkaloid, Liver Alkaloid, Plant Fragment, or Rumen Sample)
Prevention and Management of Poisoning
Grazing Management
Drug Intervention
LUPINES (LUPINUS SPP.)
Toxicology
Cattle Grazing
Biomarkers of Poisoning (Clinical Effects, History of Ingestion, Serum Analysis, Liver, Urine, Crooked Calf Syndrome (CCS))
Prevention, Management, and Treatment
POISON HEMLOCK (CONIUM MACULATUM)
Toxicology
Biomarkers of Poisoning
Prevention and Treatment
WATER HEMLOCK (CICUTA SPP.)
Toxicology
Biomarkers of Poisoning (Blood Enzymes, CPK, AST, GGTP, Histological Lesions in the Heart, Long Muscles, Clinical Signs, Hi ...
Prevention and Treatment
PONDEROSA PINE NEEDLES (PINUS SPP.)
Toxicology
Biomarkers of Poisoning (Premature Parturition; Live-Premature Calf; Retained Fetal Membrane; Agathic or Tetrahydroagathic ...
Prevention and Treatment
RAYLESS GOLDENROD (ISOCOMA PLURIFLORA)
White Snakeroot (Eupatorium rugosum)
Toxicology
Biomarkers of Poisoning (Blood Enzymes, CPK, Troponin, Histology, Lesions in Heart, Long Muscles, Clinical Signs, History o ...
Prevention and Treatment
HALOGETON (HALOGETON GLOMERATUS)
Toxicology
Treatment of Poisoned Animals
Biomarkers of Poisoning (Blood or Ocular Calcium, Oxalate Analysis of Blood, History of Ingestion
Management to Prevent Poisoning
PYRROLIZIDINE ALKALOID-CONTAINING PLANTS
Toxicology
Biomarkers of Poisoning (Liver Enzymes, AST, LDH, GGTP, Cirrhosis, Histology, Pyrrole Analysis of Liver)
Prevention and Treatment
PHOTOSENSITIZING PLANTS
Toxicology
Primary
Secondary
Biomarkers of Poisoning (Bilirubin, Liver Enzymes, Skin Biopsies, History of Ingestion)
Prevention and Treatment
DEATH CAMAS
Toxicity of Death Camas to Livestock
Biomarkers of Poisoning (Alkaloid Analysis of Serum, Liver, Pathology, History of Ingestion)
Management and Prevention
KNAPWEEDS: CENTAUREA SPP.
Toxicology
Clinical Signs
Biomarkers of Poisoning (History of Ingestion, Pathology Brain Lesions, Horses Only)
Prevention and Treatment
CONCLUDING REMARKS AND FUTURE DIRECTIONS
References
Part V: Pharmaceuticals and Nutraceuticals Biomarkers
38. Biomarkers of Drug Toxicity and Safety Evaluation
Introduction
Clinical Drug Development
Application of Biomarkers in Postmarketing
Integration and Use of Safety Biomarkers in Drug Development
Application of Safety Biomarkers in Different Phases of Drug Development
Traditional Indicator for Drug Toxicity Assessment
Alkaline Phosphatase
Creatine Kinase
Aminotransferases
Lactate Dehydrogenase
Bilirubin
Creatinine and Blood Urea Nitrogen
Plasma Proteins
Total Protein
Gamma-Glutamyltranspeptidase
Hydroxybutyrate Dehydrogenase
Lactate Dehydrogenase
Sodium, Potassium, and Chloride
Urinalysis
Biomarkers of Drug Liver Toxicity
Biomarker for Monitoring Liver Injury
Alanine Aminotransferase and Its Two Isoforms
Glutamate Dehydrogenase
Serum F Protein
Arginase-I
New Biomarkers for Monitoring Liver Injury
Alpha-Glutathione S-transferase
Gamma-Glutamyl Transpeptidase
Sorbitol Dehydrogenase
Purine Nucleotide Phosphorylase
Malate Dehydrogenase
Paraoxonase-1
Biomarkers in Acetaminophen Hepatotoxicity
High Mobility Group Protein B1
Caspase-Cleaved K18
Glutathione S-Transferase Alpha
Malate Dehydrogenase
Cytochrome c
Biomarkers for Drug-Induced Liver Injury in Tuberculosis–Human Immunodeficiency Virus–Infected Patients
Biomarker of Drug-Induced Kidney Toxicity
Urinary β2-Microglobulin
Biomarkers of Drug-Induced Vascular Injury
Biomarkers for Monitoring Drug-Induced Vascular Injury
Biomarkers of Drug-Induced Cardiac Injury
Monoclonal Antibodies
Troponins
B-type Natriuretic Protein
Circulating Micro-RNAs
Biomarkers of Drug-Induced Brain Injury
Biomarkers in Drug Safety Evaluation
Importance of Understanding Specific Mechanisms of Toxicity in Drug Development
Biomarkers in Drug Safety Evaluation
Safety Biomarkers for Preclinical and/or Clinical Perspectives
Kidney
Kidney Injury Molecule-1
β2-Microglobulin
Cystatin C
Clusterin
Trefoil Factor 3
Other Kidney Biomarkers
Lipocalin-2
N-Acetyl-β-d-Glucosaminidase
Interleukin-18
Matrix Metalloproteinase-9
Netrin-1
Monocyte Chemotactic Peptide-1
Liver
Heart
α2-Macroglobulin
Ultrasound
Others Novel Biomarkers
C-Reactive Protein
Galectin-3
Matrix Metalloproteinases
Tissue Inhibitors of Metalloproteinases
Lung
Lipid/Carbohydrate Metabolism
Central Nervous System
Genetic and Genomic Biomarkers
Biomarker Validation and Qualification
Identification of Safety Biomarkers
Preclinical Qualification
Clinical Qualification and Diagnostic Use
Translational Safety Biomarker
Concluding Remarks and Future Directions
References
39. Risk Assessment, Regulation, and the Role of Biomarkers for the Evaluation of Dietary Ingredients Present in Dietary Supple ...
Introduction
Overview of Regulation of Dietary Supplements in the United States
Federal Food, Drug, and Cosmetic Act
Regulatory Challenges
Dietary Ingredient Safety
The Risk Assessment
Hazard Identification
Hazard Characterization
Exposure Assessment
Risk Characterization
Biomarkers of Toxicity: Dietary Ingredients
Biomarkers Associated with the Presence of Banned Ingredients
Banned Dietary Ingredients Causing Neurostimulation—Ephedrine Alkaloids
Banned Dietary Ingredients Causing Neurosuppression—Mitragyna speciosa (Kratom)
Biomarkers Associated with the Presence of Endocrine Disruptors
Dessicated Thyroid Glandulars
Biomarkers Associated With the Presence of Carcinogens
Aristolochic Acid
Pyrrolizidine Alkaloids—Symphytum Spp. (Common Name, Comfrey)
Biomarkers of Toxicity: New Dietary Ingredients
Premarket Notification of a New Dietary Ingredient
Premarket Notification for New Dietary Supplement as Enhancers of Metabolism, Energy, and Weight Loss
Usnic Acid
Biomarkers of Toxicity: New Dietary Ingredients
Active Moiety of a New Dietary Ingredient
Red Yeast Rice—Monacolin K and Statin Activity
Biomarkers of Toxicity: New Dietary Ingredients
Safety Assessment of Multiple Dietary Ingredients in a Dietary Supplement
New Dietary Ingredients (Supplementation with Multiple, Combinations of Dietary Ingredients) Rauwolfia and Yohimbe Bark Extract
New Dietary Ingredients (Supplementation with Multiple, Combinations of Dietary Ingredients)—Galantamine and Huperzine A (C ...
Concluding Remarks and Future Directions
References
40. Nutriphenomics in Rodent Models: Impact of Dietary Choices on Toxicological Biomarkers
Introduction
Dietary Choices
Grain-Based Diets
Purified Diets
Diet-Induced Metabolic Disorders
High-Fat Diets for Diet-Induced Obesity Models
Diet-Induced Atherosclerosis/Hypercholesterolemia Models
Mice and Rats
Hamsters
Guinea Pigs
High-Fructose/Sucrose Diets for Hypertriglyceridemia and Insulin Resistance
Nonalcoholic Fatty Liver Disease
Diets High in Sodium (and Fructose) for Hypertension
Metabolic Disease Development and the Importance of Fiber Type
Know Your Control Diet
Potential Effects of Grain-Based Diets and Low-Fat Purified Diets on the Rodent Phenotype
Grain-Based Diets
Phytoestrogens and Development/Maturation
Phytoestrogens and Cancer
Arsenic and Heavy Metals
Other Compounds or Contaminants
Purified Low-Fat Diets and Health Status of Rodents
Kidney Calcinosis
Pubertal Onset
Metabolic Effects
Concluding Remarks and Future Directions
References
Part VI: Nanomaterials and Radiation
41. Engineered Nanomaterials: Biomarkers of Exposure and Effect
Introduction and Background
Classification and Characteristics of Nanomaterials
Carbon-Based Nanoparticles: Fullerenes
Inorganic Nanoparticles
Organic Nanoparticles
Toxicity Testing of Nanomaterials
Definition and Meaning of Biological Monitoring and Its Application to Engineered Nanomaterials
Challenges of the Development of Biomarkers of Exposure to Engineered Nanomaterials Due to Their Biokinetics
Biological Interactions Relevant to Biomarkers of Exposure to Engineered Nanomaterials at Molecular, Cellular, and Organ Level
Examples of Given Biomarkers
Biomarkers of Exposure
Biomarkers of Effect
Biomarkers of Lung Inflammation and Systemic Effects
Biomarkers of Oxidative DNA Damage and RNA Methylation
Toward Specific Biomarkers for Engineered Nanomaterials Exposure
Concluding Remarks and Future Directions
Acknowledgments
References
42. Biomarkers of Exposure and Responses to Ionizing Radiation
INTRODUCTION
KEY DEFINITIONS AND UNITS
RADIATION PROTECTION SYSTEM
SOURCES OF RADIATION EXPOSURE
HISTORICAL PERSPECTIVE
Personal Experience
BIOMONITORING OF EXPOSURE TO RADIATION
Radiation-Attributable Disease
Late-Occurring Health Effects Attributable to Radiation Exposure
PATH FORWARD
Dedication and Acknowledgments
References
Part VII: Carcinogens Biomonitoring and Cancer Biomarkers
43. Biomonitoring Exposures to Carcinogens
Introduction
Assessing Exposures
Chemicals and Metabolites
DNA and Protein Adducts
Assessing Effects
Chromosomal Aberrations and Sister Chromatid Exchanges
Micronuclei
Comet Assay
Specific Gene Mutations
Telomere Shortening
Epigenetic Changes
Assessing Susceptibility
New Scenarios to Assess Exposures and Effects: The Omics Approach
Adductomic
Transcriptomics
Epigenomic
Metabolomic
Concluding Remarks and Future Directions
References
44. Genotoxicity Biomarkers: Molecular Basis of Genetic Variability and Susceptibility
Introduction
Genotoxic Biomarker Detection Methods
In Silico Approaches
In Vitro and In Vivo Biomarkers of Genotoxicity
Gene Mutations in Prokaryotes
Micronucleus Formation
Chromosomal Aberrations
Comet Formation
Toxicogenomic Signatures
Other Genotoxic Biomarker Detection Methods
Biomarkers and Mechanism of Action
Gene Mutations and Their Mechanism of Action
Chromosomal Aberrations and Their Mechanisms of Action
Molecular Basis of Genetic Variability and Susceptibility
Genetic Variability
Susceptibility
Concluding Remarks and Future Directions
References
45. Epigenetic Biomarkers in Toxicology
Introduction
DNA Methylation
Pathways of DNA Methylation
DNA Methyltransferases
Transcriptional Repression and DNA Methyl-Binding Proteins
Histone Modifications
Histone Acetylation
Histone Methylation
Histone Phosphorylation
Histone Ubiquitination and Sumoylation
Polycomb and Trithorax Proteins
Noncoding RNA
Epigenetics and Disease
Abnormalities of DNA Methylation
Abnormalities in Histone Modifications
Epigenetics and Cancer
Epigenetics and Autoimmune Disease
Systemic Lupus Erythemosus
Type 1 Diabetes Mellitus
Epigenetics and Metabolic Syndrome
Concluding Remarks and Future Directions
References
46. Risk Factors as Biomarkers of Susceptibility in Breast Cancer
Introduction
Epidemiology
Pathology
Risk Factors
Life Stage–Related Risks
Gestation and Development of the Fetus
Puberty
Pregnancy
Lactation and Breastfeeding
Family History
Genetic Modifications
History of Benign Conditions
Prognostic Factors
Detection and Screening
Tumor Marker Analysis
Testing for Genetic and Molecular Changes
Syndromes Predisposing to Breast Cancer
Hereditary Breast and Ovarian Syndrome
High Penetrance Genes Predisposing to Breast Cancer
Therapeutic Implications of Genetic Biomarkers
Other Highly Penetrant Breast Cancer Predisposing Genes
Moderate Penetrance Breast Cancer Genes
Low Penetrance Breast Cancer Polygenes
DNA Methylation, Definitions, and Measurement Methods
Impact of Methylation Biomarkers
DNA Methylation Markers and Primary Prevention
DNA Methylation Markers for Secondary Prevention and Early Detection
DNA Methylation Markers and Tertiary Prevention and Role in Prognosis
Male Breast Cancer Susceptibility Factors
Epidemiology
Pathology
Risk Factors
Cancer Aggressiveness Risk Factors
Impact of Cancer Aggressiveness Risk Factors for Patient Management and Health Policies
References
47. Pancreatic and Ovarian Cancer Biomarkers
Introduction
Currently Used Clinical Biomarkers
Cancer Antigen 19-9 in Pancreatic Cancer
Cancer Antigen-125 in Ovarian Cancer
Diagnostic Biomarkers
Pancreatic Cancer
Ovarian Cancer
Biomarkers in the Evaluation of a Pelvic Mass
Prognostic Biomarkers
Pancreatic Cancer
Ovarian Cancer
Predictive Biomarkers
Pancreatic Cancer
Ovarian Cancer
Concluding Remarks and Future Directions
References
48. Prostate Cancer Biomarkers
Introduction
Screening and Early Detection for Prostate Cancer
Contemporary Clinical Biomarkers in Prostate Cancer
Prostate-Specific Antigen
Additional Serum Testing
Prostate Cancer Antigen 3
Candidate Biomarkers for Prostate Cancer
ConfirmMDx Test
Cell Cycle Progression Signature Test
Oncotype DX Test
Transmembrane Protease Serine 2-Erythroblast Transformation Specific–Related Gene Fusion Rearrangement
Glutathione S-transferase P1
α-Methylacyl Coenzyme A Racemase
Chromogranin A
Prostate-Specific Membrane Antigen
Prostate Stem Cell Antigen
Early Prostate Cancer Antigen
B7-H3
Sarcosine
Caveolin-1
Serum Calcium
Hypermethylation of PDZ, LIM Domain Protein 4 Gene, and PDLIM5
Exosomes
Ki-67
Golgi Phosphoprotein 2 and 3
DAB2 Interacting Protein
Other Emerging Prostate Cancer Biomarkers
Concluding Remarks and Future Directions
References
Part VIII: Disease Biomarkers
49. Biomarkers of Alzheimer's Disease
Introduction
Genetic Risk Factors for Alzheimer's Disease
Mechanisms of Synaptic Dysfunction and Neuronal Loss
Long-Term Potentiation Impairments and Excitotoxicity Through Glutamatergic Dysfunction
Posttranslational Modifications of Tau Cause Aggregation and Synaptic Starving
Biomarkers
Prognostic Biomarkers
Decreased Cerebrospinal Fluid Aβ42
Increased Positron Emission Tomography Amyloid Imaging
Elevated Cerebrospinal Fluid Total-Tau and Phospho-Tau
Elevated Cerebrospinal Fluid p-tau:Aβ42 Ratio
MicroRNA Biomarkers
Diagnostic Biomarkers
Brain Atrophy
18F-2-Deoxy-2-Fluoro-d-Glucose Positron Emission Tomography
Single-Photon Emission Computed Tomography Perfusion Imaging
F2-Isoprostanes
Concluding Remarks and Future Directions
References
50. Biomarkers of Parkinson's Disease
Abbreviations
Introduction
Genetic Biomarkers
α-Synuclein
LRRK2
Parkin
PINK1
DJ-1
Biochemical Markers
α-Synuclein and Abnormal Protein Accumulations
Mitochondrial Dysfunction and Oxidative Stress Markers
DJ-1
Urate
8-OHG and 8-OHdG
Neuroinflammation Markers
Apoptosis Markers
Neuroimaging Markers
Concluding Remarks and Future Directions
Acknowledgments
References
51. Biomarkers for Drugs of Abuse and Neuropsychiatric Disorders: Models and Mechanisms
Introduction
Drugs of Abuse
Neuronal Basis of Drug Dependence
Neurotoxicity of Psychostimulants
Amphetamine
Oxidative Stress Due to High Dopamine Content in Methamphetamine-Induced Neurotoxicity
Fos B Transcription Factor Is Responsible for the Synaptic Modification Following Meth Abuse
Meth Neurotoxicity Involving DNA Damage
Meth Excitotoxicity Is Mediated by Glutamate Release and Activation of Glutamate Receptors
Meth Neurotoxicity Is the Result of Blood–Brain Barrier Dysfunction and Hyperthermia
Mitochondrial and Endoplasmic Reticulum–Dependent Death Pathway in Meth-Induced Apoptosis
Neuroinflammatory Mechanism in Meth-Induced Neurotoxicity
Cocaine
Epigenetic Regulation (ΔFos-B and Cyclic Adenosine Monophosphate Response Element Binding Protein) in Cocaine-Induced Neuro ...
Cocaine-Induced Changes in Synaptic Plasticity Is Mediated Through Brain-Derived Neurotrophic Factor
Opioids
Opioid Actions Are Orchestrated Through Inhibitory G Protein (Gi/Go)
Intracellular Targets of Opioid Activation: cAMP, Ca2+, MAPK, CREB, AP-1 Transcription Factors
Neurotoxic Effects of Opioids Linked with Oxidative Stress, Mitochondrial Dysfunction, and Apoptosis
Cognitive Deficits During Opioid Addiction Are Due to Inhibition of Neurogenesis
Cannabinoids/Marijuana
Cannabinoid Signaling Is Mediated by Cannabinoid Receptors and Endocannabinoids
Endogenous Cannabinoid Ligand Interaction With Cannabinoid Type 1 Receptor Regulates Expression of Genes Involved in Long-T ...
Alcohol
Modulation of γ-Aminobutyric Acid Type A and N-Methyl-d-aspartate Receptors by Ethanol Contributes to Anxiety, Arousal, and ...
Astrocytes Are the Main Site of Ethanol-Induced Oxidative Stress and Neurotoxicity
Generation of Proinflammatory Molecules Activates Mitogen-Activated Protein Kinase Signaling by Ethanol
Abused Substances and Neuropsychiatric Disorders
Schizophrenia
Bipolar Disorder/Mood Disorder
Anxiety Disorders
Animal Models in Neuropsychiatry
Animal Models of Depression
Animal Models of Anxiety
Animal Models of Schizophrenia
Biomarkers of Bipolar Disorder and Schizophrenia
Biomarkers of Schizophrenia
Concluding Remarks and Future Directions
References
52. Osteoarthritis Biomarkers
Abbreviations
INTRODUCTION
PATHOPHYSIOLOGY AND SIGNALING PATHWAYS IN OSTEOARTHRITIS
CLASSIFICATION OF OSTEOARTHRITIS BIOMARKERS
BIOMARKERS OF COMFORT, MOBILITY, FUNCTION, AND INFLAMMATION AND PAIN
Humans
Animals
Observation of Ortolani and Cranial Tibial Drawer Examination
OSTEOARTHRITIS BIOMARKERS IN SERUM, SYNOVIAL FLUID, AND URINE
Biomarkers of Inflammation
Cytokines/Adipocytokines, Chemokines, and Neuropeptides
Prostaglandin E2 and EP4 Receptor
Fractalkine
Erythrocyte Sedimentation Rate
Biomarkers of Osteoarthritis Progression and Cartilage Degeneration
Cartilage Oligomeric Matrix Protein (COMP)
Matrix Metalloproteinases (MMPs)
Hyaluronan
Lubricin
Follistatin-Like Protein 1 (FSTL 1)
Ghrelin
TSG-6
Cartilage Damage and Imaging Biomarkers
Newly Identified Biomarkers
DNA-Methylation and MicroRNAs (miR)
CORRELATION OF CIRCULATORY BIOMARKERS WITH RADIOGRAPHIC/IMAGING BIOMARKERS AND SYMPTOMS OF OSTEOARTHRITIS
CONCLUDING REMARKS AND FUTURE DIRECTIONS
Acknowledgments
References
53. Pathological Biomarkers in Toxicology
Introduction
Diagnostic Pathology
Enzootic Hematuria
Cyclopia
Taxus (Yew) Poisoning
Ergotism
Morphologic (Gross and Microscopic) and Clinical Pathology
Solar Injuries
Skin Photosensitization
Skin Neoplasia
Iatrogenic Acromegaly in Dogs
Endocrine Alopecia
Alopecia and Inhibition of Hair Growth (Mimosine Toxicity)
Acute Bovine Pulmonary Edema and Emphysema
Toxic Myopathy—Ionophore Toxic Syndrome
Drug-Eluting Stents
Hepatopathies
Glycogen Hepatopathy
Hepatotoxicity Secondary to Enzyme Induction
Peroxisome Proliferator-Activated Receptor Hepatopathy (Characteristic Lesion by Light and Electron Microscopy) in Rodents
Iron Toxicosis
Copper Poisoning in Sheep
Nephropathies
Polycystic Kidney Disease in Rats
Alpha 2-Microglobulin Nephropathy
Tubulointerstitial Nephritis
Ethylene Glycol (Oxalate) Poisoning
Melamine Poisoning
Lead Toxicity
Nephrotoxicity and Acute Renal Injury
Polioencephalomalacia
Sodium Chloride Toxicity
Nigropallidal Encephalomalacia in Horses
Phospholipidosis
Concluding Remarks and Future Directions
References
54. Oral Pathology Biomarkers
Introduction
Anatomical and Histological Considerations of the Oral Cavity
Teeth
Histology of Oral Mucosa
Oral Physiology and Pharmacodynamics
Biomarkers in the Oral Cavity
Common Drugs and Toxicants showing Oral Manifestations
Select Diseases of the Oral Cavity
Oral Biomarkers of Exposure and Effects of Select Drugs/Toxicants
Tetracycline/Minocycline
Mercury and Silver
Lead
Biomarkers of Select Diseases
Periodontal Disease
Immunologically Mediated Oral Diseases
Pemphigus
Pemphigoid
Oral Lichen Planus
Oral Squamous Cell Carcinoma
Saliva: A Hidden Plethora of Biomarkers
Concluding Remarks and Future Directions
References
Part IX: Special Topics
55. Biomarkers of Mitochondrial Dysfunction and Toxicity
Introduction
Mitochondrial Function: General Overview
Mitochondrial Toxicity
Xenobiotics and Mitochondrial Dysfunction
Mitochondria and Disease
Mitochondrial Dysfunction in Diabetes
Mitochondrial Dysfunction in Ischemia/Reperfusion
Mitochondrial Dysfunction in Cancer
Concluding Remarks and Future Directions
References
56. Biomarkers of Blood–Brain Barrier Dysfunction
Introduction
Structure and Function of Brain Barriers
In Vivo and In Vitro Models to Study the Blood–Brain Barrier
In Vivo Model
In Vitro Models
Toxicants Affecting the Central Nervous System Barriers
Metals
Aluminum
Copper and Iron
Lead
Manganese
Mercury
Zinc
Pesticides
Organophosphates
Organochlorines and Pyrethrins/Pyrethroids
Rotenone
Herbicides
Mycotoxins
Drugs of Abuse and Therapeutic Drugs
Methamphetamine
Cocaine
Alcohol/Ethanol
Neurodegenerative Diseases and Other Conditions
Neurodegenerative Diseases
Epilepsy
HIV Infection
Concluding Remarks and Future Directions
Acknowledgments
References
57. Biomarkers of Oxidative/Nitrosative Stress and Neurotoxicity
Introduction
Lipid Peroxidation and Markers of Oxidative Stress
Prostaglandin-Like Compounds as In Vivo Markers of Oxidative Stress
Aldehydes as Lipid Peroxidation Products
Reactivity of Lipid Peroxidation Products
Excitotoxicity and Oxidative Damage
Neuroinflammation and Oxidative Injury
Metal Toxicity and Oxidative Injury
Manganese
Mercury
Concluding Remarks and Future Directions
References
58. Cytoskeletal Disruption as a Biomarker of Developmental Neurotoxicity
Introduction
Microtubules
Effects of Organophosphorus Esters on Microtubules
Effects of Heavy Metals on Microtubules
Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Microtubules
Microfilaments
Effects of Organophosphorus Esters on Microfilaments
Effects of Heavy Metals on Microfilaments
Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Microfilaments
Intermediate Filaments
Effects of Organophosphorus Esters on Intermediate Filaments
Effects of Heavy Metals on Intermediate Filaments
Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Intermediate Filaments
Concluding Remarks and Future Directions
References
59. MicroRNA Expression as an Indicator of Tissue Toxicity and a Biomarker in Disease and Drug-Induced Toxicological Evaluation
Introduction
Regulatory Mechanisms of miRNA Biogenesis
Standard Toxicity Measurements (Tissue and Circulating Biomarkers)
Toxicology
Clinical Pathology
Overview
Hematology
Clinical Chemistry
Urinalysis
Coagulation Tests
Anatomic Pathology
Nomenclature
Database
Sample Preparation
Kinetics and Pharmacokinetics/Pharmacodynamics
MiRNA Biomarkers of Toxicity in Organ Systems
Esophagus
miRNAs as Biomarkers of Pancreatic Toxicity
Liver
Heart
Kidney
Brain
Environmental Exposure and miRNA
Role of miRNA in Arsenic-Induced Carcinogenesis
Concluding Remarks and Future Directions
References
60. Citrulline: Pharmacological Perspectives and Role as a Biomarker in Diseases and Toxicities
List of Abbreviations
Introduction
Citrulline or Arginine Supplementation
Salient Aspects of Nitric Oxide
Biochemical Aspects
Pharmacokinetics and Pharmacodynamics of Citrulline
Pharmacodynamics of Citrulline
Citrulline in Sickle Cell Anemia
Citrulline and Cardiovascular Effects
Citrulline in Hypertension
Citrulline in Diabetes
Citrulline as Immunomodulator
Citrulline Therapy May Decrease Severity of Sepsis
Citrulline in Arginase-Associated T-Cell Dysfunction
Citrulline: Role in Dementia of Alzheimer's Disease and in Multi-Infarct Dementia
Citrulline: Can It Improve Aerobic Function/Energy Production?
Citrulline for Urea Cycle Disorders
Citrulline for Acceleration of Wound Healing
Citrulline in Intestinal Pathology
Citrulline: Effects of Amino Acids on Hair Strength
Citrulline's Effect on Protein Synthesis
Citrulline as a Biomarker in Diseases
Citrulline as a Biomarker in Intestinal Disease
Citrulline as a Biomarker in Celiac Disease
Citrulline: Is It a Reliable Biomarker in Critically Ill Patients?
Citrulline as a Biomarker to Identify Patients at High Risk of Developing a Catheter-Related Bloodstream Infection
Citrulline as a Biomarker for Gastrointestinal Tolerance to Enteral Feeding
Anticyclic Citrullinated Peptide Antibodies: A Novel Biomarker of Rheumatoid Arthritis
Citrulline as a Biomarker in Periodontitis for Presymptomatic Rheumatoid Arthritis?
Citrulline as a Biomarker in Rheumatoid Arthritis in Patients With Active Tuberculosis
Conditions in Which Citrulline May Prove to Be a Biomarker in the Future
Conditions Detected by Anticyclic Citrullinated Peptide
Citrulline as a Biomarker for Brucellosis Presenting With Peripheral Arthritis
Citrulline as a Biomarker for Sjögren's Syndrome
Conditions Detected by Citrulline Levels
Citrulline as a Biomarker in Transplants
Citrulline as a Biomarker in Acute Respiratory Distress Syndrome
Citrulline as a Biomarker in Polycystic Ovary Syndrome
Citrulline as a Biomarker in Acute Kidney Injury
Homocitrulline as a Biomarker for Renal Failure
Citrulline as a Biomarker in Critically Ill Patients and Correlation With Severity of Inflammation
Citrulline as a Biomarker in Intrauterine Growth Retardation
Citrulline as a Biomarker of Congenital Anomalies
Citrulline as a Biomarker for OAT Deficiency in Early Infancy
Citrulline as a Biomarker in Citrin Deficiency
Citrulline as a Biomarker in Toxicities
Citrulline as a Biomarker of Kainic Acid–Induced Neurotoxicity
Citrulline as a Biomarker of Anticholinesterase Poisoning
Citrulline as a Biomarker of Ammonia Toxicity?
Concluding Remarks and Future Directions
References
Part X: Applications of Biomarkers in Toxicology
61. Analysis of Toxin- and Toxicant-Induced Biomarker Signatures Using Microarrays
Biomarkers and Biomarker Signatures in Disease: a Variety of Marker Classes and Possible Inferences
Selecting Sample Sources for the Measurement of Biomarkers
Individual Biomarkers and Aggregated Biomarker Signatures
Approaches to Biomarker Signature Characterization
Multiplexed High Content Solid Phase Immunoassays
Flow Cytometry and Fluorescence-Activated Cell Sorting
Immunohistochemistry and Tissue Microarrays
Mass Spectrometry Analysis
Grating-coupled Surface Plasmon Resonance Microarray Analysis
Biomarker Signatures in the -Omics era
Genomic and Transcriptomic Analyses
Proteomics
Metabolomics
Lipidomics
Genotype–Phenotype Associations
Data Analysis Considerations
Concluding Remarks and Future Directions
Acknowledgments
Conflict of Interest Declaration
References
62. Biomarkers Detection for Toxicity Testing Using Metabolomics
Introduction to Metabolomics
Metabolomics Experimental Overview
Study Design and Planning
Sample Collection and Quenching
Sample Preparation
Instrumental Analysis
Raw Data Processing
Statistical Analysis of Processed Data
Identification of Putative Biomarkers
Biomarker Validation
Metabolomics Experimental Overview Conclusions
Applications of Metabolomics Biomarkers in Toxicity Testing
Metabolomics in Toxicology Consortia
Metabolomics Biomarkers in Hepatotoxicity
Metabolomics Biomarkers in Renal Toxicity
Metabolomics Biomarkers of Toxicity From Chemical Agents
Metabolomics Biomarkers in Neurotoxicity
Metabolomics Biomarkers of Biotoxins
Metabolomics Biomarkers of Nanomaterials
Metabolomics Biomarkers of Drugs of Abuse
Concluding Remarks and Future Directions
References
63. Transcriptomic Biomarkers in Safety and Risk Assessment of Chemicals
Introduction
Transcriptomics
Transcriptomic Methods
Types of Transcriptomic Biomarkers
Transcriptomics in Biomarker Discovery
Cardiovascular Biomarkers
Hepatic Biomarkers
Biomarkers of Anabolic Agents
Infectious Agents and Sepsis
Tobacco and Risk Continuum
Environmental Chemical Risk Assessment
Concluding Remarks and Future Directions
References
64. Percellome Toxicogenomics Project as a Source of Biomarkers of Chemical Toxicity
Introduction
Materials and methods for Percellome Data Generation and example studies
Experimental animals and dose setting
Microarray data generation
Comprehensive Selection of Responding mRNAs
Estragole Study
Pentachlorophenol Study
Merging of TGP Data to Percellome Database
New Repeated Dosing Study
Results and Discussion
Estragole Study
PCP Study
Merging the TGP Data to Percellome Database
New Repeated Dosing Study
Concluding Remarks and Future Directions
Acknowledgments
References
65. Proteomics in Biomarkers of Chemical Toxicity
Introduction
Heavy Metals
Arsenic
Cadmium
Chromium (VI)
Lead
Mercury
Pesticides and Herbicides
Methyl Parathion
Diazinon
Paraquat
Glyphosate
Organic Pollutants
Benzo(a)pyrene
2,3,7,8-Tetrachlorodibenzo-p-Dioxin
Concluding Remarks and Future Directions
References
66. Biomarkers for Testing Toxicity and Monitoring Exposure to Xenobiotics
Introduction
Requirements Expected in a Biomarker for Toxicology Testing and Biomonitoring Xenobiotics Exposure
Biological Samples Used for Biomonitoring Exposure Through Biomarkers
Biomarkers of Exposure for Monitoring Xenobiotics Exposure
Adducts: A Relevant Case of Biomarkers of Exposure
Biomarkers of Effect for Toxicology Testing
Molecular Biomarkers for In Vitro Testing Toxicity
Concluding Remarks and Future Directions
References
67. Biomarkers in Epidemiology, Risk Assessment and Regulatory Toxicology
Introduction
Biomarkers
New Advances in Biomonitoring, Exposure Science, Toxicology, Risk Assessment, and Decision-Making
Biomarkers of Exposure
Methylmercury
Biomarkers of Effect
Cholinesterase Inhibition
Selenium
Biomarkers of Susceptibility
Cancer-Related Biomarkers
Formaldehyde
Concluding Remarks and Future Directions
References
Index
A
B
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D
E
F
G
H
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J
K
L
M
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O
P
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T
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Back Cover

Citation preview

BIOMARKERS IN TOXICOLOGY SECOND EDITION Edited by

RAMESH C. GUPTA, DVM, MVSC, PHD, DABT, FACT, FACN, FATS Professor and Head, Toxicology Department Breathitt Veterinary Centre Murray State University Hopkinsville, Kentucky United States

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 © 2019 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-814655-2 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Mica H. Haley Acquisition Editor: Erin Hill-Parks Editorial Project Manager: Kristi Anderson Production Project Manager: Mohanapriyan Rajendran Cover Designer: Victoria Pearson Typeset by TNQ Technologies

This book is dedicated to my daughter Rekha, wife Denise, and parents the late Chandra and Triveni Gupta.

Contributors Arturo Anado´n Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain

Subash Chandra Gupta Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, India

Vellareddy Anantharam Parkinson’s Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA, United States

Saurabh Chatterjee Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC, United States Catheryne Chiang Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States

Anthony E. Archibong Department of Microbiology, Immunology & Physiology, Meharry Medical College, Nashville, TN, United States Irma Ares Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain

Anirudh J. Chintalapati St. John’s University, College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences, Toxicology Division, New York, United States

Adam D. Aulbach Charles River Laboratories, Mattawan, MI, United States

P. Cohn New Jersey Department of Health (retired), Trenton, NJ, United States

Nikee Awasthee Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, India

Robert W. Coppock Toxicologist and Associates, Ltd, Vegreville, AB, Canada

Aryamitra Banerjee Study Director, Toxicology Research Laboratory, University of Illinois at Chicago, Chicago, IL, United States

Lucio G. Costa Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, United States; Department of Medicine & Surgery, University of Parma, Parma, Italy

Leah D. Banks Department of Biochemistry, Cancer Biology, Neuroscience & Pharmacology, Meharry Medical College, Nashville, TN, United States Frank A. Barile St. John’s University, College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences, Toxicology Division, New York, United States

Tirupapuliyur V. Damodaran Department of Biological and Biomedical Sciences, North Carolina Central University, Durham, NC, United States Clinton D’Souza Division of Environmental Health and Toxicology, Nitte University Center for Science Education and Research (NUCSER), Deralakatte, India

Sudheer R. Beedanagari Bristol Myers Squibb, New Brunswick, NJ, United States Charalampos Belantis Department of Urology, University General Hospital of Heraklion, University of Crete, Medical School, Heraklion, Crete, Greece

Wolf-D. Dettbarn United States

Vanderbilt University, Nashville, TN,

Amy A. Devlin US Food and Drug Administration, Silver Spring, MD, United States

Enrico Bergamaschi Laboratory of Toxicology and Occupational Epidemiology, Department of Public Health Science and Pediatrics, University of Turin, Italy

Robin B. Doss Toxicology Department, Breathitt Veterinary Center, Murray State University, Hopkinsville, Kentucky, United States

Sadikshya Bhandari Molecular and Cell Biology, University of Connecticut, Storrs, CT, United States

Shiwangi Dwivedi Division of Environmental Health and Toxicology, Nitte University Center for Science Education and Research (NUCSER), Deralakatte, India

Sneha P. Bhatia Research Institute for Fragrance Materials, NJ, United States Karyn Bischoff Cornell University, Diagnostic Toxicologist, New York State Animal Health Diagnostic Center, Ithaca NY, United States David J. Borts Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, United States Emily Brehm Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States

Margitta M. Dziwenka Faculty of Medicine & Dentistry, Division of Health Sciences Laboratory Animal Services, Edmonton, AB, Canada Jorge Este´vez Instituto de Bioingenierı´a, Universidad Miguel Herna´ndez de Elche, Spain Daniel S. Fabricant President and CEO, Natural Products Association, Washington D.C., United States A.M. Fan California Environmental Protection Agency (retired), Oakland/Sacramento, CA, United States

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CONTRIBUTORS

Vanessa A. Fitsanakis Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, OH, United States

Holly E. Hatfield Department of Biochemistry, University of Cincinnati Medical School, Cincinnati, OH, United States

John Flaskos Laboratory of Biochemistry and Toxicology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

Wallace A. Hayes University of South Florida College of Public Health, Tampa, FL, United States; Michigan State University, East Lansing, MI, United States

Jodi A. Flaws Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States

Ioannis Heretis Department of Urology, University General Hospital of Heraklion, University of Crete, Medical School, Heraklion, Crete, Greece Antonio F. Herna´ndez Department of Legal Medicine and Toxicology, University of Granada School of Medicine, Granada, Spain

Swaran J.S. Flora National Institute of Pharmaceutical Education and Research, Raebareli, U.P., India Sue M. Ford Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, St. John’s University, Jamaica, NY, United States Jessica S. Fortin College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States Domniki Fragou Laboratory of Forensic Medicine & Toxicology, School of Medicine, Aristotle University of Thessaloniki, Greece Shayne C. Gad Principal of Gad Consulting Services, Raleigh, NC, United States Bianca Galateanu Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania Dale R. Gardner United States Department of Agriculture, Agricultural Research Service, Poisonous Plant Research Laboratory, Logan, UT, United States George Georgiadis Department of Urology, University General Hospital of Heraklion, University of Crete, Medical School, Heraklion, Crete, Greece Fernando Gil Department of Legal Medicine and Toxicology, University of Granada School of Medicine, Granada, Spain Saryu Goel

Nonclinical Expert, Leesburg, VA, United States

Mary Gulumian Toxicology Research Projects NIOH, School of Pathology, University of the Witwatersrand South Africa P.K. Gupta Director Toxicology Consulting Group, Former Principal Scientist and Chief Division of Pharmacology and Toxicology, IVRI, Advisor, World Health Organization (Geneva), Bareilly, India Ramesh C. Gupta Toxicology Department, Breathitt Veterinary Center, Murray State University, Hopkinsville, Kentucky, United States Rekha K. Gupta School of Medicine, University of Louisville, Louisville, KY, United States Sharon Gwaltney-Brant Veterinary Information Network, Mahomet, IL, United States Alan J. Hargreaves School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom Kelly L. Harris Department of Biochemistry, Cancer Biology, Neuroscience & Pharmacology, Meharry Medical College, Nashville, TN, United States Kenneth J. Harris Department of Biochemistry, Cancer Biology, Neuroscience & Pharmacology, Meharry Medical College, Nashville, TN, United States

Corey J. Hilmas Senior Vice President of Scientific and Regulatory Affairs, Natural Products Association, Washington D.C., United States Darryl B. Hood College of Public Health, Ohio State University, Columbus, OH, United States Pasi Huuskonen School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland Stewart B. Jacobson Shin Nippon Biomedical Laboratories USA, Ltd, Everett, WA, United States Sandra A. James-Yi Associate Principle Scientist, Product Safety/Nutritional Toxicology, Mary Kay Inc., Addison, Texas, United States Huajun Jin Parkinson’s Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA, United States Jun Kanno Japan Bioassay Research Center, Japan Organization of Occupational Health and Safety, Hadano, Japan Arthi Kanthasamy Parkinson’s Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA, United States Anumantha G. Kanthasamy Parkinson’s Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA, United States Shilpa N. Kaore Raipur Institute of Medical Sciences, Raipur, India Navinchandra M. Kaore Raipur Institute of Medical Sciences, Raipur, India Bhupendra S. Kaphalia Department of Pathology, University of Texas Medical Branch, Galveston, TX, United States Vesa Karttunen School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland Gurjot Kaur Human and Environmental Toxicology, Department of Biology, University of Konstanz, Konstanz, Germany Ravneet Kaur

Aveley, Western Australia

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CONTRIBUTORS

Prasada Rao S. Kodavanti Neurotoxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, United States

Charalampos Mavridis Department of Urology, University General Hospital of Heraklion, University of Crete, Medical School, Heraklion, Crete, Greece

Urmila P. Kodavanti Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Durham, North Carolina, United States

Vincent P. Meador Covance Laboratories, Inc., Global Pathology, Madison, WI, United States

George A. Kontadakis Laboratory of Optics and Vision and Ophthalmology Department, University of Crete, Heraklion, Greece Gopala Krishna United States

Nonclinical Consultants, Ellicott City, MD,

Priya A. Krishna Nonclinical Consultants, Ellicott City, MD, United States Kavya A. Krishna Nonclinical Consultants, Ellicott City, MD, United States Maria Kummu Research Unit of Biomedicine, Pharmacology and Toxicology, Faculty of Medicine, University of Oulu, Oulu, Finland George D. Kymionis Jules Gonin Eye Hospital, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland Rajiv Lall Vets Plus Inc., Menomonie, WI, United States P. Lin National Institute of Environmental Health Sciences, National Health Research Institutes, Taiwan Bommanna G. Loganathan Department of Chemistry and Watershed Studies Institute, Murray State University, Murray, KY, United States Jarkko Loikkanen Finland

European Chemicals Agency, Helsinki,

Marcello Lotti Dipartimento di Scienze Cardio-ToracoVascolari e Sanita` Pubblica, Universita` degli Studi Padova, Padova, Italy Michael A. Lynes Molecular and Cell Biology, University of Connecticut, Storrs, CT, United States

Roger O. McClellan Independent Advisor, Toxicology and Risk Analysis, Albuquerque, New Mexico, United States

Lars Friis Mikkelsen Ellegaard Go¨ttingen Minipigs A/S, Dalmose, Denmark Dejan Milatovic

Charlottesville, VA, United States

Ida R. Miller Mukherjee Institute of Psychiatry and Human Behavior, Bambolim, Goa, India Anupama Mukherjee Oral Pathology, Microbiology and Forensic Odontology, Goa Dental College and Hospital, Panaji, India Pushpinder Kaur Multani Johnson & Johnson, Janssen Research & Development, Malvern, PA, United States Pa¨ivi Myllynen Kirsi Myo¨ha¨nen Finland

NordLab, Oulu, Finland European Chemicals Agency, Helsinki,

Rekek Negga Department of Biology, King University, Bristol, TN, United States Carolina Negrei Departament of Toxicology, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Meliton N. Novilla Shin Nippon Biomedical Laboratories USA, Ltd, Everett, WA, United States; School of Veterinary Medicine, Purdue University, West Lafayette, IN, United States Stephanie Padilla Integrated Systems Toxicology Division, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, United States Carlos M. Palmeira Center for Neurosciences and Cell Biology of the University of Coimbra and Department of Life Sciences of the University of Coimbra Largo Marqueˆs de Pombal, Coimbra, Portugal

Brinda Mahadevan Medical Safety & Surveillance, Abbott Laboratories, Columbus, OH, United States

Kip E. Panter United States Department of Agriculture, Agricultural Research Service, Poisonous Plant Research Laboratory, Logan, UT, United States

Jitendra K. Malik Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India

Markku Pasanen School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

Charalampos Mamoulakis Department of Urology, University General Hospital of Heraklion, University of Crete, Medical School, Heraklion, Crete, Greece

Daniel J. Patrick United States

Jane A. Mantey Department of Biochemistry, Cancer Biology, Neuroscience & Pharmacology, Meharry Medical College, Nashville, TN, United States Marı´a Rosa Martı´nez-Larran˜aga Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain Marı´a Ara´nzazu Martı´nez Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain

Charles River Laboratories, Mattawan, MI,

Sofia Pavanello Dipartimento di Scienze Cardio-ToracoVascolari e Sanita` Pubblica, Universita` degli Studi Padova, Padova, Italy Henrik Duelund Pedersen Ellegaard Go¨ttingen Minipigs A/S, Dalmose, Denmark Olavi Pelkonen Research Unit of Biomedicine, Pharmacology and Toxicology, Faculty of Medicine, University of Oulu, Oulu, Finland Michael A. Pellizzon NJ, United States

Research Diets, Inc., New Brunswick,

Jason Pitt University of Evansville, Evansville, IN

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CONTRIBUTORS

Argyro Plaka Laboratory of Optics and Vision and Ophthalmology Department, University of Crete, Heraklion, Greece Aramandla Ramesh Department of Biochemistry, Cancer Biology, Neuroscience & Pharmacology, Meharry Medical College, Nashville, TN, United States Saniya Rattan Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States Jenni Repo School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland Matthew R. Ricci United States

Research Diets, Inc., New Brunswick, NJ,

Anabela P. Rolo Center for Neurosciences and Cell Biology of the University of Coimbra and Department of Life Sciences of the University of Coimbra Largo Marqueˆs de Pombal, Coimbra, Portugal

Miguel A. Sogorb Instituto de Bioingenierı´a, Universidad Miguel Herna´ndez de Elche, Spain Ajay Srivastava States

Vets Plus Inc., Menomonie, WI, United

Szabina A. Stice Division of Biotechnology and GRAS Notice Review, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD, United States Markus Storvik School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland David T. Szabo PPG Industries Incorporated, Pittsburgh, PA, United States Joa˜o S. Teodoro Center for Neurosciences and Cell Biology of the University of Coimbra and Department of Life Sciences of the University of Coimbra Largo Marqueˆs de Pombal, Coimbra, Portugal

Magdalini Sachana Organization for Economic Cooperation and Development (OECD), Paris, France

Aristidis M. Tsatsakis Center of Toxicology Science & Research, Medical School, University of Crete, Heraklion, Greece

Heidi Sahlman School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

John Tsiaoussis Laboratory of Anatomy-HistologyEmbryology, Medical School, University of Crete, Heraklion, Crete, Greece

Nitin Saini Johnson & Johnson, Janssen Research & Development, Malvern, PA, United States

Kirsi Va¨ha¨kangas School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

Vandana Saini

Washington, United States

Kai Savolainen Nanosafety Research Centre, Finnish Institute of Occupational Health, Helsinki, Finland Ratanesh Kumar Seth Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC, United States Abha Sharma National Institute of Pharmaceutical Education and Research, Raebareli, U.P., India Anurag Sharma Division of Environmental Health and Toxicology, Nitte University Center for Science Education and Research (NUCSER), Deralakatte, India Elina Sieppi Research Unit of Biomedicine, Pharmacology and Toxicology, Faculty of Medicine, University of Oulu, Oulu, Finland Rui Silva Center for Neurosciences and Cell Biology of the University of Coimbra and Department of Life Sciences of the University of Coimbra Largo Marqueˆs de Pombal, Coimbra, Portugal Anita Sinha Vets Plus Inc., Menomonie, WI, United States Iordanis Skamagkas Department of Urology, University General Hospital of Heraklion, University of Crete, Medical School, Heraklion, Crete, Greece Samantha J. Snow Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Durham, North Carolina, United States

Sumit Singh Verma Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, India Eugenio Vilanova Instituto de Bioingenierı´a, Universidad Miguel Herna´ndez de Elche, Spain Suryanarayana V. Vulimiri National Center for Environmental Assessment, Environmental Protection Agency (EPA), Washington DC, United States Genoa R. Warner Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States Kevin D. Welch United States Department of Agriculture, Agricultural Research Service, Poisonous Plant Research Laboratory, Logan, UT, United States Christina Wilson-Frank Purdue University, College of Veterinary Medicine, Animal Disease Diagnostic Laboratory, Department of Comparative Pathobiology, West Lafayette, IN, United States S.H. You Institute of Food Safety and Risk Management, National Taiwan Ocean University, Keelung, Taiwan Snjezana Zaja-Milatovic PAREXEL International, Alexandria, VA, United States Ioannis E. Zisis Department of Urology, University General Hospital of Heraklion, University of Crete, Medical School, Heraklion, Crete, Greece Csaba K. Zoltani Emeritus US Army Research Lab, Aberdeen Proving Ground, MD, United States

Foreword The first edition of Biomarkers in Toxicology, edited by Ramesh Gupta, was published in 2014. The whole area of biomarkers, not only in toxicology, is rapidly developing, partly because of the availability of highly sophisticated analytical equipment, and so the second edition of this book is greatly welcomed. The second edition contains 12 new chapters, and most of the rest have been updated. Merriam Webster defines a biomarker as a distinctive biological or biologically derived indicator (as a metabolite) of a process, event, or condition (as, for example, aging, disease, or oil formation). There are other definitions, for example, in Environmental Health Criteria 222 Biomarkers. In Risk Assessment http://www.inchem.org/ documents/ehc/ehc/ehc222.htm#1.0 biomarkers are defined thus “A biomarker is any substance, structure or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease.” The subject of the present book is biomarkers in toxicology, but it should be remembered that biomarkers include substances used in the detection of numerous diseases, including the autoimmune diseases, which are not generally thought to be toxicological in origin. However, biomarkers are crucial to toxicology and allied disciplines such as epidemiology and risk assessment. The earliest toxicological biomarkers of exposure date from before precise analytical techniques were available and include the KaysereFleischer ring (described 1902/3), usually indicative of copper accumulation in the cornea in cases of Wilson’s disease, as well as lead lines in the gums associated with lead toxicity. The cherry red color noted as a (rather unreliable) clinical sign in carbon monoxide poisoning may also be described as a biomarker. One of the earliest biomarkers relying on biochemical analytical techniques was measurement of cholinesterase activity, initially whole blood cholinesterase and later plasma pseudocholinesterase (butyrylcholinesterase) and red blood cell acetylcholinesterase. Cholinesterase measurements were introduced at defense laboratories after World War II as a screening test for excessive exposure to organophosphate compounds: a 20% depression in activity was considered to mandate cessation of exposure of individuals to organophosphate nerve agents (the basis of the 20% figure is obscure, but seemed protective). Pseudocholinesterase and red blood cell acetylcholinesterase measurements now have numerous uses in worker protection, clinical diagnosis of poisoning, and human and experimental animal studies (including regulatory ones) in relation to the use of organophosphate and other anticholinesterase pesticides. Since World War II biomarkers of toxicity have ballooned in importance and number in worker and consumer protection and clinical and experimental toxicology and are also widely used in regulation of chemicals in animal and, less commonly, human experimental studies. Toxicological biomarkers are also used in allied disciplines, for example, epidemiology, and may be used to estimate loads of exposure in populations being investigated. In toxicology, biomarkers are often divided into biomarkers of exposure, of effect, and of susceptibility, and all of these are dealt with in this book, which is extremely wide-ranging. The book has an initial introductory part, including discussion of rodent, nonhuman primate, and zebrafish and Caenorhabditis elegans models for toxicological testing. There are two new chapters in this part: firstly, Drosophila melanogaster, Eisenia fetida, and Daphnia magna for toxicity testing and biomarkers and secondly, adverse outcome pathways and biomarkers. Part II, systems toxicity biomarkers, comprises chapters on biomarkers of toxicity in relation to all important organs and organ systems. There is an additional chapter on reproductive and developmental toxicity biomarkers, and another on ototoxicity biomarkers. Part III, renamed chemical agents, solvents, and gases toxicity biomarkers, deals with biomarkers in relation to the toxicity of specific groups of compounds and comprises chapters on pesticides, as well as polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), brominated flame retardants, polycyclic aromatic hydrocarbons (PAHs), bisphenol A, melamine and cyanuric acid, and metals; there is a very useful chapter on biomarkers of chemical mixture toxicity. Part IV (biotoxins biomarkers) has three chapters on, respectively, freshwater cyanotoxins, mycotoxins, and poisonous plants: biomarkers for diagnosis (of poisoning). Part V covers pharmaceuticals and nutraceuticals, with chapters on drug toxicity biomarkers and nutriphenomic biomarkers together with a new chapter on biomarkers of toxicity for dietary ingredients contained in dietary supplements. Part VI covers nanomaterials and radiation, with two chapters, one on biomarkers of exposure and effect of engineered nanomaterials and the other on biomarkers of exposure and effects of radiation.

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FOREWORD

Part VII is entitled carcinogens biomonitoring and cancer biomarkers and contains six chapters. These are on biomonitoring exposures to carcinogens, genotoxicity biomarkers, epigenetic biomarkers in toxicology, breast cancer biomarkers, pancreatic and ovarian cancer biomarkers, and prostate cancer biomarkers. Part VIII is called disease biomarkers and deals with biomarkers of Alzheimer’s disease, biomarkers of Parkinson’s disease, biomarkers for drugs of abuse and neuropsychiatric disorders: models and mechanisms, osteoarthritis biomarkers, pathological biomarkers in toxicology and oral pathology biomarkers. Of these, the chapters on osteoarthritis and oral pathology are new. Part IX is called special topics. This part of the book contains chapters on biomarkers of mitochondrial dysfunction and toxicity, biomarkers of blood-brain barrier dysfunction, biomarkers of oxidative/ nitrosative stress and neurotoxicity, cytoskeletal disruption as a biomarker of developmental neurotoxicity, membrane transporters and transporter substrates as biomarkers for drug pharmacokinetics, pharmacodynamics, and toxicity/adverse events, and citrulline: pharmacological perspectives and role as a biomarker in diseases and toxicity. Of these chapters, that on the blood-brain barrier dysfunction is new and is particularly welcome as the blood-brain barrier is very important in protecting the central nervous system against toxicants. The last part of the book is on applications of biomarkers. It contains three new chapters: biomarkers detection for toxicity testing using microarray technology, metabolomics, and proteomics. Also there are chapters on transcriptomic biomarkers, percellome toxicogenomics, biomarkers in computational toxicology, biomarkers in biomonitoring of xenobiotics and biomarkers in toxicology, risk assessment, and environmental chemical regulations. The 67-chapter book has an outstanding array of authors from the United States, Canada, Denmark, Finland, Greece, India, Italy, Japan, Portugal, Romania, and Spain. Professor Gupta deserves our gratitude for assembling such a distinguished group of experts to produce so comprehensive a book on this rapidly growing and very important field. Timothy C Marrs Edenbridge United Kingdom

C H A P T E R

1 Introduction Ramesh C. Gupta Toxicology Department, Breathitt Veterinary Center, Murray State University, Hopkinsville, Kentucky, United States Developing and validating highly sensitive methods for measurement of biomarkers and understanding the resultant data are complex processes that require a great deal of time, effort, and intellectual input. Furthermore, understanding drug metabolism seems essential in some cases, as the metabolite of a drug can be used as a biomarker, and the drug and/or its metabolite has to be patented by the United States Patent Office and by a similar governmental office/agency in other countries. In the past, many drugs were developed with biomarker assays that guided their use, and this trend is likely to continue in the future for drug discovery and development. With the judicious use of biomarkers, as in evidence-based medicine, patients are most likely to benefit from select treatments and least likely to suffer from their adverse effects. On the contrary, utilization of a bad biomarker can be as harmful to a patient as a bad drug. Therefore, biomarkers need to be validated and evaluated by an accredited laboratory, which participates in a proficiency testing program, to provide a high level of confidence to both clinicians and patients. In the toxicology field, biomarkers should be specific, accurate, sensitive, validated, biologically or clinically relevant, and easy and fast to perform to be useful as predictive tools for toxicity testing and surveillance and for improving quantitative estimates of exposure and dose. Therefore, biomarkers are utilized for biomonitoring data that are useful in a variety of applications, from exposure assessment to risk assessment, management, and regulations (Ganzleben et al., 2017). In the early 1990s, Dr. Maria Cristina Fossi from the University of Siena, Italy, emphasized the approach for the development and validation of nondestructive biomarkers over destructive biomarkers in the field of toxicology. She described the ideal biomarker as being measurable in readily available tissues or biological products and obtainable in a noninvasive way; related

Biomarkers can broadly be defined as indicators or signaling events in biological systems or samples of measurable changes at the molecular, biochemical, cellular, physiological, pathological, or behavioral levels in response to xenobiotics. The Biomarkers Definitions Working Group of the National Institutes of Health (NIH) has defined the biomarker as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes or pharmacological responses to a therapeutic agent.” In the field of toxicology, biomarkers have been classified as markers of exposure, effect, and susceptibility. Measurement of biomarkers reflects the time course of an injury and provides information on the molecular mechanisms of toxicity. These biomarkers provide us the confidence of accurate diagnosis, prognosis, and treatment. The biomarkers of early chemical exposure can occur in concert with biomarkers of early disease detection, and that information aids in avoiding further chemical exposure and in strategic development of a novel treatment, including personalized medicine (i.e., treating the patient, and not the disease). In essence, with the utilization of specific biomarkers, an ounce of prevention can be worth a pound of treatment. Biomarkers are used in drug development, during preclinical and clinical trials, for efficacy and safety assessment. Biomarkers can reveal valuable information regarding diagnosis, prognosis, and predict treatment efficacy or toxicity; serve as markers of disease progression; and serve as auxiliary endpoints for clinical trials (Stern et al., 2018), with the ultimate goal of delivering safe and effective medicines to patients (Lavezzari and Womack, 2016: Gerlach et al., 2018). In addition, a biomarker in drug development should be ethically acceptable (Hey, 2017). Safety biomarkers can be used to predict, detect, and monitor drug-induced toxicity during both preclinical studies and human clinical trials.

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1. INTRODUCTION

to exposure and/or degree of harm to the organism; directly related to the mechanism of action of the contaminants; highly sensitive with techniques that require minimal quantities of sample and are easy to perform and cost-effective; and suitable for different species. The development and validation of new techniques in the laboratory may provide the basis for a valuable field method. But, before a new biomarker’s application, some basic information is required, such as dosee response relationships, and biological and environmental factors, which can influence the baseline values of responses. It is important to mention that, when dealing with a biochemical or metabolic biomarker, species differences can be the biggest challenge for any toxicologist. Biomarkers have applications in all areas of toxicology, especially in the fields of pesticides, metals, mycotoxins, and drugs. In the case of veterinary toxicology, biomarkers of plant toxins deserve equal attention. Farmers, pesticide application workers, and greenhouse workers are exposed to pesticides by direct contact and their family members can be exposed via secondhand exposure. Measurement of residues of pesticides, and their metabolites and metals in urine, serves as the most accurate and reliable biomarkers of exposure in agriculture, industrial, and occupational safety and health settings. Recent evidence suggests that in utero or early life exposure to certain pesticides, metals, and other environmental contaminants may cause neurodegenerative (Alzheimer’s, Parkinson’s, schizophrenia, Huntington’s, ALS, and others) and cardiovascular diseases, diabetes, and cancer later in life. In these diseases and many others, specific and sensitive biomarkers play important roles in early diagnosis, and this can serve as the cornerstone for timely therapeutic intervention. Mycotoxin-related toxicity, carcinogenesis, and other health ailments are encountered in man and animals around the world. In developing countries, where regulatory guidelines are not strictly followed, adverse health effects (especially reproductive and developmental effects) are devastating. In these scenarios, early biomarkers of exposure play a pivotal role in avoiding further exposure to the contaminated food/feed and thus safeguard human and animal health. With the current knowledge of system biology, proteomics, metabonomics, toxicogenomics, and various mathematical and computational/chemometric modelings, undetectable biomarkers can be discovered and these biomarkers can predict how tissues respond to toxicants and drugs and/or their metabolites, and how the tissue damage and repair processes compromise the tissue’s function. Imaging and chemometric biomarkers are of greater sensitivity and carry more information than conventional biomarkers, as they detect (1) low

levels of chemical exposure (exposure biomarker) and (2) an early tissue response (endogenous response biomarker). The priority will always be for the development of a noninvasive approach over an invasive approach, and nondestructive biomarkers over destructive biomarkers, but this may not be possible in all cases. In 2011, the Joint SOT/EUROTOX Debate proposed that “biomarkers from blood and urine will replace traditional histopathological evaluation to determine adverse responses,” identifying and comparing the strengths and limitations of histopathology with serum and urine biomarkers. Unlike histopathological techniques, blood and urine biomarkers are noninvasive, quantifiable, and of translational value. Of course, the complete replacement of histopathological biomarkers with blood and urine may not be possible in the near future, as in some instances histopathological biomarkers will still be used because of recent developments in invaluable molecular pathology techniques. For the quest of developing the most sensitive and reliable biomarkers, integration of novel and existing biomarkers with a multidisciplinary approach appears fruitful. Furthermore, a multibiomarker approach seems more informative and accurate than a single biomarker approach. By now, microRNAs (miRNAs) have been well recognized as reliable and robust biomarkers for early detection of diseases, birth defects, pathological changes, cancer, and toxicities (Quiat and Olson, 2013; Wang et al., 2013; Bailey and Glaab, 2018). Because they are stable in biofluids, such as blood, there is rapidly growing interest in using miRNAs as diagnostic, prognostic, and predictive biomarkers, and the outlook for the clinical application of miRNA discoveries is promising, especially in molecular medicine. Soon, incorporating pharmacological and toxicological targeting of miRNAs into the development of innovative therapeutic strategies will be routine. Still, more innovative biomarkers need to be developed that will be highly sensitive (biotechnology-based techniques), require minimum quantities of sample, and will promise highthroughput screening. At the recent meetings of the Society of Toxicology, the EUROTOX, and International Congress of Toxicology, a large number of toxicologists emphasized the importance of biomarkers in health, disease, and toxicity. Accordingly, Biomarkers in Toxicology, second edition has been prepared to meet the challenges of today’s toxicologists, pharmacologists, environmentalists, and physicians in academia, industry, and government. This reference book is of particular interest to those in governmental agencies, such as NIH, USEPA, USFDA, USDA, NIOSH, OSHA, CDC, REACH, EFSA, etc. This is the most comprehensive biomarkers book to date as it covers every possible aspect of exposure, effects, and susceptibility to chemicals. There are many novel topics

I. TOXICITY TESTING MODELS AND BIOMARKERS

REFERENCES

in this volume that are not covered in any previous book. This edition identifies and establishes the most sensitive, accurate, unique, and validated biomarkers that can be used as indicators of exposure and effect(s) of chemicals, and chemical-related long-term diseases, such as cardiovascular, metabolic and neurodegenerative diseases, and cancer. Sixty-seven chapters are organized under eight sections with a user-friendly format, and each chapter is enriched with current literature and references for further reading. This book begins with general concepts of toxicity and safety testing and biomarker development using various animal and animal alternative models, adverse outcome pathways, followed by biomarkers of system/organ toxicity, chemicals, solvents, gases, and biotoxins. There are several chapters on biomarkers of pharmaceuticals, nutraceuticals, petroleum products, chemical mixtures, radiation, engineered nanomaterials, epigenetics, genotoxicity, and carcinogens. In the disease section, chapters cover the biomarkers of Alzheimer’s, Parkinson’s, neuropsychiatric disorders, osteoarthritis, and some other pathological conditions. Under special topics, chapters are included on mitochondrial dysfunction and toxicity, the bloodebrain barrier, oxidative/nitrosative stress, developmental neurotoxicity, miRNAs as indicators of tissue injury, and citrulline in diseases and toxicity. Lastly, a large number of chapters are dedicated to the application of biomarkers in toxicology, including the latest strategies and technologies in the development of biomarkers, biomarkers in drug development, safety evaluation, and toxicity testing and integration of biomarkers in biomonitoring of chemical exposure and risk assessment, especially in the context of industrial,

5

environmental, and occupational medicine and toxicology. The editor remains indebted to the contributors of this book for their hard work and dedication. These contributors are highly qualified and considered authorities in the fields of toxicology, pharmacology, pathology, biochemistry, and human and veterinary medicine. He expresses his gratitude to Ms. Denise Gupta and Ms. Robin B. Doss for their untiring support in technical assistance and text and reference checking. Finally, the editor would like to thank Ms. Kristi Anderson, Ms. Kattie Washington, Ms. Kathy Padilla, and Mr. Mohana Priyan Rajendran (the editorial staff at Academic Press/Elsevier) for their immense support at every stage of the production of this book.

References Bailey, W.J., Glaab, W.E., 2018. Accessible miRNA as novel toxicity biomarkers. Int. J. Toxicol. 37 (2), 116e120. Ganzleben, C., Antignac, J.-P., Barouki, R., et al., 2017. Human biomonitoring as a tool to support chemicals regulation in the European Union. Int. J. Hyg. Env. Health 220, 94e97. Gerlach, C.V., Derzi, M., Ramaiah, S.K., et al., 2018. Industry perspective on biomarker development and qualification. Clin. Pharmacol. Ther. 103 (1), 27e31. Hey, S.P., 2017. Ethical challenges in biomarker-driven drug development. Clin. Pharmacol. Ther. 103 (1), 23e25. Lavezzari, G., Womack, A.W., 2016. Industry perspective on biomarker qualification. Clin. Pharmacol. Ther. 99 (2), 208e213. Quiat, D., Olson, E.N., 2013. MicroRNA in cardiovascular disease: from pathogenesis to prevention and treatment. J. Clin. Invest. 123 (1), 11e18. Stern, A.D., Alexander, B.M., Chandra, A., 2018. Innovation incentives and biomarkers. Clin. Pharmacol. Ther. 103 (1), 34e36. Wang, K., Yuan, Y., Li, H., et al., 2013. The spectrum of circulating RNA: a window into systems toxicology. Toxicol. Sci. 132 (2), 478e492.

I. TOXICITY TESTING MODELS AND BIOMARKERS

C H A P T E R

2 Rodent Models for Toxicity Testing and Biomarkers Shayne C. Gad Principal of Gad Consulting Services, Raleigh, NC, United States

INTRODUCTION

Accordingly, we will proceed to understand the current uses of these three rodent species as predictive models for effects in humans, as well as how they are measured and what their normal ranges are. As these potential pieces of data are overviewed and considered, it is important to remember that each of these biomarkers is a part of the overall picture as to what the model is predicting as per potential adverse effects in humans. Meaningful safety assessment requires that all the data be incorporated in an integrated safety assessment. Because dose/toxicodynamic relationships will vary with level of exposure of test animals, it is also necessary that multiple (traditionally at least three) “dose” levels be evaluated. The picture becomes both more complex but also clearer as to relevance as new biomarkers are identified and became understandable. These include proteomics (Amacher, 2010), new clinical chemistry parameters, immune system responses, and real time functional physiologic system measurements by telemetrized instrumentation (Gad, 2013). Table 2.1 (Gad, 2013) presents the current most relevant associations of biomarkers with renal and liver toxicity, whereas Table 2.2 (adopted from Gad, 2013) presents an overview of the association between classical clinical chemistry parameters and specific target organ toxicities (Table 2.3). Table 2.4 summarizes causes associated with hematological findings in the rat. Over the last 10 years, diligent efforts under the rubric of the Critical Path Initiative have led to the identification of a more specific set of clinical chemistry biomarkers for key potential target organs.

Three rodent species are widely used in toxicology: the rat, the mouse, and the hamster. Two of these, the rat and mouse, are the most widely used in experimental biology and medicine. These have formed the basis for exploring the efficacy of drugs and for the identification and evaluation of toxicities associated with exposure to drugs, industrial and agricultural chemicals, and understanding the mechanisms of their toxicity since toxicology became an identified discipline. A large (and growing) set of biomarkers are known for use in identifying and determining the relative (and relevant) risks to humans or other target species. These include: • • • • • • • •

Body weights Clinical pathology (hematology) Clinical chemistry Organ weights Gross histologic changes at necropsy Immunogenicity Microscopic evaluation of tissues Changes in physiologic functions and electrophysiology • Effects on specific genomic markers Biomarkers are measurements of test model (animal) parameters that can provide important quantitative data about the biological state of the test model, which are predictive of effects in humans. These biomarkers in toxicology are preferably shared by both test animals and humans and in a manner that the relationship of findings in one species to another is known.

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2. RODENT MODELS FOR TOXICITY TESTING AND BIOMARKERS

Heart

Troponins

Zethelius et al. (2008)

Kidney

KIM-1, Albumin, Beta-2Microglobulin

Hoffmann et al. (2010), Ozer et al. (2010), and Vaidya et al. (2010)

Liver

DILI (Drug Induced Liver Injury)l ALT, BUN, coagulation factor

Shi et al. (2010)

Other recently identified biomarkers for specific targets include: 1. Mitochondrial Dysfunction. Increased uptake of calcium (because ATP depletion) by mitochondria activates phospholipases, resulting in accumulation of free fatty acids. These cause changes in the permeability of mitochondrial membranes, such as the mitochondrial permeability transition. 2. Progressive Loss of Phospholipids. Increased degradation by endogenous phospholipases and inability of the cell to keep up with synthesis of new phospholipids (reacylation, an ATP-dependent process). 3. Cytoskeletal Abnormalities. Activated proteases lyse cytoskeletal elements and cell swelling causes detachment of cell membrane from cytoskeleton; stretching of the cell membrane results in increased membrane damage. TABLE 2.1

Classic Associations in Toxicology

Liver Toxicity

Renal Toxicity

Increased plasma activity of liver marker enzymes, e.g., alanine and aspartate aminotransferases

Increased water consumption and urine volume. Urine parameters may change, e.g., enzymes and cellular debris.

Decreased plasma total protein concentration

Increased plasma concentrations of urea and creatinine. Proteinuria.

Increased coagulation times due to decreased synthesis of coagulation factors

Severe renal toxicity may lead to decreased erythrocyte parameters due to effects on erythrocyte synthesis

Increased liver weight due to enzyme induction or accumulation of lipid or glycogen

Increased kidney weight

Change in color or size at necropsy

Change in color or size at necropsy

Histological findings such as necrosis or centrilobular hypertrophy due to enzyme induction

Histological change, e.g., basophilic tubules or necrosis, papillary necrosis, or glomerular changes.

4. Reactive Oxygen Species. Produced within the cell by infiltrating neutrophils and macrophages, especially after restoration of blood flow to an area (reperfusion injury). Cell injury triggers release of a number of inflammatory cytokines and chemokines that amplify the host immune response and attract neutrophils to the site. 5. Lipid Breakdown Products. Unesterified free fatty acids, acyl carnitine, and lysophospholipids. These have a detergent effect on membranes and may exchange with membrane phospholipids, causing permeability changes.

THE RAT Use in Toxicological Research Ideally, safety testing of products intended for use in humans, or to which humans could be exposed, should be done in humans. The data from humans would apply without reservation to complex human physiology and cellular/biochemical mechanisms and human risk assessment. Unfortunately, humans cannot be used for this purpose. Therefore, the choice of an appropriate species for toxicology studies should be based on a comparison of the pharmacokinetics, target pharmacodynamics, and metabolism of the test compound in different laboratory species and man. In the absence of this data, this choice is often based on practicality and economics. The rat has become a species of choice because of the metabolic similarities, as well as their small size, relatively docile nature, short life span, and short gestation period. The extensive use of the rat in research has led to the development of a large historical database of their nutrition, diseases, and general biology. Characteristics Although the rat is a species of choice in toxicology research because of the many physiological similarities and anatomical characteristics, differences exist that must be considered when designing and conducting studies with this animal. Rats are obligate nose breathers; as such an inhaled test material is subject to nasal filtration and absorption. The placenta is considerably more porous in the rat. This difference may increase the chance of fetal exposure to an administered test material or increase the overall level of fetal exposure to an administered test material. The overall distribution of intestinal microflora is different in the rat, which may lead to differences in the metabolism of an orally administered test material. These and other differences in the rat may lead to positive signs of toxicity to a test material that may not be present in a different species. There are

I. TOXICITY TESTING MODELS AND BIOMARKERS

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THE RAT

TABLE 2.2 Association of Changes in Biochemical Parameters With Actions at Particular Target Organs (Gad, 2013) Parameter

Blood

Heart

Lung

Albumin

Kidney

Liver

Y

Y

ALP

[

ALT (formerly SGPT)

[ [

AST (formerly SGOT)

[

[

BUN

[

Calcium

[

Cholinesterase

[

CPK

Produced by the liver; very significant reductions indicate extensive liver damage [

[

Elevations usually associated with cholestasis; bone alkaline phosphatase tends to be higher in young animals Elevations usually associated with hepatic damage or disease [

Present in skeletal muscle and heart and most commonly associated with damage to these

Usually elevated due to cholestasis, due to either obstruction or hepatopathy

Y

Estimates blood filtering capacity of the kidneys; does not become significantly elevated until the kidney function is reduced 60%e75% Can be life threatening and result in acute death

Y

Found in plasma, brain, and RBC Most often elevated due to skeletal muscle damage but can also be produced by cardiac muscle damage; can be more sensitive than histopathology

[

Also estimates blood filtering capacity of kidney as BUN does [

Alterations other than those associated with stress uncommon and reflect an effect on the pancreatic islets or anorexia

[

Elevated in cholestasis; this is a microsomal enzyme, and levels often increase in response to microsomal enzyme induction

[

e

[

[

Increase usually due to skeletal muscle, cardiac muscle, or liver damage; not very specific

Y

Y

Absolute alterations usually associated with decreased production (liver) or increased loss (kidney); can see increase in case of muscle wasting (catabolism)

[Y

Liver enzyme that can be quite sensitive but is fairly unstable; samples should be processed as soon as possible

GGT

[ [

KIM-1 [

Protein (total)

SDH

Trophonin

Notes

[

Glucose

LDH

Pancreas

[

Creatinine

HBDH

Intestine

[

Beta-2-Microglobulin Bilirubin (total)

[

Bone

[

[

ALP, alkaline phosphatase; BUN, blood urea nitrogen; CPK, creatinine phosphokinase; GGT, gamma glutamyl transferase; HBDH, hydroxybutyric dehydrogenase; LDH, lactic dehydrogenase; RBCs, red blood cells; SDH, sorbitol dehydrogenase; SGOT, serum glutamic oxaloacetic transaminase (also called AST [aspartate amino transferase]); SGPT, serum glutamicpyruvic transaminase (also called ALT [alanine amino transferase]); [, increase in chemistry values; Y, decrease in chemistry values.

I. TOXICITY TESTING MODELS AND BIOMARKERS

10 TABLE 2.3

2. RODENT MODELS FOR TOXICITY TESTING AND BIOMARKERS

Liver Enzymes

“Liver Enzyme”

Nomenclature

Plasma-Tissue Sources

Cellular Location

AST (SGOT)

Aspartate Aminotransferase

Liver, Heart, Skeletal Muscle, Kidney, Brain, RBCs

Mitochondria, Cytoplasm

ALT (SGPT)

Alanine Aminotransferase

Mostly liver, Heart, Skeletal Muscle

Cytoplasm

Alk Phos (AP)

Alkaline Phosphatase

Bile ducts, GI tract, Bone, Placenta

Membranes

GGT

Gamma-glutamyl transferase

Liver, Kidney, Heart

Membranes

GDH

Glutamate Dehydrogenase

Liver, Kidney Skeletal Muscle

Mitochondria

SDH

Sorbitol Dehydrogenase

Mostly liver

Cytoplasm

LDH

Lactate Dehydrogenase

Heart, Skeletal Muscle, RBCs, Lung, Liver, All tissues

Cytoplasm

RBC, red blood cell.

TABLE 2.4

Some Probable Conditions Affecting Hematological Changes (Gad, 2013)

Parameter

Elevation

Depression

Parameter

Red blood cells (RBCs)

1. 2. 3. 4.

1. Anemias a. Blood Loss b. Hemolysis c. Low RBC production

Platelets

Hematocrit

1. Increased RBC 2. Stress 3. Shock a. Trauma b. Surgery 4. Polycythemia

1. Anemias 2. Pregnancy 3. Excessive hydration

Neutrophils

1. Acute bacterial infections 2. Tissue necrosis 3. Strenuous exercise 4. Convulsions 5. Tachycardia 6. Acute hemorrhage

Hemoglobin

1. Polycythemia (increased in 1. Anemias production of RBC) 2. Lead Poisonings

Lymphocytes

1. Leukemia 2. Malnutrition 3. Viral infections

Mean cell volume

1. Anemias 2. B-12 deficiency

1. Iron deficiency

Monocytes

1. Protozoal infections

Mean corpuscular hemoglobin

1. Reticulocytosis

1. Iron deficiency

Eosinophils

1. 2. 3. 4.

White blood cells

1. Bacterial infections 2. Bone marrow stimulation

1. 2. 3. 4.

Basophils

1. Lead poisoning

Vascular shock Excessive diuresis Chronic hypoxia Hyperadreno corticism

Bone marrow depression Cancer chemotherapy Chemical intoxication Splenic disorders

also differences (though generally less striking) between different strains of rats and sometimes even between the animals supplied by difference sources. Strain Differences Breeding rats for specific characteristics has produced some physiological differences between strains of rats. Some of these differences are known to affect how the various strains react to toxicants. Among others, strain

Elevation

Depression 1. Bone marrow depression 2. Immune disorder

Allergy Irradiation Pernicious anemia Parasitism

specific differences have been found in sensitivity to thiourea (Dieke and Richter, 1945), sensitivity to acetaminophen nephrotoxicity (Newton et al., 1985a,b), the incidence of spontaneous glomerular sclerosis (Bolton et al., 1976), sensitivity to the carcinogenic actions of 7,12-dimethylbenz(a)anthracene (Boyland and Sydnor, 1962), the effects of trimethyltin on operant behavior and hippocampal glial fibrillary acidic protein (GFAP) (MacPhail et al., 2003), differences in renal

I. TOXICITY TESTING MODELS AND BIOMARKERS

11

STUDY DESIGNS

carcinogenesis (Hino et al., 2003), differences in cytochrome P4501A1 gene expression caused by 2,3,7,8tetrachlorodibenzo-p-dioxin in the liver (Jana et al., 1998), susceptibility to 4-nitroquinoline 1-oxide induce carcinoma (Kitano et al., 1992), and differences in the levels of drug-metabolizing enzymes (Page and Vesell, 1969). In recent years, research and breeding programs have been focused on producing inbred and outbred strains focused on specific disease models and susceptibility to the development of certain carcinomas. When choosing a strain for use, it is important to consider these differences. Of importance for carcinogenicity studies, strain differences have been found in the incidence of spontaneous tumors. Table 2.5 gives the incidence of spontaneous tumors found in commonly used strains in carcinogenicity studies. The historical incidence is important to the analysis of a study in that a high spontaneous rate may mask a small test materialerelated increase in tumor incidence. Because of lower spontaneous tumor rates, the Wistar has become the most popular strain in toxicological research. Normal Physiological Values General values for selected physiological parameters are given in Tables 2.6 and 2.7. Normal values will vary based on the strain of animal, supplier, feed, and housing conditions. These tables should be used as a point of reference only.

STUDY DESIGNS The length and design of toxicology studies used to predict human risk are governed by guidelines issued by regulatory bodies such as the US Food and Drug

Administration (FDA), the International Conference on Harmonization (ICH), the Environmental Protection Agency (EPA), and their counterparts worldwide. Toxicology studies are divided into a series of three sets of studies that are required for each phase of clinical trials. For initial approval to begin clinical trials, the following studies are required. The length of dosing in the toxicology studies varies depending on the intended length in clinical trials. A test compound intended to be a repeat dose study for up to 28 days in duration initially requires a two phase study in which a maximum tolerated dose (MTD) following a single administration is determined followed by a second phase during which the test compound is administered daily at dose levels based on the MTD for 5e7 days (Table 2.8). Following the completion of the MTD study a 14 or 28 Day Repeat Dose study should be conducted (Table 2.9). These studies assess the effects of a test compound at dosages that do not cause immediate toxic effects. In support of Phase 2 clinical trials, longer-term subchronic and chronic toxicity studies (Table 2.10) should be conducted. Subchronic and chronic toxicity studies are designed to assess the test compound effects following prolonged periods of exposure. The highest dosage level in each of these studies should produce a toxic effect such that target organs may be identified. The lowest dosage level should provide a margin of safety that exceeds the human clinical dose and ideally allows for the definition of no observable effect level. Alternatively, when effects related to the pharmacological mechanism of the test compound or when observed effects may be related to treatment with the test compound but may not be of toxicologic significance, a no observable adverse effect level (NOAEL) may be determined. In addition to the subchronic and chronic toxicity studies in support of Phase 2 clinical trials, reproductive

TABLE 2.5 Incidence of Common Spontaneous Tumors in Fischer 344 and CD (SD)IGS Rats % Tumors in Untreated Rats CD(SD)IGS

CD (SD)

Fisher

Wistar

Organ

Tumor Type

Male

Female

Male

Female

Male

Female

Adrenal gland

Pheochromocytoma

10.0

2.3

11.3

2.3

11.9

3.2

3.2

1.3

Mammary gland

Fibroadenoma

1.4

44.5

1.3

16.7

0.8

7.1

1.2

30.2

Pancreas

Islet cell adenoma

3.6

1.4

4.0

0.3

1.5

0.2

5.3

1.9

Pituitary gland

Adenoma pars distalis

33.6

56.8

35.7

50.3

12.4

28.2

41.1

65.8

Testis

Interstitial cell tumor

Thyroid gland

C-Cell Adenoma

1.8 10.5

7.0 5.0

5.0

74.6 5.7

12.5

Male

Female

4.3 8.2

10.1

10.7

Adapted from Charles, R., 2011. Spontaneous Neoplastic Lesions in the Crl:CD BR Rat. Charles River Laboratories, Inc., Massachusetts; Mitsumori, K., Watanabe, T., Kashida, Y., 2001. Variability in the Incidence of Spontaneous Tumors in CD (SD) IGS, CD (SD), F244 and Wistar Hannover Rats in Biological Reference Data on CD(SD) IGS Rats, Yokohama, CD(SD) IGS Study Group.

I. TOXICITY TESTING MODELS AND BIOMARKERS

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2. RODENT MODELS FOR TOXICITY TESTING AND BIOMARKERS

TABLE 2.6

TABLE 2.6 Selected Normative Datadcont’d

Selected Normative Data

HUSBANDRY

Compliance (mL/cm H2O)

0.3e0.9

Room temperature (
C)

18e26

Resistance (cm H2O/mL s)

0.1e0.55

Relative humidity (%)

30e70

Pattern

Obligate nasal

Ventilation (air change/h)

10

Light/dark cycle (h)

12e14/12e10

RENAL Urine volume þ

Na excretion

Minimum cage floor size

þ

15e30 mL/24 h 200 mmol/L/24 h

Housed individually (cm2)

350

K excretion

150 mmol/L/24 h

Breeding with pup (cm2)

800

Urine osmolarity

2000 mOsm/kg H2O

Group housed (cm2 adult)

250

Urine pH

7.3e8.5

Urine specific gravity

1.01e1.07

GENERAL Life span (years)

2.5e3.0

Urine creatinine

6 mmol/L/24 h

Surface area (cm2)

0.03e0.06

Glomerular filtration rate

1.0 mL/min/100 g body weight

Chromosome number (diploid)

42

Water consumption (mL/ 100 g/day)

10e12

Food consumption (g/day) Average body temperature

a

20e40 (
C)

37.5

REPRODUCTION Puberty (males and females)

50  10 days

Breeding season

All year

Type of estrous cycle

Polyestrous

Length of estrous cycle

4e5 days

Duration of estrous

10e20 h

Mechanism of ovulation

Spontaneous

Time of ovulation

7e10 h after onset of estrous

Time of implantation

Late day 4 or 5a

Length of gestation

21e23 days

Litter size

8e16 pups

Birth weight

5e6 g

Eyes open

10e12 days

Weaning age/weight

21 days/40e50 g

CARDIOVASCULAR Arterial blood pressure Systolic (mmHg)

116e145

Diastolic (mmHg)

76e97

Heart rate (beats/min)

296e388

Cardiac output (mL/min)

10e80

Blood volume (mL/kg)

64

PULMONARY Respiration (breaths/min)

100e140

Tidal volume (mL)

1.1e2.5

The estrous cycle length may vary from 4 to 5 days between strains. Time of implantation may vary based upon the length of the estrous cycle and is dependent upon Day 0 or the first day sperm is found in the vagina. Data from Baker, H.J., Lindsey, J.R., Weisbroth, S.H., 1979. Housing to control research variables. In: Baker, H.J., Lindsey, J.R., Weisbroth, S.H. (Eds.), The Laboratory Rat, vol. 1. Academic Press, New York, pp. 169e192; Hofsteller, J., Svekow, M.A., Hickman, D.L., 2006. Morphophysiology. In: Svekow, M.A., Weisbroth, S.H., Franlin, C.L. (Eds.), The Laboratory Rat, second ed. vol. I. Academic Press, New York, pp. 93e125; Peplow, A., Peplow, P., Hafez, E., 1974. Parameters of reproduction. In: Vo, I., Melby, E., Altmon, N. (Eds.), Handbook of Laboratory Animal Science. CRC Press, Boca Raton, pp. 107e116; Waynforth, H., Flecknell, P., 1980. Experimental and Surgical Technique in the Rat, second ed. Elsevier Academic Press; Sharp, P., LaRegina, M., 1998. The Laboratory Rat. Academic Press, Philadelphia; Van Zutphen, L.F.M., Baumans, V., Beynen, A.C., 1993. Principles of Laboratory Animal Science. Elsevier, Amsterdam.

safety studies may also be required. Reproductive toxicity studies are typically required for a test compound intended to be administered to women of childbearing age or may affect male reproduction. These studies include an assessment of the potential effects of the test compound on general fertility and reproductive performance (Segment I), developmental toxicity (Segment II), or affect perinatal and postnatal development (Segment III). The highest dose in reproductive studies should be chosen so that administration causes some minimal toxicity. Typically, a dose range finding pilot study in a small number of animals should be conducted prior to initiating the definitive reproductive toxicology studies. Examples of protocols designed to meet the ICH guidelines are presented in Tables 2.9, 2.9A, and 2.10. In support of Phase 3 clinical trials, two carcinogenicity studies may be required (Table 2.8), one in rats and one in mice. Typically 18 months to 2 years in duration, this type of study is designed to assess the potential of the test compound to induce neoplastic lesions. The highest dosage in a carcinogenicity study should cause minimal toxicity when administered via the intended route for clinical use. The preclinical studies required

I. TOXICITY TESTING MODELS AND BIOMARKERS

13

ROUTES OF TEST ARTICLE ADMINISTRATION

TABLE 2.7

Growth Rates in Selected Rat Strains Age (days) Crl:CD (SD)IGSBR

Crl:(WI)BR

Crl:(LE)BR

CDF(F-344)/CrlBR

Weight (g)

M

F

M

F

M

F

M

F

Up to 50

Up to 23

Up to 23

Up to 23

Up to 25

Up to 21

Up to 21

Up to 23

Up to 23

51e75

24e28

24e29

24e28

26e30

22e25

22e26

24e29

24e29

76e100

29e34

30e35

29e32

31e34

26e29

27e31

30e34

30e35

101e125

35e37

36e39

33e35

35e40

30e34

32e36

35e39

36e42

126e150

38e42

40e44

36e40

41e47

35e37

37e43

40e45

43e55

151e175

43e45

45e50

41e44

48e56

38e42

44e50

46e50

56e72

176e200

46e49

51e56

45e48

57e64

43e46

51e55

51e57

73e105

201e225

50e52

57e70

49e52

65e81

47e49

56e69

58e63

105þ

226e250

53e56

71e84

53e56

82e105

50e55

70e86

64þ

251e275

57e59

84e105

57e61

106þ

56e58

87e102

276e300

60e65

106þ

62e67

59e64

103þ

301e325

66e71

68e73

65e70

326e350

72e77

74e79

71e80

351e375

78e87

80e87

81e90

376þ

88þ

88þ

91þ

Adapted from Charles, R., 2004. Growth Rates in Selected Rat Strains. Charles River Laboratories, Inc., Massachusetts.

in support of the clinical trials are dependent on the intended route and frequency of administration of the test compound and the intended age group to be treated (Tables 2.11e2.14).

ROUTES OF TEST ARTICLE ADMINISTRATION Oral Routes Rodents have several unique characteristics to be considered regarding the oral administration of test compounds. One of the most important characteristics is the lack of an emetic response. The lack of this response allows for a higher dose of a potential emetic compound to be administered and evaluated. Many compound and excipients may cause emesis in dogs or other large animal species and may lead to a low level of exposure and erratic blood levels. A second factor to consider is that rodents are nocturnal and eat most of their food at night. When maintained on a 12 h lighte dark cycle, rats have been found to consume 75% of their daily food intake during the dark cycle (Wong and Oace, 1981). This should be taken into consideration when designing an oral gavage study and determining when the animal may be dosed. Early in the light cycle,

animals are more likely to have a full stomach and complications associated with dosing may occur if large volumes of test article are administered. In addition, a full stomach may affect gastric emptying and the rate of absorption of an orally administered test compound. Techniques for oral administration of test compounds include mixing in the diet, via gavage or stomach tube, via capsule, or in drinking water. The most widely used methods of oral administration are the dietary and gavage techniques. Dietary Versus Gavage Methods The choice between dietary and gavage dosing techniques is typically based on several factors. A scientific decision can only be made with a knowledge of the pharmacokinetics of the test compound administered by both methods. Other considerations that may be used in making this decision are as follows. The dietary method can be used if a compound can be mixed with the diet, is stable under storage conditions in the diet, and is palatable to the animal. A major advantage of the dietary method is that it requires less manpower to perform the study. The diet mixing process can be performed weekly or, if stability allows, less often. The mixing and feeding process is less labor-intensive than gavaging rats on a daily basis.

I. TOXICITY TESTING MODELS AND BIOMARKERS

14 TABLE 2.8

2. RODENT MODELS FOR TOXICITY TESTING AND BIOMARKERS

Maximum Tolerated Dose Study in Rats

Phase A

TABLE 2.9 14 or 28 Day Repeat Dose Toxicity Study in Rats

Oral MTD Study

Dose Level 1

Main Study*

Males

Females

3

3

Vehicle control

Toxicokinetics

Males

Females

Males

Females

10

10

e

e

Dose Level 2

3

3

Low dose

10

10

9þ3

9 þ 3a

Dose Level 3

3

3

Mid dose

10

10

9 þ 3a

9 þ 3a

Dose Level 4

3

3

High dose

10

10

9 þ 3a

9 þ 3a

a

Observations: Twice daily (mortality/moribundity).

Phase B

7-Day Oral Range Finding Study Main Study

Toxicokinetics

Males

Females

Males

Females

Control

5

5

e

e

Low dose

5

5

9

9

Mid dose

5

5

9

9

High dose

5

5

9

9

Experimental Design:

In Phase A, the dose level will be increased until the maximum tolerated dose (MTD) is determined. The MTD is a dose that produces neither mortality nor more than a 10% decrement in body weight nor clinical signs of toxicity. In Phase B, animals will be dosed daily for 7 days at fractions of the single dose MTD to estimate a repeat dose MTD. Dose Route/Frequency: As requested. Phase A: Once. Phase B: Once per day for 7 consecutive days. Observations: Twice daily in both phases (mortality/moribundity). Detailed Clinical Observations: Daily in both phases. Body Weights: Daily in both phases. Food Consumption: Daily. Clinical Pathology (Phase B only): Hematology, clinical chemistry, and urinalysis evaluations on all surviving main study animals at termination. Necropsy (Phase B only): Tissues saved for possible future histopathological evaluation. Organ Weights (Phase B only): Adrenals, brain, heart, kidneys, liver, lungs, ovaries with oviducts, pituitary, prostate, salivary glands, seminal vesicles, spleen, thyroid with parathyroid, thymus, testes, uterus. Toxicokinetics: Blood collected on days 1 and 7 (three cohorts consisting of three animals/sex/treatment group bled twice to equal six time points), calculation of Cmax, Tmax, AUC0-24, and T1/2.

Several disadvantages also exist in using the dietary method. Methods must be developed and validated to prove homogeneity and stability. This is not as easy a process as with a suspension or solution. The dietary method is also less exact than the gavage method, in that the concentration of compound mixed in the feed is based on predicted feed consumption and body weights. In addition, if the feed is not palatable to the animal, or the test compound makes the animal ill, feed consumption may be reduced thereby reducing exposure to the test compound. In addition, the facility and control animals may be exposed to the test compound through dust or vapors.

Detailed Clinical Observation: Weekly. Functional Observational Battery: Pretest and Day 14 or 25. Body Weights: Weekly. Food Consumption: Weekly. Ophthalmology: All animals prior to test article administration; all surviving main study animals at study termination. Clinical Pathology: Hematology, clinical chemistry, and urinalysis evaluations on all surviving main study animals at termination. Toxicokinetics: Blood collected on Days 1 and 14 or 27 (three cohorts consisting of three animals/sex/treatment group bled twice to equal six time points); TK modeling. The use of subsets of all the animals in a test group is called “spare sampling,” intended to avoid the need for additional (“Satellite”) groups of animals. Note that although six time points are commonly collected, more may be required or taken to adequately characterize a drug’s pharmacokinetics. Necropsy: All main study animals; toxicokinetic animals euthanized and discarded. Organ Weights: Adrenals, brain, heart, kidneys, liver, lungs, ovaries with oviducts, pituitary, prostate, salivary glands, seminal vesicles, spleen, thyroid with parathyroid, thymus, testes, uterus. Slide Preparation/Microscopic Pathology: All animals in the vehicle control and high dose groups and all found dead animals: full set of standard tissues; low and mid dose group target organs (to be determined); gross lesions from all animals. a

Three additional animals/sex/treatment group included as replacement animals. * Should also refer to table note “a”.

The gavage method may be used when the test compound is not stable in the diet or may not be palatable to the animals. In addition, the gavage method is preferable when evaluating toxicokinetics or pharmacokinetics. As with dietary mixtures, test compound administered via gavage as a solution or suspension should be analyzed for homogeneity, stability, and concentration. Methods for solution or suspension may be easier to develop than those required for dietary mixtures. For Good Laboratory Practices (GLP) studies, evaluation of homogeneity, stability, and concentration should be conducted for every study. If the same methodology and batch size are used for multiple studies, homogeneity may be established once. Stability of the test compound in solution or suspension should be determined under the testing conditions in the proposed vehicle. Typically, stability for toxicology studies is established for between 7 and 14 days. If the test compound is not found to be stable, stability of shorter duration may be established. Lastly, concentration analysis should be established for each dose level and should be periodically evaluated during longer-term studies. With the gavage method of dosing, a more precise amount of the test compound can be delivered and

I. TOXICITY TESTING MODELS AND BIOMARKERS

15

ROUTES OF TEST ARTICLE ADMINISTRATION

TABLE 2.9A

28 Day Repeat Dose Toxicity Study With Immunophenotyping in Rats Main Study

Vehicle control Low dose Mid dose High dose

Males

Females

10

10

10 10 10

TABLE 2.10

Main Study

Toxicokinetics Males

Females

10

9þ3

9þ3

10

9þ3

9þ3

10

9þ3

9þ3

a a a

Subchronic and Chronic Toxicity Study in Rats

Vehicle control

Toxicokinetics

Males

Females

Males

Females

15

15

e

e a

9þ3a

Low dose

15

15

9þ3

Mid dose

15

15

9þ3a

9þ3a

High dose

15

15

9þ3a

9þ3a

a a a

Observations: Twice daily (mortality/moribundity).

Detailed Clinical Observation: Weekly. Functional Observational Battery: Pretest and Day 25. Body Weights: Weekly. Food consumption: Weekly. Ophthalmology: All animals prior to test article administration; all surviving main study animals at study termination. Clinical Pathology: Hematology, clinical chemistry, and urinalysis evaluations on all surviving main study animals at termination. Immunotoxicology: Immunophenotyping of blood leukocytes by flow cytometry on all surviving main study animals at termination. NK cell assay on blood leukocytes of all surviving main study animals at termination. May include identification of any antidrug antibodies (ADAs). Toxicokinetics: Blood collected on Days 1 and 27 (three cohorts consisting of three animals/sex/treatment group bled twice to equal six time points). Necropsy: All main study animals; toxicokinetic animals euthanized and discarded. Organ Weights: Adrenals, brain, heart, kidneys, liver, lungs, ovaries with oviducts, pituitary, prostate, salivary glands, seminal vesicles, spleen, thyroid with parathyroid, thymus, testes, uterus, two lymph nodes (e.g., mesenteric, axillary, popliteal, etc.) including the lymph node draining the route of administration. Slide Preparation/Microscopic Pathology: All animals in the vehicle control and high dose groups and all found dead animals: full set of standard tissues (add Peyer’s patch, extra lymph node); low and mid dose group target organs; gross lesions from all animals.

Observations: Twice daily (mortality/moribundity).

Detailed Clinical Observation: Weekly. Body Weights: Weekly. Food Consumption: Weekly. Ophthalmology: All animals prior to test article administration; all surviving main study animals at study termination. Clinical Pathology: Hematology, clinical chemistry, and urinalysis evaluations on all surviving main study animals at termination. Toxicokinetics: Blood collected on Days 1 and 90 (three cohorts consisting of three animals/sex/treatment group bled twice to equal six time points); TK modeling. Necropsy: All main study animals; toxicokinetic animals euthanized and discarded. Organ Weights: Adrenals, brain, heart, kidneys, liver, lungs, ovaries with oviducts, pituitary, prostate, salivary glands, seminal vesicles, spleen, thyroid with parathyroid, thymus, testes, uterus. Slide Preparation/Microscopic Pathology: All animals in the vehicle control and high dose groups and all found dead animals: full set of standard tissues; low and mid dose group target organs; gross lesions from all animals. a

Three additional animals/sex/treatment group included as replacement animals.

Food consumption and body weight predictions are based on historical laboratory data for early time points in a study. As the study progresses, growth and food consumption curves can be established for each group

a

Three additional animals/sex/treatment groups included as replacement animals; the control animals will not be evaluated for toxicokinetics.

may reduce the amount of test compound required to complete the study. This becomes important when evaluating the effects of a pharmaceutical, as the required dose levels and exposure levels to show safety may be lower than that required for a pesticide or chemical. A disadvantage of the gavage method is that it involves handling of the rat for each dosing. Handling of the rat has been shown to increase corticosterone levels (Barrett and Stockham, 1963) and may affect study results. Additionally, daily intubation may lead to death due to esophageal puncture or inhalation pneumonia. Dietary Method When utilizing the dietary method, the test compound is mixed with the diet and administered to the animals either ad libitum or the diet is presented to the animals for a fixed amount of time each day. The dosage received by an animal is regulated by varying the concentration of test compound in the diet based on the predicted food consumption and body weight.

TABLE 2.11 Study of Fertility and Early Embryonic Development to Implantation in Rats Males

Females

Vehicle control

25

25

Low dose

25

25

Mid dose

25

25

High dose

25

25

Dose Route/Frequency: Males dosed began 28 days before mating and continued until euthanasia. Females dosed began 14 days before mating and continued through Day 7 of gestation (implantation).

Observations: Twice daily (mortality/moribundity). Clinical Examinations: Observations for clinical signs, body weights, and food consumption measurements recorded during the study period. Beginning at initiation of test article administration, females examined daily to establish estrous cycle. Uterine Examinations: Performed on dams on Day 13 of gestation. Gravid uterine weight and the weight of the ovaries recorded. Total number of corpora lutea and implantations, location of resorptions, and embryos recorded. Females subjected to necropsy, and reproductive organs and gross lesions fixed for possible microscopic evaluation. Evaluation of Males: Following disposition of females, the males were euthanized and subjected to a necropsy. The testes and epididymides weighed, and analysis of sperm parameters (concentration, motility, and morphology) performed. Reproductive organs and gross lesions fixed for possible microscopic evaluation. Statistical Analysis: Standard.

I. TOXICITY TESTING MODELS AND BIOMARKERS

16 TABLE 2.12

2. RODENT MODELS FOR TOXICITY TESTING AND BIOMARKERS

Embryo-Fetal Development in Rats

TABLE 2.14 Carcinogenicity Study in Rats

Time Mated Females

Main Study

6-Month Satellite

Males

Females

Males

Females

Vehicle control

60

60

20

20

25

Low dose

60

60

20

20

25

Mid dose

60

60

20

20

Dose Route/Frequency: Dosing initiated on Day 6 of gestation and continued to include Day 17 of gestation.

High dose

60

60

20

20

Observations: Twice daily (mortality/moribundity). Clinical Examinations: Daily Gestation Days 6 through 20. Body weights/Food Consumption: Gestation Days 0, 6, 9, 12, 15, 18, and 20. Cesarean Section/Necropsy: Litters will be delivered by cesarean section on Day 20 of gestation. Gravid uterine weight will be recorded. Total number of corpora lutea, implantations, early and late resorptions, live and dead fetuses, and sex and individual body weights of fetuses will be recorded. External abnormalities of fetuses will be recorded. Approximately one-half of the fetuses will be processed for visceral abnormalities, and the remaining fetuses will be processed for skeletal abnormalities. Dams will be subjected to a necropsy and gross lesions and target organs (if known) will be saved.

Study Desgin: Group as per Table 2.14.

Vehicle control

25

Low dose

25

Mid dose High dose

and group mean data can be used to predict future food consumption. Different concentrations of the test compound and diet should be made for each sex. Test compounds and diets are mixed in two steps: (1) the compound and about 10% of the total amount of diet are blended in a premix, then (2) the premix and the remainders of the diet are mixed. The total amount of diet TABLE 2.13

Observations: Twice daily (mortality/moribundity). Detailed Clinical Observations: Once weekly. Body Weights: Weekly for first 13 weeks, monthly thereafter. Food Consumption: Weekly for first 13 weeks, monthly thereafter. Ophthalmology: All animals pretest and all survivors prior to terminal sacrifice. Clinical Pathology: Main Study: Hematology at termination. 6-Month Satellite: Hematology, clinical chemistry, and urinalysis evaluations on all surviving satellite animals at termination. Necropsy: All animals. Slide Preparation/Microscopic Pathology: All animals, full set of standard tissues, all masses, and all lesions. Statistical Analysis: Standard.

to be mixed is first weighed out, the 10% is separated into the premix. To make the premix, the entire test compound and an aliquot of the diet (from the 10%) are put into a mortar. These ingredients are ground with a pestle

Pre- and Postnatal Development, Including Maternal Function in Rats P Generation (F0)

F1 Generation

Males

Females

Males

Females

Vehicle control

NA

25

25

25

Low dose

NA

25

25

25

Mid dose

NA

25

25

25

High dose

NA

25

25

25

Number in Study: P Generationd100 females, F1 Generationd100 males, 100 females.

Dose Route/frequency: Once daily to P animals from Gestation Day (GD) 6 to Postnatal Day (PND) 21. F1 animals not dosed. Observations: Twice daily (mortality/moribundity). Clinical Observations: P femalesddaily during treatment/F1 adultsdweekly. Body Weights: P femalesdGD 0, 6, 10, 14, 17, and 20, PND 0, 7, 10, 14, and 21. F1 malesdWeekly through termination. F1 femalesdWeekly until evidence of copulation detected, then GD 0, 7, 10, and 13. Food Consumption: P femalesdOn corresponding body weight days during gestation/lactation. Vaginal Smears: All F1 females during a 21-day cohabitation period until evidence of copulation is detected. Litter Evaluations: All F1 offspring, count, body weight, sex, clinical observations on PND 0, 4, 7, 14, 21; behavioral and developmental evaluation of four males and four females from each litter for static righting, pinna detachment, cliff aversion, eye opening, air drop righting reflex, neuropharmacological evaluation, auditory response. One male and one female (selected for the next generation) tested for sexual maturation (vaginal opening, preputial separation), motor activity/emotionality, and passive avoidance. Cesarean Section: On GD 13, F1 females for location of viable and nonviable embryos, early and late resorptions, number of total implantations, and corpora lutea. Sperm Evaluation: May be conducted on F1 males if evidence of reduced fertility is noted (additional cost). Necropsy: Gross lesions/target organs fixed for possible microscopic evaluation (additional cost). All P females at PND 22 as well as all F1 weanlings not selected for F1 generation. All F1 females at GD 13. All F1 males after termination of F1 cesarean sections.

I. TOXICITY TESTING MODELS AND BIOMARKERS

ROUTES OF TEST ARTICLE ADMINISTRATION

until the mixture appears homogeneous. The mixture and the remainder of the premix are then layered in a small capacity mixer and mixed for 5e10 min. The time for this mixing process can be varied if analysis shows the total mixture is not homogeneous. For the final mix, the premix and the remainder of the diet are layered in a large capacity mixer. The mixing time will vary with the type of blender and can be varied if the analysis shows the total mixture is not homogeneous. Several types of blenders are available for the mixing process; these include open-bowl “kitchen” mixers, V or PK blenders, and Turbula mixers. Metal parts should be ground to eliminate electrostatic forces. In addition, alternative methods of dietary administration such as microencapsulation may be used for volatile, reactive, or unpalatable chemicals. Gavage Method In the gavage procedure, the test compound is administered by passing a feeding tube or gavage needle attached to a syringe down the esophagus into the stomach. Test Article Preparation If not already a liquid, the test compound is prepared for administration by adding it to the appropriate vehicle. The choice of vehicle will depend on the characteristics of the compound and whether it is to be administered as a suspension or a solution. In addition, consideration must be given to the effects of the vehicle on the rat (Gad and Chengelis, 1998). Common vehicles used include water and food grade oils such as corn oil. Suspensions are made when aqueous vehicles are desired and the test compound is not soluble. Suspending agents such as methylcellulose are added to increase the viscosity and hold the compound in suspension. Other agents such as Tween 80, ethanol, polyethyleneglycol 400 (PEG 400), and others may be used as wetting or stabilizing agents. Equipment Soft catheters made of silastic or polyethylene (e.g., infant feeding tubes), stainless steel gavage needles with smooth ball-shaped tips, or polyethylene gavage needles with ball-shaped tips are commonly used. All are commercially available and are relatively inexpensive. Although the soft catheter minimizes the chance of esophageal trauma, liquid can leak past the catheter and back up the esophagus and be aspirated. The ballshaped tips of the stainless steel gavage needles reduce the chances of tracheal injections; however, if an animal struggles while the needle is in the esophagus, the rigid needle increases the chances of perforating the esophagus. The polyethylene gavage needle incorporates the best of both the soft catheter and the stainless steel

17

needle, but because of the flexible nature of the needle, the risk for tracheal injection is increased. Conybeare and Leslie (1980) found that deaths in gavage studies were a result of aspiration of small amounts of irritant solutions or acidic, hypertonic solutions. They also found that the use of a ball-tip 4 mm in diameter helped to eliminate deaths related to dosing. With gentle handling, the animals will be acclimated to the techniques used and dosing will become easier. Aspiration and tracheal administration of test compound as well as esophageal trauma have been associated with gavage dosing and may lead to difficulty in interpretation of the study. The catheter and the needles all have risks inherent in their use; therefore, care should be taken when using these tools and animal technicians should be properly trained. The choice of the appropriate catheter or needle should be left up to the technician and should be whatever the technician has been trained and is most comfortable with. Technique The description below is appropriate for either a gavage needle or catheter; for simplicity, only the needle will be mentioned in the description. Prior to picking up the animal, the syringe should be attached to the needle and filled with the appropriate amount of test compound to be delivered. Any air bubbles should be eliminated and the needle wiped clean of residual test compound. This is done so that the animal does not taste the test compound and residual test compound is not aspirated as the needle is passed down the esophagus. If the dosing liquid is distasteful, the animal may struggle after repeated dosing and increase the chances of being injured. To position the animals for gavage, it should be grasped by the skin of the back and neck ensuring that the head, neck, and back are in a straight line. Alternatively, the animals can be grasped about the shoulders, with the index finger and thumb on either side of the head. The objective is to firmly hold the animals to be able to control any struggling if it occurs and to also prevent the animal from being able to bite the technician. For even more control, the animal may be placed on a table or brought up against the operator’s chest. Once the animal is in position, the needle can be inserted into the mouth of the animal, moved over the tongue, and down into the esophagus. The length of the needle should be inserted into the animal. A slight rotation of the needle may help with insertion into the esophagus. If the needle is inserted into the trachea, the animal may struggle. The syringe should be grasped lightly such that, if the animal does struggle, the chances of an esophageal tear are minimized. If the animal continues to struggle, the needle should be withdrawn to allow the animal to calm down, and then dosing should

I. TOXICITY TESTING MODELS AND BIOMARKERS

18

2. RODENT MODELS FOR TOXICITY TESTING AND BIOMARKERS

be attempted again. Alternatively, if a catheter is used, as the tube is placed into the mouth, it should be placed to the side between the molars. This is done because the tube may by bitten or transected if passed too close to the front teeth. With the needle in place, the test compound should be slowly expelled into the animal. If administered to rapidly, reflux may occur and the test compound may back up into the esophagus, resulting in an inaccurate dose being given and possible aspiration of the test compound. Once the dose has been delivered, the needle should be withdrawn and the animal observed for any signs of distress or respiratory difficulty. An experienced technician should be able to dose between five and seven animals per minute without causing discomfort to the animals and with minimal dosing-related deaths. Gavage liquids are commonly administered at a volume of 5e10 mL/kg body weight. The volume should be enough to be delivered accurately, but not so much that it will adversely affect the animal. The maximum volume should be no more than 20 mL/kg. If using volumes greater than 10 mL/kg, it may be advisable to fast the animals for several hours prior to dosing. This will ensure that the stomach is empty prior to dosing and able to handle the larger volume. This option should be considered carefully, as fasting can affect the rate of absorption and clearance from the stomach. In addition, the choice of housing and bedding should be considered when dosing with large volume as rats have the tendency to eat the bedding, which may hinder gavage dosing. In addition, the volume chosen can have an effect on the results of the study and volumes greater the 10 mL/kg should only be used when issues of solubility and exposure exist. Ferguson (1962) found that a change in dose volume of from 5% to 1% of body weight could reduce mortality rate from approximately 95%e5%, respectively, at equivalent doses. Neonatal Administration Neonatal intragastric injections can be made orally with thin silicone tubing (Gibson and Becker, 1967; Smith and Kelleher, 1973) or by intragastric injection with a 27-gauge needle through the abdominal wall (Worth et al., 1963; Bader and Klinger, 1974). The oral method using silicone tubing is performed in a similar manner to the previously described method in adult rats. The intragastric injection through the abdominal wall is performed by first locating the stomach in the upper left quadrant of the abdomen and then carefully inserting the needle through the abdominal wall into the stomach taking care that the animal does not move. The syringe should be gently aspirated to ensure proper placement and then the injection completed and the needle withdrawn.

Capsule To eliminate the possibility of dosing errors and to deal with compounds that cannot be delivered through conventional means, methods have been developed for the administration of capsules into the esophagus of the rat. The test compound may be prefabricated into a small capsule or the test article may be weighed and placed into commercially available capsules. An individual capsule is then placed into a specially designed cup in the end of a gavage needle, and the needle is then inserted into the esophagus of the rat. The capsule is then pushed out of the cup into the esophagus using either air or a rod inside the needle. The needle is then withdrawn and the capsule moves down into the stomach by peristaltic action. Only a small amount of test compound can be administered as a single dose using this method, but multiple capsules can be administered sequentially in the same dosing session. Water As an alternative to dietary administration, compounds that are water soluble, palatable to the rat, and stable in water may be administered via the drinking water. This method offers similar advantages as adding a test compound to the diet. Additionally, compounds will be more easily mixed and analyses will be more easily developed than when a compound is in the diet. However, spillage of water makes measurement of the actual dose received difficult. Intravenous Route One of the most common methods of administration of test compound is via intravenous (iv) injection or infusion. The iv route is often the route of choice for compounds that have poor bioavailability via the oral route or have a short half-life. Several issues must be considered when administering a test compound intravenously. The compound must be soluble in an acceptable iv vehicle or excipient, must be able to be administered as a solution, and should be sterile or sterile filtered prior to administration. In addition, when designing a study, the pharmacokinetic profile of the test compound administered intravenously should be considered. Study activities such as clinical observations and functional observational battery should be planned around the expected time of greatest plasma concentration. A variety of veins may be used for iv injections (Diehl et al., 2001). These include the lateral tail (caudal), jugular, femoral, saphenous, lateral marginal, dorsal metatarsal, sublingual, and dorsal penile vein. Although most of these are superficial, and easily available for injection, several require the use of anesthesia or more than one technician and may be of limited use in repeat

I. TOXICITY TESTING MODELS AND BIOMARKERS

ROUTES OF TEST ARTICLE ADMINISTRATION

dose studies. Although anesthesia may be acceptable for acute studies or surgical model, its repeated use may have an effect on the toxicity of a test compound. Lateral Tail Vein The lateral tail veins are currently the most widely used for iv injections in the rat. The veins are easily visible, especially in young animals and injections can be performed by one person without the use of anesthesia. The technician performing the function should be well trained and care should be taken to ensure that the lateral veins are being accessed and not the dorsal or ventral artery of the vein. Bolus Injection The animal should be placed in an appropriate restrainer. This typically consists of a solid tube in which the animal is placed into headfirst and has a stop that is placed behind the animal with a hole that allows the tail to hang out the back. The restraint tube is designed to be secure enough that the animal cannot move, back out, or turn, but can still breathe comfortably. Once secure, the tail should be cleaned and the vein may be dilated with heat. This may be accomplished by placing the tail in warm water (40e45
C), placed under a heat lamp, or wrapped with warm gauze. Care must be taken to avoid using excessive heat as tissue damage may result. Minasian (1980) describes a tourniquet made from a plastic syringe and thread. If used, this should not be left on for an extended period of time. When performing an injection, the end of the tail should be held firmly and taut with the thumb and index finger of one hand. A 23-gauge needle attached to an appropriately sized syringe should be held with the bevel up at a shallow angle parallel to the vein. The skin of the tail is then pierced and the needle advanced until resistance is no longer felt. The plunger of the syringe should then be aspirated to ensure proper placement of the needle. The use of a needle with a clear or transparent hub will facilitate confirmation of correct placement. Blood backflow into the needle confirms entry into the vein. Alternatively, a butterfly needle with an extension line may be used. The butterfly needle with an extension set precludes the need to hold the tail, needle, and syringe. When using this type of setup, the butterfly needle is attached to an extension set and syringe that is filled with the test compound. The tail may be taped to the table, and the butterfly needle is then inserted into the vein and placement is verified by aspiration on the syringe. Once confirmed, the butterfly needle may also be taped in place. This prevents the needle from pulling out of the vein during dosing. This type of setup can be very useful when administering large volumes of test article as a slow bolus over several minutes or when the test compound may be irritating or mildly caustic.

19

Taping the animal’s tail in place prevents the animal from pulling the tail out of the fingers of the technician. If repeated dosing is to be performed, the initial venipunctures should be performed as close to the tip of the tail as possible. During the injection, if the needle comes out of the vein, a bleb will form under the skin. The needle should be repositioned immediately to prevent infiltration of the solution around the vein. Infiltration of an irritating solution can cause necrosis and make future injections difficult or impossible. Injection of 2 mL/ 100 g body weight can be accomplished without stress to the rat. Barrow (1968) found that injections of volumes over this amount produced respiratory difficulty and pulmonary edema. Tail Vein Infusions Tail vein infusions are convenient because catheter placement can be accomplished without anesthesia. A 23-gauge or smaller needle connected to an extension set is inserted into the tail. The needle and extension set is then secured to the tail with tape. The extension set is attached to a syringe that is placed on a pump and the test compound can be infused. The tail may be taped to a wooden stick or tongue depressor to further protect the needle from being dislodged. Over the needle, catheters are also commercially available and offer the advantage that the needle is removed once the catheter is placed in the vein and may help to prevent further penetration of the vein wall and subsequent perivascular dosing (Rhodes and Patterson, 1979). Advantages that this technique has over permanent indwelling catheters are that the catheter is removed following dosing and will not become occluded and the animal doses not have to undergo anesthesia and a surgical procedure to place the catheter. Permanent catheters have a tendency over time to develop a fibrin flap or become clotted, thus losing patency. A major disadvantage is that the animals have to be restrained during the infusion, which may causes stress and alter the results of the study. When using this technique, the duration of the infusion should be limited so that the length of time the animal is restrained is limited. An alternative technique using the lateral tail vein involves placing a catheter in the vein and wrapping the tail in a similar manner as previously described, then a lightweight protective cover attached to a tether system is placed around the tail to hold the catheter or needle in place. Jugular Vein Although this route has been used for bolus injections, it is most widely used as a site for cannulation from indwelling catheters. The indwelling catheter requires surgical implantation under anesthesia.

I. TOXICITY TESTING MODELS AND BIOMARKERS

20

2. RODENT MODELS FOR TOXICITY TESTING AND BIOMARKERS

Bolus Injection Although injections can be made by exposing the jugular vein by incision, this method is not acceptable for repeated dosing. The jugular vein can be accessed for test compound administration without exposing the vein. The animal can either be anesthetized or restrained on the back. The head is positioned to either the left or the right for access to the respective jugular vein. A 23-gauge needle fitted to a syringe with the bevel up is inserted in a cephalocaudal direction into the angle made by the neck and shoulder. The needle should enter the vein anterior to the point at which it passes between the pectoralis muscle and the clavicle. When about onehalf the length of the needle has penetrated the skin, the bevel should be in the lumen of the vessel. Insertion of the needle through the muscle stabilizes the needle and minimizes bleeding. Caution should be used when using this technique as it is considered to be a “blind stick” into the vessel, and damage to the vessel may occur. Repeated access of the vessel is not recommended. Infusion For the purpose of continuous infusion of the test compound over extended periods of time or for repeated short-term infusions, implanted catheters in the jugular vein may be used. For implantation of a jugular catheter, the animal is first anesthetized and placed in dorsal recumbency, and the surgical site is prepared. A midline incision is then made in the neck, and a section of the jugular vein is dissected free. Manipulation of the vein should be limited to prevent vasospasm. A cephalic ligature is then tied and the vein elevated. A small incision is then made in the vein, and the catheter is passed into the vein and tied in place. The other end of the catheter is then tunneled subcutaneously (sc) to between the scapula where the catheter is exteriorized. The catheter should be filled with an anticoagulant solution such as heparin when not in use. When correctly positioned, the tip of the cannula will be at the junction of both vena cava. If placing catheters into young animals, enough of the catheter should be inserted to allow for growth of the animal. Care should be taken that the catheter is not inserted too far as the tip may be pushed into the right ventricle of the heart. Improper placement of the catheter may lead to administration of the test compound directly into the heart, which can cause complications. Similar to the jugular vein, administration of test article via the femoral vein requires an implanted catheter. For implantation of a femoral catheter, the animal is first anesthetized and placed in dorsal recumbency, and the surgical site is prepared. A midline incision is then made in the inguinal area and a section of the

femoral vein is dissected free. Manipulation of the vein should be limited to prevent vasospasm. A ligature is then tied and the vein elevated. A small incision is then made in the vein and the catheter is passed into the vein and tied in place. The other end of the catheter is then tunneled sc to between the scapula where the catheter is exteriorized. The catheter should be filled with an anticoagulant solution such as heparin when not in use. When correctly positioned, the tip of the cannula will be position in the vena cava. For longer-term infusions, the femoral vein catheter may be preferable as patency is easier to maintain and the risk of damage to the heart from the catheter is avoided. Several commercial vendors offer surgical support services and for an additional fee will implant either jugular or femoral catheters. These vendors will typically have a specific methodology for implant, but will accept requests for modifications such as catheter type, exteriorization site, etc. The typical catheter implanted may be manufactured from polyethylene, polypropylene, or silastic. In recent years, manufacturers have developed catheters impregnated or ionically bound with heparin. These materials may help to prolong the life of the catheter. The useful lifetime for jugular and femoral catheters is quite variable; the lumen of the cannula may eventually become obstructed by a blood clot or fibrous mass. The position of the tip of the catheter is important. Clot formation is less likely to occur if the tip of the catheter is placed in the venous stream rather than in the jugular vein (Popovic and Popovic, 1960). It is recommended for repeated short-term infusions, and when test article is not being infused, a slow infusion of saline will help to prolong the life of the catheter. Prior to use, the patency of the catheter should be checked by removing the anticoagulant lock, check for blood draw back, and then flushing with saline or Lactated Ringers solution. Alternatively, patency can be checked by injecting 3e6 mg of pentobarbital solution (0.05e0.10 mL of a 60 mg/mL solution) into the catheter (Weeks, 1972). If the catheter is patent, the rat will lose its righting reflex and become ataxic within 10e15 s of injection. The rat will recover in 10e15 min. Rats will destroy the catheter if it is left unprotected or in easy reach of the forepaws. By exteriorizing the catheter between the scapula, the rat will not be able to chew on the catheter. For the purpose of continuous infusion, several manufacturers have developed tether systems and catheter sheaths made of metal that prevent the animal from chewing on the catheter. These systems typically consist of a jacket with an attached tether through which the catheter is passed. The catheter then attaches to a swivel that prevents the catheter from becoming kinked. The swivel then attaches to a second catheter that can be attached to a syringe or

I. TOXICITY TESTING MODELS AND BIOMARKERS

ROUTES OF TEST ARTICLE ADMINISTRATION

pump for administration of the test compound (Guo and Zhou, 2003). When performing long-term infusion studies, the effects of the catheter and harness should be considered. Infections, septicemia, a variety of visceral lesions, endothelial lesions, and increased platelet consumption have been observed in cannulated animals (Hysell and Abrams, 1967; Meuleman et al., 1980; Vilageliu et al., 1981). Decreased or erratic weight gains and decreased liver and thymus weights have been observed in tethered animals. These changes may be attributed to the stress involved in chronic tethering of the animals. An alternative to an exteriorized catheter is to attach a sc port to the catheter that can be accessed via a transcutaneous needle stick. This type of setup helps to prevent infections that can occur with transcutaneous catheters. One of the pitfalls of this sc port is that the port may only be accessed a finite number of time. In addition, care has to be taken to ensure the port and catheter are properly flushed of all test compound and blood as clots can easily form. Administration of small volumes of test compound may be accomplished using a sc implanted osmotic pump. This type of pump is connected to the catheter after being filled with the test compound and implanted in a sc pocket. This allows for continuous administration of small amounts of compound without the need for a jacket and tether system. Saphenous, Lateral Marginal, and Metatarsal Veins These veins in the leg and foot are easily visualized and can be injected without anesthesia; however, assistance is required. Shaving the area over the saphenous or lateral marginal vein makes visualization easier. During injection it is necessary for one technician to restrain the animal and occlude the vessel to cause it to dilate. Wiping the skin over the vein with 70% alcohol or with gauze soaked in hot water will help to dilate the vessel and increase the possibility of success. The second technician then performs the injection, in which a 26e27-gauge needle should be used. Dorsal Penis Vein When administering test article via the dorsal penis vein, it is preferable to use anesthesia. Lightly anesthetizing the animals with an inhaled anesthetic such as isoflurane or CO2/O2 will prevent the animal from struggling and increase the possibility of a successful injection. This procedure requires two technicians to perform the injection. One technician holds the animal by the skin on the back and the feet and tail. The vertebral column is then hyperextended. The second technician then grasps the tip of the penis between the thumb and forefinger, and injects the test solution into the dorsal vein using a 26e30-gauge needle.

21

Sublingual Vein Although the method of sublingual vein injection has the disadvantage of requiring anesthesia, it only requires one technician. Ideally, the animal should be anesthetized with an inhaled anesthetic such as isoflurane or CO2/O2, but injectable anesthesia may also be used. The animal should be placed in dorsal recumbency with the head toward the operator. The test compound may be administered by holding the tongue between the thumb and forefinger; using a 26e30gauge needle, the vein is entered at a very shallow angle and the injection is performed. After completion of the injection, the bleeding can be stopped using direct pressure. Once the bleeding has stopped, a small cottonwool pledget should be placed over the vein and the tongue placed back in the mouth. The animal will spit the cotton out on regaining consciousness.

Intraperitoneal Route Test compounds injected into the peritoneal cavity will be absorbed into the portal circulation and transported to the liver. As a result, the compound will be subjected to the metabolic activity of the liver prior to being circulated to the remainder of the animal. Based on the level of blood flow and circulatory surface area in the peritoneal cavity, compounds injected intraperitoneally (ip) will be absorbed quickly. Intraperitoneal administration of test compounds in the rat can be performed by one person. The animal should be picked up by the scruff of the neck and back and held firmly in dorsal recumbency. This position will allow for proper access to the peritoneal cavity. The belly of the animal should be visually divided into quadrants and a needle (1 and measurements of biochemical molecules/fragments of protein, DNA, and COMP (>1 unit in serum); CTX-II; serum hyaluronic acid; and pentosidine. In addition, these biomarkers include degree of joint space narrowing (JSN) or change in cartilage volume by MRI and correlated well with

urinary CTX-II levels. Efficacy of intervention biomarkers may be measured prior to therapy to predict treatment efficacy or measure short-term changes that occur as a result of pharmacologic or other interventions (e.g., measuring concentrations of a biomarker of cartilage degradation, such as CTXII). Some examples of diagnostic biomarkers include COMP, CTXII, NTX-I. For detailed BIPED classification and examples of biomarkers, see Rousseau and Delmas (2007). Recently, Bay-Jensen et al. (2016) classified OA biomarkers into three broad categories: (1) inflammatory biomarkers, such as cytokines, chemokines, or cell type markers relevant to OA pathology; (2) biomarkers reflecting the turnover of the ECM of OA cartilage; and (3) biomarkers that target autoantibodies, signaling molecules, or growth factors (discussed above in pathophysiology). It is noteworthy that a biomarker can fall into more than one category.

BIOMARKERS OF COMFORT, MOBILITY, FUNCTION, AND INFLAMMATION AND PAIN The diagnosis of OA often relies on clinical and radiological examinations of late and irreversible stages. Sensitive serum biomarkers specific for early stages (potentially reversible) of OA are lacking. It is suggested that combining biochemical markers with tissue and cell imaging techniques and bioinformatics (i.e., machine learning, clustering, and data visualization) may facilitate the development of biomarkers enabling earlier detection of OA (Mobasheri, 2012; Mobasheri et al., 2017).

Humans In OA in humans, several scoring systems are used to measure joint mobility, flexibility, and pain, such as Knee Osteoarthritis Scoring System (KOSS), modified Knee Injury and Osteoarthritis Index (mKOOS) global score, Clinical American College of Rheumatism (ACR) criteria, Whole-Organ Magnetic Resonance Imaging Score (WORMS) of the knee, Boston Leeds Osteoarthritis Knee Score (BLOKS), MRI Osteoarthritis Knee Score (MOAKS), modified Western Ontario and McMaster Universities Arthritis Index (mWOMAC) subscale, and Visual Analog Scales (VAS) (Kornaat et al., 2005; Peterfy et al., 2004; Hunter et al., 2008, 2011; Belo et al., 2009; Runhaar et al., 2014). The global mKOOS test is composed of four categories (symptoms, stiffness, discomfort, and function/daily living activities). Additionally, mWOMAC subscale scores consist of 24 items divided into three subscales assessing discomfort (5 items), stiffness (2 items), and physical function (17

VIII. DISEASE BIOMARKERS

OSTEOARTHRITIS BIOMARKERS IN SERUM, SYNOVIAL FLUID, AND URINE

items). Additionally, self-reported feelings of joint health, comfort, mobility, and function are the primary outcome measures of OA (Lopez et al., 2017). OA severity is often determined using weightbearing anteroposterior radiographs of the affected joints, which are evaluated according to the Kellgren and Lawrence classification. The grade of OA is described as follows: Grade 0, no radiographic findings of OA; Grade 1, minute osteophytes of doubtful clinical significance; Grade 2, definite osteophytes with unimpaired joint space; Grade 3, definite osteophytes with moderate JSN; and Grade 4, definite osteophytes with severe JSN and subchondral sclerosis.

Animals Dogs and horses are evaluated for overall pain, pain upon limb manipulation, and exercise-associated lameness using the Glasgow scoring system on a monthly basis for a study period of at least 4e5 months (Gupta et al., 2009, 2012). Overall pain is graded on a scale of 0e10: 0, no pain; 5, moderate pain; and 10, severe and constant pain. Pain upon limb manipulation is evaluated during the extension and flexion of all four limbs for a period of several minutes. Pain level is graded on a scale of 0e4: 0, no pain; 1, mild; 2, moderate; 3, severe; and 4, severe and constant. Pain and lameness are measured after physical exercise and graded on a scale of 0e4: 0, no pain; 1, mild; 2, moderate; 3, severe; and 4, severe and constant. In horses, pain is measured using similar criteria and scales. Additionally, flexibility and range of motion in the affected joints are measured using a Goniometer (May et al., 2015). Hielm-Bjo¨rkman et al. (2009) used a five-point scale to measure pain in OA dogs [0, no sign of pain; 1, mild pain (dog turns head in recognition); 2, moderate pain (dog pulls limb away); 3, severe pain (dog vocalizes or becomes aggressive); and 4, extreme pain (dog does not allow palpation)]. In dogs, Ground Force Plate (GFP) (Kistler Instrument, Amherst, NY, USA) is utilized to quantitatively measure the lameness-associated pain in each leg of each dog (Gupta et al., 2012). The Kistler’s GFP system consists of plates, lasers, and a computer. The GFP measures two major parameters: (1) peak vertical force or g force (Newton/Kg body weight), and (2) impulse area (Newton/Kg body weight). In a similar way, GFP can quantitatively measure the pain level in horses. Observation of Ortolani and Cranial Tibial Drawer Examination Along with the external evaluations of pain, such as gait, or lameness, other options may also be performed during the physical examination. The Ortolani Maneuver is a common test performed on canines that are

933

predisposed to hip dysplasia such as German shepherds or larger breed canines (Siberian huskies, Rottweilers, Newfoundlands, and Labradors). Ortolani is performed on the hip joint by flexing the knee and hip to 90 degrees, placing the index finger on the greater trochanters, and abducting the hip (Ortolani’s sign, 2007). As the hip is abducted, or moved away from the body, a positive Ortolani will be shown with a “clunk” sound or feeling as the femoral head relocates anteriorly to the acetabulum, or hip socket. The Ortolani is typically used on patients that have been sedated to receive a true positive or negative sign. In moderate arthritis, dogs may exhibit a negative Ortolani sign. Cranial tibial drawer is another test that can be performed on physical examination to indicate arthritic changes and diagnose the rupture of the cranial cruciate ligament (CCL). In this procedure, the canine is in a laterally recumbency with the veterinarian located behind the patient. The thumb of one hand is placed on the caudal aspect of the femoral condular region, and the index finger of the same hand is placed over the patella. The thumb of the other hand is placed on the head of the fibula, and the index finger is placed on the tibial crest (Devine, 1993). A positive tibial drawer is elicited with the ability to move the tibia cranially or forward in respect to the fixed femur. Currently, experimental/small animals are heavily used to unravel pathways of pain and develop models to measure OA-associated pain (electrophysiology, von Frey hair algesiometry, acoustic frequency and duration recording for vocalization measurement, and nerve injury marker activating transcription factor-3 (Malfait et al., 2013)).

OSTEOARTHRITIS BIOMARKERS IN SERUM, SYNOVIAL FLUID, AND URINE There are many biomarkers of inflammation and cartilage degeneration associated with OA that can be determined in synovial fluid, serum, or urine, and they have potential as a diagnostic utility. Synovial changes in OA are regarded by many as a secondary response to the degradation of cartilage (Felson, 2013) though there are others who advocate them as a primary driver for OA, which may be partly responsible for pain and disease progression (Benito et al., 2005; Berenbaum, 2013). Many of the biomarkers of OA are listed in Table 52.1; and some of these are described in brief below.

Biomarkers of Inflammation Biomarkers of joint inflammation can be detected in serum, synovial fluid, and urine much earlier than irreversible joint damage and radiographic changes (Das et al., 2015).

VIII. DISEASE BIOMARKERS

934 TABLE 52.1

52. OSTEOARTHRITIS BIOMARKERS

Biomarkers of Osteoarthritis (OA)

OA-associated inflammation and pain

OA-associated cartilage degeneration/loss

Biomarkers

References

Cytokines (IL-1b, IL-6, IL-8, IL-10, IL-17, and IL-22), adipocytokines/adipokines (leptin, resistin, and visfatin)

Stannus et al. (2010), Koskinen et al. (2011), Berry et al. (2011), Goldring and Otero (2011), Francin et al. (2014), Daghestani and Kraus (2015), Deligne et al. (2015), Fowler-Brown et al. (2015), Bay-Jensen et al. (2016), Wan and Zhao (2017)

Tumor necrosis factor-a (TNF-a)

Stannus et al. (2010), Jaime et al. (2017), Wan and Zhao (2017)

Bradykinin B1 receptor

Duclos et al. (2016)

Chemokines (C-X-C and CeC)

Abramson and Attur (2009), Endres et al. (2010), Daghestani and Kraus (2015)

Toll-like receptors (TLR1 and TLR2)

Scanzello et al. (2008), Sillat et al. (2013)

Neuropeptides

Zhang et al. (2013), Heikkila¨ et al. (2017)

Activating transcription factor-3 (AFT-3)

Malfait et al. (2013)

Prostaglandin E2 (PGE2)

Lin et al. (2006), Heikkila¨ et al. (2017)

Fractalkine (CX3CL1)

Huo et al. (2015), Bay-Jensen et al. (2016)

Nuclear factor-kappa B (NF-kB)

Shakibaei et al. (2007), Goldring and Otero (2011), Rasheed et al. (2016)

C-reactive protein (CRP)

Daghestani and Kraus (2015), Bay-Jensen et al. (2016), Hillstrom et al. (2016)

Coll2-1, and Coll2-1 NO2

Henroitin et al. (2013, 2014)

sRAGE

Bierhaus et al. (2006), Chayanupatkul and Honsawek (2010)

Protease granzyme A

Jaime et al. (2017)

TSG-6

Maier et al. (1996), Bardos et al. (2001), Bayliss et al. (2001), Milner and Day (2003), Wisniewski et al. (2014)

AMPK and SIRT1

Liu-Bryan (2015)

Erythrocyte sedimentation rate (ESR)

Murdock et al. (2016)

Uric acid

Denoble et al. (2011)

COMP, COMP neoepitope, and ykl-40

Petersson et al. (1998), Clark et al. (1999), Williams and Spector (2008), Benedetti et al. (2010), Nagala et al. (2012), Shahi et al. (2013), Sharif et al. (2004), Varma and Dalal (2013), Das et al. (2015), Skio¨ldebrand et al. (2017)

Cartilage collagenases

Ehrlich et al. (1978), Goldring and Otero (2011)

Bone sialoprotein (BSP)

Petersson et al. (1998)

MMPs (MMP-1, MMP-3, MMP-10, MMP-13)

Pelletier et al. (2010), Koskinen et al. (2011), Hsueh et al. (2014)

C-telopeptide of type I collagen (CTXI)

Wu et al. (2017)

N-telopeptide of type I collagen (NTXI)

Wu et al. (2017)

N-terminal procollagen III propeptides (PIIINP)

Wu et al. (2017)

Type II C-telopeptide

Jung et al. (2004)

Type II collagen

Bay-Jensen et al. (2016)

Type II collagen neoepitope

Bay-Jensen et al. (2016)

Coll2-1 (a type II collagen fragment)

Henroitin et al. (2013)

Hyaluronic acid/Hyaluronan

Plickert et al. (2013), Singh et al. (2015), Das et al. (2015)

Adiponectin

Francin et al. (2014)

VIII. DISEASE BIOMARKERS

OSTEOARTHRITIS BIOMARKERS IN SERUM, SYNOVIAL FLUID, AND URINE

TABLE 52.1

935

Biomarkers of Osteoarthritis (OA)dcont’d

Proteomic and metabolomic analysis for OA biomarkers

MicroRNAs in OA

Biomarkers

References

Aggrecan neoepitope, aggrecan, and aggrecanase 1 (ADAMTS-4) and aggrecanase 2 (ADAMTS-5)

Dufield et al. (2010), Swearingen et al. (2010a,b), Goldring and Otero (2011), Hsueh et al. (2014), Li et al. (2014); Peffers et al. (2014)

Biglycan, decorin, and matrilin-1

Poole et al. (1996), Wiberg et al. (2003), Peffers et al. (2014)

Ghrelin

Wu et al. (2017)

Clusterin and Lubricin (proteoglycan 4)

Swan et al. (2011), Elsaid et al. (2012), Ritter et al. (2014), Svala et al. (2017)

Fibulin-3 peptides (Fib3-1 and Fib3-2)

Henroitin et el (2012)

Follistatin-like protein 1 (FSTL1)

Wang et al. (2011)

Fibromodulin and fibronectin

Hsueh et al. (2014), Peffers et al. (2014)

Uric acid

Denoble et al. (2011)

Radiographic biomarkers (K-L Grade Criteria)

Kellgren and Lawrence (1957), Bauer et al. (2006), Mobasheri (2012), Hall et al. (2014), Andronescu et al. (2015), Palmer et al. (2017), Ramı´rez-Flores et al. (2017)

MRI- and ultrasound-biomarkers

Peterfy et al. (2004), Gamero et al. (2005), Garvican et al. (2010a,b), Guermazi et al. (2013), Eckstein et al. (2014), Hall et al. (2014), Peffers et al. (2014), Nieminen et al. (2017), Roemer et al. (2014), Palmer et al. (2017), Ramı´rez-Flores et al. (2017)

Cartilage thickness and cartilage damage score

Bagi et al. (2017), Wirth et al. (2017)

Cartilage volume, using local-area cartilage segmentation (LACS) software method

Schaefert et al. (2017)

Cartilage

Guo et al. (2008), Castro-Perez et al. (2010), Lourido et al. (2014), Sanchez et al. (2017)

Synovial fluid

Ruiz-Romero and Blanco (2009, 2010), Han et al. (2012)

Serum/plasma

Castro-Perez et al. (2010), Zhai et al. (2010)

Urine

Hsueh et al. (2014)

miR-9, miR-16, miR-22, miR-33a; miR-92a-3p; miR-98; miR-370; miR-140; miR-146a, miR-222, miR-373; miR-16-5p; miR-26a-5p; miR-634; and others

Li et al. (2011), Beyer et al. (2015), Kostopoulou et al. (2015), Li et al. (2015), Song et al. (2015), Cui et al. (2016), Rasheed et al. (2016), Wang et al. (2016), Cong et al. (2017), Mao et al. (2017), Si et al. (2017), van Meurs (2017)

Cytokines/Adipocytokines, Chemokines, and Neuropeptides In a number of studies, profiles of cytokines/adipocytokines, leptins (Stannus et al., 2010; Koskinen et al., 2011; Berry et al., 2011; Goldring and Otero, 2011; Deligne et al., 2015; Fowler-Brown et al., 2015; Bay-Jensen et al., 2016; Hillstrom et al., 2016; Wan and Zhao., 2017), chemokines (Endres et al., 2010; Daghestani and Kraus, 2015; Bay-Jensen et al., 2016), and neuropeptides (Zhang et al., 2013) have been described in serum and/or synovial fluid in relation to OA inflammation.

Prostaglandin E2 and EP4 Receptor Prostaglandin E2 (PGE2), a proinflammatory mediator, is produced from arachidonic acid by cyclooxygenase (COX) enzymes. PGE2 plays a pivotal role in the development of joint inflammation and pain in arthritis by binding of PGE2 to the EP4 receptor, one of the four G-proteins (Lin et al., 2006). Fractalkine Fractalkine (CX3CL1) plays an important role in inflammation and chronic pain, and its levels were

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found to be high in serum and synovial fluid and correlated with WOMAC pain and WOMAC total scores (Huo et al., 2015). Erythrocyte Sedimentation Rate In a clinical setting, an increase in erythrocyte sedimentation rate is commonly used as a biomarker of inflammation in OA patients (Murdock et al., 2016).

Biomarkers of Osteoarthritis Progression and Cartilage Degeneration There is a great need to identify biomarkers of OA progression because the disease process is highly unpredictable. Biomarkers of cartilage degeneration (COMP, MMPs, clusterin, lubricin, bone sialoprotein, N-terminal propeptides of procollagen type I (P1NP), and beC-terminal telopeptide (b-CTX), and many others) can be detected in synovial fluid, serum, and other bodily fluids (Petersson et al., 1998; Abramson and Attur, 2009). Noncollagenous and nonproteoglycan components of cartilage can also be detected in body fluids following their release due to cartilage turnover (Garvican et al., 2010a,b; Abramson and Attur, 2009). Early detection of OA and OA progression can be evaluated macroscopically, histologically, immunohistochemically, and via imaging (MRI). In recent years, MRI has demonstrated its relevance for the evaluation of structural changes during the development and progression of knee OA (Guermazi et al., 2013). In a recent investigation, Nieminen et al. (2017) demonstrated that histopathological information relevant to OA can be reliably obtained from contrast-enhanced microcomputed tomography (CE mu CT images). This new grading system may be used as a reference for 3D imaging and analysis techniques intended for volumetric evaluation of OA pathology in research and clinical applications. Availability of new technologies and tools for improved morphological and pathophysiological understanding of OA-related changes in joints are an aid to early diagnosis and prognosis and in the monitoring of treatment (Boesen et al., 2016; Wyatt et al., 2017). Some important biomarkers of OA progression are discussed below. Cartilage Oligomeric Matrix Protein (COMP) COMP is a pentameric noncollagenous glycoprotein (435,000 Da) belonging to the heterogeneous family of thrombospondin, which can bind to collagen types I, II, and IX. It has been reported that COMP can bind up to five collagen molecules, thereby retaining them in close proximity. By this process, COMP facilitates the collagenecollagen interactions and microfibril formation. It is reported that COMP is mainly produced by articular chondrocytes, and COMP levels in serum

and synovial fluid might be related to cartilage degeneration (Clark et al., 1999; Williams and Spector, 2008; Benedetti et al., 2010). Das et al. (2015) identified COMP as a potential biomarker of OA, which has shown significant clinical promise as a tool for early detection, therapeutic monitoring, prognostication, and drug development. Increase in serum COMP level by > 1 unit increased the probability of radiographic progression by 15% (Sharif et al., 2004). The findings of Varma and Dalal (2013) and Shahi et al. (2013) suggested that serum COMP level, along with clinical profile including family history, joint injury and other visible symptoms, and radiographs, could be used as valuable biomarkers for early diagnosis, prognosis, and management of OA. In a most recent investigation, Skio¨ldebrand et al. (2017) identified and quantified a unique COMP neoepitope in the synovial fluid from joints of healthy horses and those with different stages of OA. The findings revealed that the increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a novel candidate biomarker for the early molecular changes in articular cartilage associated with OA. Matrix Metalloproteinases (MMPs) MMPs, also known as “Matrixins,” are capable of degrading ECM proteins. It is well established that a number of MMPs (MMP-1, MMP-3, MMP-10, and MMP-13) are significantly involved in degeneration/ loss of cartilage in OA (Pelletier et al., 2010; Koskinen et al., 2011). Increased levels of some of the MMPs in synovial fluid and serum have been correlated with cartilage degeneration and therefore have potential to serve as biomarkers of OA progression. Hyaluronan Hyaluronic acid (HA; hyaluronate, hyaluronan) is an anionic, nonsulfated glycosaminoglycan and is an important component of articular cartilage present as a coat around each chondrocyte. Molecular weight (MW) of HA can range from 5000 to 20,000,000 DA. The average MW of HA in human synovial fluid is 3e4 million DA. HA is synthesized by a class of integral membrane proteins called hyaluronan synthases (HAS), of which vertebrates have three types (HAS1, HAS2, and HAS3). HA is degraded by a family of enzymes called hyaluronidases. In a number of studies, HA has been recognized as a biomarker of OA (Venable et al., 2008; Plickert et al., 2013; Das et al., 2015; Singh et al., 2015). For further details on HA in relation to OA, readers can referred Gupta (2016). Lubricin The glycoprotein lubricin (also known as proteoglycan 4) is secreted by chondrocytes, synoviocytes, and

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meniscus cells. It contributes to the boundary lubrication of the articular cartilage surface and is found in synovial fluid. As a boundary lubricant, lubricin reduces the coefficient of friction of the articular surface. Svala et al. (2017) demonstrated for the first time that the reduced sialation of lubricin in synovial fluid from OA joints may affect the boundary lubrication of the superficial layer of articular cartilage and could be one of the early events in the progression of OA. Reduced expression of lubricin in synovial synoviocytes and superficial zone chondrocytes with subsequent loss of joint lubrication are early events in the inflammation associated with OA. Using mass spectrometry, Ritter et al. (2014) identified several proteins (including clusterin and lubricin) in human plasma as predictors of OA progression. Intra-articular injections of lubricin were reported to improve damaged cartilage and preserve viability of superficial zone chondrocytes (Swan et al., 2011; Elsaid et al., 2012). Follistatin-Like Protein 1 (FSTL 1) FSTL 1 is a glycoprotein implicated in OA pathogenesis, which secretes into synovial fluid and serum from OA articular cartilage. Wang et al. (2011) found that the FSTL 1 mRNA and protein levels were substantially elevated in the synovial tissues from OA patients. The FSTL 1 expression was strong in the cytoplasm of the synovial and capillary endothelial cells of the synovial tissues, but it was weak in the chondrocytes of the articular cartilage from OA patients. Interestingly, the serum and synovial fluid FSTL 1 levels were maximally higher in female OA patients than in male patients. The serum FSTL 1 levels of OA patients had significant correlations with K-L grade, JSN, WOMAC stiffness scale, and the WOMAC function subscale. The findings suggested that FSTL 1 could be a biomarker from synovium and might represent a biomarker of the burden of disease (severity of joint damage). However, FSTL 1 has two limitations: (1) it is not specific to OA, as it plays roles in kidney, heart, and other organs; and (2) it poorly correlates with chondrocytes of the articular cartilage. Furthermore, there are conflicting reports that FSTL 1 has potential preventive effects on joint destruction by inhibiting the production of MMPs and cytokines both in synovial cells in vitro and in mouse models in vivo (Tanaka et al., 2003; Kawabata et al., 2004). Ghrelin Ghrelin is a peptide hormone, which is involved in energy homeostasis, autoimmunity, inflammation, and many other physiological/pathophysiological processes. Wu et al. (2017) found that significant increases in serum levels of ghrelin were associated with increased knee symptoms, intrapatellar fat pad (IPFP)

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signal intensity alteration, and serum levels of MMP-3, MMP-13, N-telopeptide of type I collagen (NTXI), and N-terminal procollagen III propeptide (PIIINP), suggesting that ghrelin may have a role in knee OA. TSG-6 Tumor necrosis factor-stimulated gene-6 (TSG-6) is a member of the hyaluronan-binding proteins. TSG-6 plays a role in joint inflammation because of its inducible expression by the proinflammatory cytokines IL1b and TNF-a in human synoviocytes and chondrocytes, and the presence of increased levels of this protein in the synovial fluids and cartilage of a majority of arthritis patients has been reported (Bayliss et al., 2001; Milner and Day, 2003; Wisniewski and Vilcek, 2004). TSG-6 serves as a biomarker of OA progression and is of particular interest for aiding development of disease modifying OA drug (DMOAD) and in identifying high-risk patients who would benefit mostly from the use of DMOAD (Kraus et al., 2011; Wisniewski et al., 2014). Potent antiinflammatory and chondroprotective activities of this protein have also been demonstrated in experimental arthritis models (Bardos et al., 2001).

Cartilage Damage and Imaging Biomarkers Although many joint structures are affected in OA, articular cartilage is one of the main tissues involved in the OA disease process (Roemer et al., 2014). Articular damage or loss in OA is detected by radiography and measuring decreases in joint space width (JSW) on the radiograph, the so-called “gold standard.” However, radiographic evidence is seen only after significant cartilage degradation has already taken place (Mobasheri, 2012). Radiographic measures are less than adequate for multiple reasons. First, radiographs indicate changes in bone and only indirectly measure alterations in cartilage (Bauer et al., 2006). Second, the measurement of articular cartilage change, namely JSN, is itself confounded by meniscal cartilage lesions and meniscal extrusion. Third, bone marrow perturbations and synovial abnormalities may go undetected. Fourth, radiographic features characteristic of OA appear only after significant deterioration has occurred in both the hard and soft tissues within and around the joint and changes may occur relatively slowly. Fifth, radiographic features are usually poorly correlated with joint function. Bauer et al. (2006) stated that imaging techniques might themselves be considered as biomarkers for the pathologic joint abnormalities that define OA. Imaging measures should be accurate, precise (reliable), specific, sensitive to a longitudinal change, and acceptable to regulatory agencies (Eckstein et al., 2014). Recently, Palmer et al. (2017) reported that delayed gadolinium magnetic

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resonance imaging of cartilage (dGEMRIC) could detect glycosaminoglycan loss in the cartilage of asymptomatic individuals with cam morphology. These investigators found a positive correlation between dGEMRIC and the magnitude of JSW narrowing. Baseline dGEMRIC is able to predict the development of radiographic OA. MRI offers the potential to identify patients who may benefit from early intervention to prevent the development of OA. Recent studies demonstrated that articular cartilage thickness loss/cartilage degeneration score, and osteophyte formation, measured by MRI and m-computed tomography were found to be associated with concurrent and subsequent radiographic progression and with concurrent symptomatic progression, and therefore they can be used as biomarkers of OA progression (Bagi et al., 2017; Wirth et al., 2017). Schaefert et al. (2017) validated a previously developed local-area cartilage segmentation software method to measure cartilage volume using MRI scans. Cartilage volume change was strongly correlated with radiographic and pain progressions. In canines, Ramı´rez-Flores et al. (2017) found a correlation between synovial fluid effusion and osteophytosis in the stifle joint, using the results of orthopedic, radiographic, ultrasonographic, and arthroscopic examinations. Inflammation, effusion, and capsular thickening can be clinically evident in some joints with OA and are more frequently observed using sensitive measures such as ultrasound and MRI (Hall et al., 2014).

Newly Identified Biomarkers Currently, there is no universal biomarker for OA, and the available biomarkers lack specificity and sensitivity for early diagnosis and monitoring progression of OA. Therefore, new biomarkers are needed. New biomarkers are also needed to distinguish between catabolic and maintenance events because many existing biomarkers reflect normal cartilage turnover, tissue repair, or ECM remodeling. A new biomarker for diagnosing OA should be evaluated by comparison against an established gold standard in an appropriate spectrum of subjects. For OA, an accepted “gold standard” diagnostic test is the radiograph, and typically, K-L grade
2 is required for a diagnosis of OA (Altman et al., 1986). De Senny et al. (2011) characterized four novel biomarkers in the serum of knee-OA patients (V65 vitronectin fragment, C3f peptide, CTAP-III, and m/z 3762 protein). Mobasheri (2012) and Mobasheri et al. (2017) recognized that enrichment of the deaminated epitope of D-COMP suggests that OA disease progression is associated with posttranslational modifications that may show specificity for particular joint sites. Fibulin-3 peptides (Fib3-1 and Fib3-2) have been proposed as

potential biomarkers of OA along with FSTL 1 (described above), a new serum biomarker with the capacity to reflect the severity of joint damage. The “membrane attack complex (MAC)” component of complement has also been implicated in OA. Recently, Mobasheri et al. (2017) identified new biomarkers based on novel technologies, such as “omics.” These authors referred to several new biomarkers, such as adipocytokines including leptin and adiponectin. ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4), aggrecan ARGS neoepitope fragment (ARGS) in synovial fluid, and plasma chemokine (CeC motif) ligand 3 (CCL3) were reported as potential new biomarkers of knee OA. New and refined proteomic technologies and novel assays including a fluoro-microbead guiding chip for measuring Ctelopeptide of type II collagen (CTX-II) in serum and urine and a novel magnetic nanoparticle-based technology (termed magnetic capture) for collecting and concentrating CTX-II also have great potential to be biomarkers of OA.

DNA-Methylation and MicroRNAs (miR) Recently, Rogers et al. (2015) examined the role of inflammation-related genes in OA from the perspective of genetics, epigenetics, and gene expression. There is no compelling evidence that DNA variation in inflammatory genes is an OA risk factor. However, there is compelling evidence that epigenetic effects involving inflammatory genes are a component of OA and that alteration in the expression of these genes is also highly relevant to the disease process. van Meurs (2017) stated that current technological advances in the area of “omics” have elevated multiple molecular levels, from detailed sequencing of the genome, to epigenetic markers, such as DNA-methylation and microRNAs (miRNAs), transcriptomics, and metabolomics. The objective is to identify biomarkers that predict OA’s early onset and progression. miRNAs have great promise as biomarkers because they are relatively stable in circulation. The exact role of miRNAs in OA is still under investigation, but as such, they could potentially be good biomarkers (van Meurs, 2017). Published studies have focused on one or two miRNAs, based on the hypothesis that they target a gene or metabolism that plays an important role in OA pathogenesis. For example, miR-33a, which is known to be important in cholesterol metabolism (Kostopoulou et al., 2015), was found to also regulate cholesterol metabolism in chondrocytes through the TGF-b1/Akt/SREBP-2 pathway, as well as cholesterol efflux-related genes ABCA1 and ApoA1. miR-370 and miRNA-373 were found to regulate expression of SHMT-2 and MECP-2 in chondrocytes (Song et al., 2015). miR-16-5p has been shown to

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regulate SMAD5 expression in cartilage (Li et al., 2015) and miRNA-26a-5p was found to regulate iNOS expression in cartilage (Rasheed et al., 2016). miR-634 has been shown to target PIK3R1 in chondrocytes (Cui et al., 2016). Beyer et al. (2015) identified let-7e as a potential predictor for severe knee or hip OA, but an increase in circulating levels of let-7e could be due to other conditions, such as metabolic dysfunction or ischemic stroke. miR-140 levels are significantly reduced in human OA cartilageederived chondrocytes and synovial fluid. Overexpressing miR-140 in primary human chondrocytes promoted collagen II expression and inhibited MMP-13 and ADAMTS-5 expression (Si et al., 2017). These authors demonstrated that intra-articular injection of miR-140 can alleviate OA progression by modulating ECM homeostasis in rats and may have potential as a new therapy for OA. In an OA rat model, Wang et al. (2016) confirmed the effects of miR-98 on apoptosis in cartilage cells in vivo. MiR-98 expression is reduced in the cartilage cells of OA patients and the overexpression of miR-98 inhibits cartilage cell apoptosis, whereas inhibition of miR-98 leads to cartilage cell apoptosis. Recently, Cong et al. (2017) identified 46 differentially expressed miRNAs involved in autophagy, inflammation, chondrocyte apoptosis, chondrocyte differentiation and homeostasis, chondrocyte metabolism, and degradation of the ECM. Additionally, these authors identified a wide range of miRNAs that have been shown to be differentially expressed in OA. The function of upregulated miRNAs primarily target transcription. These miRNAs may be useful as diagnostic biomarkers and/ or may provide new therapeutic targets in OA.

CORRELATION OF CIRCULATORY BIOMARKERS WITH RADIOGRAPHIC/ IMAGING BIOMARKERS AND SYMPTOMS OF OSTEOARTHRITIS Although circulatory biomarkers, particularly of inflammation, are poorly correlated with radiographic or imaging evidence of OA, they are often used for early detection of this disease (Orita et al., 2011). Patra and Sandell (2011) reported that biochemical markers offer potential nonradiographic alternatives to detect early, nonsymptomatic OA. Accumulating evidence supports that COMP predicts MRI cartilage loss and appears as a useful biomarker of early OA (Williams and Spector, 2008). Combinations of cartilage-derived and bone-derived biomarkers have been used in the context of OA presence, severity, and bone turnover in cartilage integrity to assess the impact of treatment (Clark et al., 1999; Patra and Sandell, 2011). Circulatory

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biomarkers of inflammation have been used to profile OA progression attesting to the inflammation of OA joints. Stannus et al. (2010) found that the circulating levels of IL-6 and TNF-a are associated with knee radiographic OA and knee cartilage loss in older adults. Berry et al. (2011) investigated the relationship of serum markers of cartilage metabolism to imaging and clinical outcome measures of knee joint structures. Pelletier et al. (2010) reported a decrease in serum levels of MMPs in prediction of the disease-modifying effects of OA drugs assessed by quantitative MRI in patients with knee OA. Of course, there are many other studies that have described correlation between body fluid biomarkers and joint inflammation and cartilage degeneration/loss.

CONCLUDING REMARKS AND FUTURE DIRECTIONS OA is a disease of the entire joint but primarily causes degeneration of articular cartilage. Currently, there are no reliable, quantifiable, and easily measured biomarkers that provide an earlier diagnosis, inform on the prognosis of disease, and monitor responses to therapeutic modalities. Available biomarkers also lack sensitivity and specificity. Biomarkers of both cartilage proteoglycan and noncollagenous, nonproteoglycan components of cartilage can be detected following their release as a result of turnover and disease. Therefore, continuing quests are underway to identify, test, validate, and qualify biomarkers of OA. Genetic and genomic biomarkers could be essential to stratify OA patients and assess their risk of developing OA later in life with different underlying driving etiologies (van Meurs, 2017). Many molecular “omic” advancements will be made in the near future to the genetic and epigenetic data. Imaging continues to play an important role in OA research, where several exciting new technologies and computer-aided analysis methods are emerging to complement the conventional imaging approaches. New biomarkers are needed to distinguish between catabolic and maintenance events because many existing biomarkers reflect normal cartilage turnover, tissue repair, or ECM remodeling. Circulatory microRNAs are not only offering early detection of disease but also providing basis for the development of novel targeted therapies for OA. There are still many challenges that we have yet to overcome in the near future.

Acknowledgments The authors would like to thank Ms. Robin B. Doss and Ms. Denise M. Gupta for their technical assistance in preparation of this chapter.

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53 Pathological Biomarkers in Toxicology Meliton N. Novilla1,2, Vincent P. Meador3, Stewart B. Jacobson1, Jessica S. Fortin4 1

Shin Nippon Biomedical Laboratories USA, Ltd, Everett, WA, United States; 2School of Veterinary Medicine, Purdue University, West Lafayette, IN, United States; 3Covance Laboratories, Inc., Global Pathology, Madison, WI, United States; 4College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States

INTRODUCTION Simply defined, a biomarker is “a specific physical trait or measurable biologically produced change in the body connected with health or disease conditions” (Hunt, 2009). In toxicology practice, biomarkers are associated with some aspect of normal or abnormal biological function resulting from exposure to drugs, food additives, biopharmaceuticals, medical devices, environmental chemicals, and plants. From the three broad biomarker categories of exposure, effect, and susceptibility (Timbrell, 1998; Barr and Buckley, 2011), this chapter focuses on biomarkers of effect, limited to morphologic and clinicopathologic alterations in organs and tissues from which diagnoses are based. Brief descriptions of historical advancements are given, from the systematic practice of autopsies around 1346 and the invention of the light microscope in 1600 and its use in medicine beginning in the 1800s (Rosai, 1997). The 1800s to the recent past, considered to be the diagnostic era, was the period during which pathologists were principally involved in delivering diagnoses of disease conditions (Hunt, 2009). Pathologists arrived at their conclusions from the anamnesis, gross examination of specimens and later histopathologic examination after the light microscope was introduced into medical practice. From the 1960s to the 1990s, the inclusion of special pathology techniques, such as electron microscopy, confocal laser scanning microscopy, immunohistochemistry, and in situ hybridization, facilitated the transition of diagnostic pathology toward a prognostic and therapeutically oriented discipline (Rosai, 1997; Schnitt, 2003; Hunt, 2009). Now, sophisticated and novel technologies that relate to biomarkers of effect, exposure, and susceptibility to disease are becoming available, as

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00053-0

evidenced by the many chapters in this book. These biomarkers aid in disease diagnosis, understanding pathogenesis, defining prognosis, and helping to guide selections of the best options for therapy. This chapter provides examples of pathological biomarkers, with emphasis on morphologic (gross and microscopic) and clinicopathologic changes. Biomarker examples of the effects of investigational drugs, biologicals, medical devices, environmental contaminants, and poisonous plants are given when the biomarker has been validated and applied in preclinical and clinical studies conducted in humans and animals. However, because of space limitations, not all information can be included; hence, apologies are extended to colleagues whose work has been omitted or inadvertently missed.

DIAGNOSTIC PATHOLOGY Among the most significant historical advances in the evolution of medicine was the systematic practice of the autopsy/necropsy beginning in the 14th century, followed by the use of the light microscope in the 20th century. Restrictions against opening the human body after death were eased during the Black Death pandemic (1347e1350). Prior to this development, dissection (vivisection) had been carried out since ancient times for reasons related to animistic and naturalistic philosophies rather than to determine the cause of the disease. Hippocrates (468e377 BC), founder of a school of medicine in ancient Greece, taught that disease resulted not from divine or supernatural origin but from natural causes (King and Meehan, 1973). Hippocrates was the first great naturalistic physician and his concepts influenced the course of scientific medicine ever after. However, it

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would be almost 1800 years after his death before the autopsy found its place in medicine. Prior to these milestone events, diagnoses were based on clinical signs and symptoms. It has been reported that before the first histologic examination of colon cancer in humans, diagnosis was based on clinical observations of bowel symptoms, cachexia, and abdominal mass, which were deemed sufficient to confirm the diagnosis, as late as 1990 (cited by Hunt, 2009). Many years elapsed before autopsies became accepted practice; hence the medical practitioner had to have observed the cardinal signs of inflammation: rubor, tumor, calor, dolor et functio laesa, learned to recognize gross abnormalities from apparently normal organs and tissues, and applied knowledge of prevailing maladies at the time. Gross findings were considered in disease diagnosis after physicians performed autopsies on dying patients “to know more clearly the illnesses of their bodies” (cited by Rosai, 1997). The systematic performance of the autopsy ushered the birth of the discipline of pathology in human medicine and, ipso facto, in veterinary medicine. Rosai (1997) reported that those who practiced the discipline of pathology employed morphological techniques to explain symptoms and signs, determine the cause of death, guide therapy, and predict the evolution of disease. With the introduction of the light microscope and other diagnostic techniques in the 20th century, pathology practice evolved to its present state. For the toxicologic pathologist, adequate training and ever-increasing improvements in processes and procedures have resulted in high-quality tissue specimens, proper identification of significant histopathologic findings, and accurate, correctly interpreted diagnoses (Crissman et al., 2004; Van Tongeren et al., 2011). According to Buck et al. (1973), the accurate diagnosis of toxicosis is based on history, clinical signs, postmortem findings, chemical analysis, and laboratory animal tests. Depending on data availability, diagnoses rendered may vary from suspected (presumptive or preliminary) to definitive or confirmatory as in the following examples.

Enzootic Hematuria Some cattle at a ranch in Cotabato province, Mindanao Island in the Philippines were clinically observed to have been excreting bloody urine by the farm veterinarian. Leptospirosis was diagnosed earlier following the isolation of Leptospira pomona by the Animal Disease Diagnostic Laboratory, University of the Philippines College of Veterinary Medicine (UPCVM), Diliman, Quezon City. However, hematuria persisted even after treatment; hence, a team consisting of a microbiologist, parasitologist, and pathologist from

the UPCVM were invited to conduct field necropsies and collect samples for additional tests. During the inspection and tour of the ranch, lush growths of bracken fern were found alongside pasture fences and hillsides on the ranch; hence, bracken fern intoxication was suspected. Fenced cattle are known to be exposed to toxins, such as ptaquiloside and other toxins, mutagens, and carcinogens, from consumption of tips of crosiers and young fronds of the bracken fern along with pasture grasses during grazing (Panter et al., 2011; Panter et al., 2012). At necropsy, the urinary bladder in three of five animals contained residual reddish urine and multiple red nodules on the bladder mucosa. The nodules correlated histologically with hemorrhagic and proliferative lesions, consistent with those described in bracken fern intoxication (Pamukcu et al., 1976; Jones and Hunt, 1983). The red nodular growths on the urinary bladder mucosa were gross pathological biomarkers. Maxie (1985) reported that more than 90% of cattle with enzootic hematuria had urinary bladder tumors. Being associated with the clinical signs of bloody urination and probable prolonged ingestion of bracken fern, the bladder tumors confirmed the suspected diagnosis of bracken ferneinduced toxicity in the cattle herd. Clinicopathological findings that might be encountered are anemia, leucopenia, monocytosis, thrombocytopenia, hypergammaglobulinemia, microhematuria, and proteinuria (Perez-Alenza et al., 2006). Regarding L. pomona infection, Chirathaworn and Kongpan (2014) reported that several inflammatory mediators were higher in susceptible animals versus resistant hosts. Immune responses with cytokines/chemokines and serum proteins (TNFa, IL-6, IL-8, and PTX3) that were induced following Leptospira infection may be correlated positively with mortality and were suggested to be biomarkers for disease severity in human with leptospirosis. There are several studies on the expression of biomarkers in bovine enzootic hematuria (BEH) bladder lesions to detect molecular changes in the different stages of the disease. Up to 13% of the bladder lesions were found to be nonneoplastic and only half of the neoplastic lesions were malignant. The urothelial carcinoma was the most frequent tumor type (Carvalho et al., 2006; Peixoto et al., 2003). Cytokeratin is a protein found in the intracytoplasmic cytoskeleton of epithelial cells commonly used as an immunohistochemistry marker to identify epithelial cells in normal or malignant state. Cytokeratin 7 (CK7) is expressed in the urothelium, and this protein is frequently used to confirm the urothelial origin of tumors (Moll, 1998). There is a conserved transmembrane protein across species namely uroplakins (UPS) that is expressed in the superficial urothelial cells. The marker uroplakin III (UPIII) is also routinely

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used to confirm the urothelial origin of tumors. However, in humans and domestic animals, a loss of expression can occur in high-grade urothelial tumors (Ambrosio et al., 2001; Ohtsuka et al., 2006; Ramos-Vara et al., 2003). An immunohistochemical study was performed on epithelial bladder tumor samples from 37 animals affected with BEH. In both high-grade and high-stage urothelial carcinomas, there was loss of UPIII and CK7 expression. Interestingly, there was immunoreactivity for p53 in high-grade and high-stage carcinomas (Cota et al., 2013).

Cyclopia Consumption of another toxic plant, Veratrum californicum, has been associated with distinctive teratogenic abnormalities in ruminants (Burrows and Tyrl, 2001). Jervoline alkaloids in this plant such as cyclopamine, cycloposine, and jervine have been incriminated in producing teratogenic effects in the offspring of ewes and less commonly cows and goats (Panter et al., 2011). Pregnant ewes ingesting the plant from the 12th to the 14th day of gestation may have prolonged gestation due to the absence of the pituitary gland in the malformed offspring. The most striking malformation is partial or complete cyclopia in which one eye or two fused eyes are in a single orbit with a median skin protuberance above. Other abnormalities such as cleft palate and limb reductions have also been described. The gross findings and history of dietary exposure are presumptive diagnostic pathological biomarkers of the toxic plant’s teratogenic effect.

Taxus (Yew) Poisoning There are no characteristic gross or microscopic tissue alterations of Taxus (yew) toxicity. These evergreen plants are ornamental shrubs that contain toxic alkaloids (taxines) and irritant oils, which have been reported to cause sudden death in humans and a variety of animals. All parts of the plant except the aril are highly poisonous, with death attributed to the cardiotoxic effects of taxine alkaloids (Wilson and Hooser, 2018). In ruminants, nonspecific myocardial hemorrhages and focal nonsuppurative interstitial myocarditis have been reported, but diagnosis is usually based on a history of known or potential exposure to the yew plant. In the dairy cow ingesting trimmings of the plant, the presence of leaves, stems, twigs, and seeds or remnants thereof in the stomach contents is diagnostic and recognized as the gross pathological biomarker of yew toxicity. In horses where extensive mastication and digestion of the plant occur, stomach contents may be difficult to assess by the naked eye. Microscopic examination for plant parts and identification of taxine alkaloids in the stomach

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contents by the gas chromatography/mass spectrometry (GC/MS) method have been used to confirm diagnosis (Tiwary et al., 2005). Cardiotoxic alkaloids from the yew leaves (Taxus baccata) have been identified and quantitated in perimortem samples of serum and gastric contents in human medicine (Musshoff et al., 1993; Strı´brny´ et al., 2010). The main substance identified by HPLC and GC-MS (gas chromatography/mass spectrometry) was 3,5-dimethoxyphenol, the aglycone of taxicatine. In cases of intoxication by yew, 3,5-dimethoxyphenol can be used as a marker.

Ergotism Gross lesions occur on the extremities of cattle grazing on pastures infected with the fungi Claviceps purpurea and Neotyphodium coenophialum or fed grain contaminated with the ergot alkaloids, ergotamine, and ergovaline, respectively. One week after consumption of C. purpurea, swelling and redness of the extremities, particularly of the posterior limb, develop and the tips of the ear and tail have gangrene, which may slough off about 2 weeks following ingestion of the ergovaline-producing fungus; necrosis (dry gangrene) of the distal extremities is observed (Jones and Hunt, 1983). The gross observations are indicative of peripheral vasoconstriction induced by the ergot alkaloids produced by the fungal organisms. Interestingly, an ELISA test to detect ergot alkaloids is available and offered by Randox Food Diagnostics. The Ergot Alkaloid ELISA kit is validated for flour and seed. The ELISA test offers excellent limits of detection for the toxin ergotamine at 1 ppb. GC/MS is routinely used for identification and confirmation of ergot alkaloids. For further details on toxicity and biomarkers of ergot alkaloids poisoning refer to Gupta et al. (2018).

Morphologic (Gross and Microscopic) and Clinical Pathology Until the light microscope was incorporated into medical practice, gross pathology was the primary means to establish a diagnosis (Hunt, 2009). However, 200 years elapsed from the invention of the very first microscope in 1600 until it was incorporated into medical practice (Hagdu, 2002). Although Anton Van Leeuwenhoek (1632e1723) was the first to use the light microscope to study organisms and Robert Hooke (1635e1703) was the first to use the microscope in the practice of medicine, integration of microscopy into medical practice did not occur until Rudolf Virchow (1821e1902) introduced the light microscope in pathology, allowing the study of cellular and later subcellular events for a better understanding of disease. The use

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of the microscope by pathologists enhanced their skills and diagnostic acumen. With the microscope, identification of causative agents and pathognomonic lesions facilitated diagnosis. For instance, light microscopic identification of Negri bodies in neurons of a dog is pathognomonic for rabies. However, few pathognomonic lesions are encountered by the toxicologic pathologist in preclinical safety and toxicity studies.

Solar Injuries Damage to the skin by ultraviolet (UV) light has resulted in sunburn, solar dermatitis, and neoplasia (Hargis and Ginn, 2006). Solar injuries begin clinically with the cardinal signs of inflammation: rubor, tumor, calor, dolor et functio laesa. Gross lesions of erythema, scaling, and crusting occur where there is little or no pigment or hair in the affected skin, whereas the haired or pigmented areas of the skin are unaffected. Microscopically, epidermal acanthosis and follicular hyperkeratosis (comedones) develop. Multiple layers of compacted stratum corneum may form cutaneous horns and the comedones can rupture (furunculosis), releasing follicular contents into the dermis. The endogenous foreign materials, including follicular stratum corneum, hair shafts, and sebum may get infected and cause an inflammatory response with secondary bacterial infection. The gross and microscopic findings and distribution pattern support the diagnosis of solar dermatitis.

Skin Photosensitization In primary skin photosensitization, gross lesions occur in nonpigmented and lightly pigmented skin and poorly haired areas of the body that include erythema and edema, followed by blisters, exudation, and necrosis and sloughing of necrotic tissue (Hargis and Ginn, 2006). Histologically, there is coagulative epidermal necrosis, which may extend to the superficial dermis, follicular epithelium, and adnexal glands. Fibrinoid degeneration and thrombosis of dermal vessels result in infarction and sloughing of necrotic tissues and secondary bacterial infection. Skin damage occurs as a result of ingestion of preformed photodynamic substances (e.g., helianthrone, furocoumarin, psoralens, phytoalexins) contained in a variety of poisonous plants, or the administration of phenothiazine, which is converted to photoreactive metabolite in the intestinal tract. The photodynamic substances are absorbed into the skin where they react with UV light, releasing energy that produces reactive oxygen molecules, degranulation of mast cells, production of inflammatory mediators, and cell damage. The gross findings on the skin associated with exposure to photoreactive substances are considered to be the pathological biomarkers of primary

photosensitization. The results of one research group suggest that changes of cell surface thiols and/or amines may be useful biomarkers to predict photosensitization potential of chemicals (Oeda et al., 2016). Further research is needed to find useful biomarkers for skin photosensitization. Secondary (hepatogenous) photosensitization occurs primarily in herbivorous animals, but any animal with massive hepatic disorder given a chlorophyll-rich ration can be affected (Hargis and Ginn, 2006). The liver fails to excrete phylloerythrin, a chlorophyll breakdown product formed in the intestinal tract due to inherited hepatic defects, primary hepatocellular damage, or bile duct obstruction from toxic plants, pyrrolizidine alkaloids, or mycotoxins. For example, gross findings associated with the mycotoxin sporidesmin, produced by the fungus Pithomyces chartarum, included bile-stained liver with prominent bile ducts, dilated with bile and surrounded by periductal edema. In addition, cholestasis and facial eczema were also observed in affected sheep.

Skin Neoplasia Chronic exposure to sunlight has been stipulated by Madewell (1981) as the best-known etiologic factor for many skin tumors in animals. After chronic exposure, the skin becomes wrinkled and thickened due to epidermal hyperplasia, fibrosis, and elastosis, which could progress to neoplasia, such as hemangiomas, hemangiosarcomas, and squamous cell carcinomas. The most frequently diagnosed malignant tumor in cattle is ocular squamous cell carcinoma. Although the cause is thought to be multifactorial, cattle with nonpigmented eyelids and conjunctiva are predominantly affected. Premalignant stages including plaques, keratomas, papillomas, and the squamous cell carcinomas are found in the junction of the cornea and sclera, the third eyelid, and on the margins of upper and lower eyelids. Scrotal hemangioma in boars is rarely reported in North America but common in tropical countries. According to Szcech et al. (1973), the probable index case in the United States was a Chester White boar from a swine-breeding establishment in Indiana. Grossly, a hemorrhagic, 1.0
1.5 cm, dark blue and pedunculated wartlike mass was located at the junction of the scrotum and perineum. Following surgical excision and histopathology, the mass was diagnosed as a capillary hemangioma. Novilla (1989) reported a high number of scrotal capillary hemangiomas, 317 (31% of 1027 boars were affected) from 17 farms in the Philippines (Fig. 53.1). Light-skinned purebred boars had more and larger scrotal tumors than dark-skinned boars had. Two- or three-way crossbred boars tended to have growths in nonpigmented areas of the scrotal skin. Grossly, the

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FIGURE 53.1 Scrotal capillary hemangiomas in Landrace Crossbred boars from the Philippines. Courtesy of Dr. D. A. Novilla, USDA, FSIS Morristown, Tennessee.

lesions varied from tiny reddish blotches or blebs to pedunculated warty and cauliflower-like masses. Histologically, the dermis contained lobules of capillary-like vascular spaces lined by mostly endothelial-type cells supported by delicate connective tissue stroma. Gross and microscopic features were consistent with previous descriptions of scrotal hemangioma (Munro et al., 1982) and presumed to be due to exposure to sunlight. According to Kycko and Reichert (2014), many candidate biomarkers were identified and proven to be not specific enough to be further investigated and developed as a diagnostic tool for skin cancer. Reports concerning proteomic biomarkers of spontaneously occurring tumors in animals provided preliminary results up to this point. Most studies resulted in protein changes involving a small number of cases, which limits the significance of the findings. The candidate biomarkers are usually nonspecific for a particular type of cancer and, in most cases, components of the acute phase response, which is not necessarily associated with cancer.

Iatrogenic Acromegaly in Dogs Dogs administered progestogens indirectly develop a syndrome of growth hormone excess (La Perle and Capen, 2012). Injection of medroxyprogesterone acetate to prevent estrus in dogs has been shown to produce off-target adverse effects. There is expression of the growth hormone gene in the mammary gland, elevated circulating levels of growth hormone, and clinical manifestations of acromegaly. Affected animals have coarse facial features with markedly folded and thickened skin of the face, enlarged abdomen, and expansion of interdigital spaces. According to Kooistra (2006), diagnosis of GH excess is established by measuring basal plasma GH levels. The

basal plasma GH level in acromegalic animals in many instances exceeds the upper limit of the reference range. The diagnosis may be supported by the measurement of elevated plasma IGF-I levels. IGF-I is bound to proteins and for this reason its level is more stable versus GH. However, there is some overlap in plasma IGF-I levels between healthy animals and individuals with acromegaly.

Endocrine Alopecia Endocrine alopecia may occur from iatrogenic hyperadrenocorticism or hyperestrogenism in dogs from administration of glucocorticoids or estrogens, respectively. Other reported causes are bilateral adrenocortical hyperplasia secondary to a pituitary tumor, functional adrenocortical hyperplastic nodule, or neoplasm inducing hyperadrenocorticism. Dogs with hyperadrenocorticism have truncal alopecia, sparing the head and limbs, distended abdomen, calcinosis cutis, and thin skin. Histologically, the epidermis, dermis, and hair follicles are atrophic with follicular hyperkeratosis and calcinosis cutis in hyperadrenocorticism. Hyperestrogenism associated with ovarian cysts in females and functional Sertoli cell tumor of the testis in males also induces endocrine alopecia (Hargis and Ginn, 2006). In hypestrogenism, gross findings include symmetrical alopecia and hyperpigmentation over the posterior trunk and limbs (Fig. 53.2). In addition, male dogs may develop pendulous prepuce and enlargement of the nipples and prostate, whereas female dogs have enlarged vulva and abnormal estrus cycle. Epidermal and follicular hyperkeratosis and follicular atrophy are observed in hyperestrogenism. A history of iatrogenic exposure to glucocorticoids or estrogen together with clinical and morphologic findings supports a diagnosis of endocrine alopecia.

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(A)

(B)

FIGURE 53.2

Hyperestrogenism, iatrogenic diethylstilbestrol, dog. Reproduced from Pathologic Basis of Veterinary Disease (Fig. 17e69), fourth ed. p. 1223. Academic Press/Elsevier.

Alopecia and Inhibition of Hair Growth (Mimosine Toxicity) Farin˜as (1951) reported the utilization of the legume Leucaena glauca, also known as ipil-ipil, Koa haole, or Santa Elena tree, as a fodder and pasture crop in the tropics. However, loss of hair has been reported after ingestion of seeds in native women and seeds and foliage in animals. The toxic nonprotein free amino acid, identified as mimosine or leucenol, was shown to act as a tyrosine analog capable of competitive inhibition of tyrosinase and inhibition of thyroxine decarboxylase (Crounce et al., 1962). Dietary administration of the purified toxic principle and ground seeds to mice produced hair growth inhibition and loss of hair, probably from inhibition of mitosis of hair follicles, especially in the matrix (Montagna and Yun, 1963). Growing (anagen) hairs were affected, and there was complete recovery following withdrawal from treatment. Grossly, the skin appeared thin and atrophic. Histologic findings of absent mitosis, destruction of matrix cells of the hair follicles, and extracellular melanin were observed. Morphologic findings together with a history of exposure to ipil-ipil seeds and foliage are suggestive of mimosine-induced alopecia.

Acute Bovine Pulmonary Edema and Emphysema Acute bovine pulmonary edema and emphysema (ABPE or “fog fever”) occurs in adult beef-type cattle grazing fall pastures on changing from sparse dry range to lush green pastures (Dungworth, 1985). Gross findings are observed primarily in the dorsocaudal lobes of the lungs, which are pale, soft, and rubbery in texture and moist on cut section. Microscopically, diffuse

interstitial pneumonia with severe alveolar and interstitial edema and interlobular emphysema are observed. There is hyaline membrane formation within alveoli and hyperplasia of type II alveolar epithelial cells in animals that survive for several days. ABPE is related to increased amounts of L-tryptophan in the ingested pasture grasses, which are metabolized to 3-methyl indole (3MI) in the rumen, absorbed into the bloodstream, and carried to the lung. In the lung, 3MI is converted by mixed function oxidases (MFOs) in Clara cells to a pneumotoxic compound that produces extensive necrosis of bronchiolar epithelial cells and type I pneumocytes and increases alveolar permeability leading to edema, interstitial pneumonia, and emphysema. Experimentally reducing the ruminal conversion of L-tryptophan to 3MI prevents the development of the condition. The history and morphologic findings support the presumptive diagnosis of ABPE.

Toxic MyopathydIonophore Toxic Syndrome Ionophore toxic syndromes have resulted from overdosage, misuse, and drug interactions of feed additives, including formulations with monensin, lasalocid, salinomycin, narasin, maduramicin, laidlomycin, or semduramicin. Target organs damaged by toxic doses of ionophores include the heart and skeletal muscles in all species studied (reviewed by Novilla, 2012). The most important change is a toxic myopathy characterized by focal areas of degeneration, necrosis, and repair in cardiac and skeletal muscles with a variable inflammatory component (Novilla and Folkerts, 1986; Van Vleet et al., 1991). The development of muscle lesions varies among domestic species. The heart is primarily affected in horses, skeletal muscle in pigs and dogs, and there is about equal tissue predilection in rats,

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chickens, and cattle. In addition, neurotoxic effects have been reported for lasalocid (Shlosberg et al., 1985; Safran et al., 1993), narasin (Novilla et al., 1994), and salinomycin (Van der Linde-Sipman et al., 1999). Neuropathic changes occurred in peripheral nerves and the spinal cord. Focal swelling, fragmentation, loss of axons, and formation of digestion chambers filled with macrophages were observed in both sensory and motor nerves, and there was vacuolation with swelling, degeneration, and fragmentation of myelin sheaths and axons in the spinal cord. It is not easy to diagnose ionophore toxicoses. Clinical signs and muscle lesions of monensin toxicoses are not pathognomonic. In the absence of proof of a gross feed mixing error with monensin (usually >5
), the diagnostic pathologist must go through a process of exclusion of potential causes of the lesion. Confirmatory diagnosis requires laboratory assays to determine the identity and amounts of the ionophore involved or concurrent use of an incompatible drug. In humans, there are three publications of intentional exposure to monensin. According to Kouyoumdjian et al. (2001), a 17-year-old Brazilian male admitted ingesting monensin premix (Rumensin, exact amount unknown), probably to develop muscle. Instead he fell ill, was hospitalized, and died from acute rhabdomyolysis with renal failure. Although the amount of monensin ingested in this case was not estimated, in another case cited by the authors, two deaths among six people who consumed baked goods made with premix were attributed to monensin exposure of at least 10 times the optimum daily dose fed to cattle. In another report from Brazil, a 16-year-old farmworker who ingested approximately 500 mg of monensin (5 g of Rumensin 100 premix) “to become stronger” developed an early and severe rhabdomyolysis followed by acute renal failure, heart failure, and death (Caldeira et al., 2001). More recently, a 58-year-old man after ingesting 300 mg monensin (self-treatment) for suspected brain toxoplasmosis presented with 8 days of vomiting and abdominal pain had severe rhabdomyolysis without renal toxicity and survived (Blain et al., 2017). Echocardiography revealed decreased ejection fraction from 69% to 56% on his echocardiogram. Clinical pathology findings included decreased total CK (from peak above 100,000 to 5192 U/L) following 15 days of aggressive hydration and sodium bicarbonate therapy. Consequent to ionophore-induced muscle damage, significant increases in enzymes of muscle origin occur (Amend et al., 1981); Van Vleet et al., 1983). Levels of aspartate transaminase, creatine phosphokinase, lactic dehydrogenase, alkaline phosphatase (ALP), blood urea nitrogen (BUN), and total bilirubin (TB) are

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elevated; calcium (Ca) and potassium (K) levels transiently decrease, whereas sodium (Na) levels are within normal limits. Cardiac troponins (both cTnI and cTnT) were reported to be highly sensitive and specific biomarker of myocardial injury in humans (O’Brien, 2008) and animals. The published range of cardiac troponin I (cTnI) in clinically normal horses was 0.0e0.06 ng/mL compared to that of 0.08e3.68 ng/mL found in six horses gavaged with a single dose of 1.0e1.5 mg monensin/kg body weight (Divers et al., 2009; Kraus et al., 2010). Because these monensin doses are close to the LD50 of 1.38 mg/kg body weight (Matsuoka et al., 1996), it was not surprising that the biomarker revealed the presence of the myocardial injury caused by toxic doses of monensin, as it would for any significant injury to heart muscle. However, the presence of heart injury is insufficient to confirm a diagnosis of monensin toxicity, so toxic exposure must be demonstrated. Ionophore toxicoses may be suspected when there is a history of a feed-related problem in a group of animals; clinical signs of anorexia, diarrhea, labored breathing, depression, locomotor disorder, colic, recumbency, and death; lesions affecting heart and skeletal muscles; or congestive heart failure. The clinical signs and lesions induced by toxic levels of ionophores are not pathognomonic. However, a history of recent introduction of newly formulated feed or supplement to a herd or flock in which signs and lesions are present may cause one to suspect that acute intoxication has occurred. Dose and time factors influence the severity and distribution of lesions. Animals that die soon after exposure may not have muscle lesions discernible by light microscopy. Lesions are likely to be found in animals that survived longer than a week. Although a presumptive diagnosis of ionophore toxicosis can be made based on history, clinical signs, lesions, and considerations of the differential diagnosis, specific assays are needed for confirmatory diagnosis. Confirmatory diagnosis requires efficient laboratory assays to determine the identity and amounts of the ionophore involved and a thorough consideration of differential diagnoses. For suspected cases of monensin toxicity in horses, the exertional myopathies, such as equine rhabdomyolysis (Monday morning disease) and hyperkalemic periodic paralysis, plant poisoning from coffee senna and white snakeroot, blister beetle intoxication, colic, and laminitis (Whitlock et al., 1978; Amend et al. , 1981), should be ruled out as differential diagnoses. Complete herd history, clinical examination, successful supportive treatment, and necropsy may help differentiate these conditions from monensin and other ionophore toxicoses (Tables 53.1 and 53.2).

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53. PATHOLOGICAL BIOMARKERS IN TOXICOLOGY

Clinical Pathology Biomarkers of Ionophore Toxicoses

Elevated

Decreased

No Change

Aspartate transaminase

Calcium

Sodium

Creatine kinase

Potassium

Lactic dehydrogenase Alkaline phosphatase Blood urea nitrogen Total bilirubin Cardiac troponin 1 Reproduced from Veterinary Toxicology (Table 80.6 Some biomarkers of ionophore toxicity), third ed. Academic Press, Elsevier, p. 50.

Drug-Eluting Stents Stents are commonly used to treat diseased or partially occluded coronary or peripheral arteries by acting as a scaffold to hold open the dilated segment of artery following balloon angioplasty. Early problems associated with use of bare metal stents included

TABLE 53.2

restenosis of the artery due to thrombosis and neointimal proliferation. Drug-eluting stents (DESs), first available in the United States in 2003, were developed to decrease restenosis and improve patient outcomes (Fig. 53.3). DESs are polymer-coated stents impregnated with antiproliferative or antiinflammatory drugs. These stents release drug (eluate) following direct contact with the arterial wall. Common drugs used with DES include sirolimus and paclitaxel, both members of the taxine family used as immunosuppressive or oncologic chemotherapeutic agents. Specific biomarkers of toxicity include evaluation of vessel injury, inflammation, endothelialization, neointimal proliferation, coverage of stent struts, and stent apposition to the artery wall, which can be characterized microscopically with hemotoxylin and eosin, elastin, and trichrome stains. Standardized scoring systems are commonly used to evaluate these parameters. Neointimal proliferation can also be estimated with standard immunohistochemical methods for cell proliferation such as Brdu (bromodeoxyuridine), proliferating cell nuclear antigen (PCNA), or Ki-67.

Summary of Cardiac and Skeletal Muscle Biomarkers

Biomarkers

Sources

Interpretation

Cardiac troponin 1 (cTn1)

Cardiac muscle

Cell injury/necrosis

Cardiac troponin 1 (cTnT)

Cardiac muscle

Cell injury/necrosis

Heart-type fatty acid binding protein (H-FABP or FABP3)

Cardiac muscle and skeletal muscle

Cell injury/necrosis

Myoglobin

Cardiac muscle and skeletal muscle

Cell injury/necrosis

Myosin light chains (Mlc)

Cardiac muscle and skeletal muscle

Cell injury/necrosis

Creatine kinase (CK)

Cardiac muscle and skeletal muscle, brain, GI tract

Cell injury/necrosis

CK-MM

Cardiac muscle and skeletal muscle

Cell injury/necrosis

CK-MB

Cardiac muscle and skeletal muscle

Cell injury/necrosis

Lactate dehydrogenase (LD)

All muscle types, liver, RBCs

Cell injury/necrosis

Aspartate aminotransferase (AST)

All muscle types, liver, RBCs

Cell injury/necrosis

Skeletal muscle troponin I (sTnI)

Skeletal muscle

Cell injury/necrosis

Atrial natriuretic peptide (ANP)

Primarily cardiac atria

Atrial wall stretch

Brain natriuretic peptide (BNP, proBNP, NT-proBNP)

Primarily cardiac ventricles

Ventricular wall stretch

LEAKAGE MARKERS

FUNCTIONAL MARKERS

Reproduced from Faqi, A.S. A Comprehensive Guide to Toxicology in Nonclinical Drug Development. Academic Press/Elsevier, p. 459.

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HEPATOPATHIES

FIGURE 53.3 Actinomycin-D stent in rabbit iliac arteries. From Schwartz et al., 2004. JACC 44, 1373e1385; photo(s) of drug-eluting stents from Dr. S. B. Jacobson.

One study has indicated an association of restenosis occurrence after DES implantation with diabetes mellitus, circulating CD45-positive platelets, and neutrophil to lymphocyte ratio (Melnikov et al., 2017).

G

HEPATOPATHIES Glycogen Hepatopathy Glycogen accumulates in hepatocytes secondary to xenobiotics that increase glycogen synthesis or decrease glycogenolysis. Glucocorticoid analog, such as dexamethasone, are potent stimulators of glycogen synthesis, especially in dogs, causing centrilobular hepatocellular hypertrophy secondary to glycogen accumulation (Fig. 53.4). Glycogen accumulation extends into midzonal regions when the effect has greater magnitude. Liver size and weight are increased because of the increased hepatocellular glycogen accumulation. Ultrastructurally, hepatocytes have large accumulations of

Inset A G

L Inset B

FIGURE 53.4 Glycogen hepatopathy caused by corticosteroid administration to a dog. Courtesy of Dr. V. Meador, Covance Laboratories Global Pathology.

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glycogen free in the cytosol that displaces cytoplasmic components to the cell periphery. Interspersed in glycogen are lipid droplets.

Hepatotoxicity Secondary to Enzyme Induction Proliferation of smooth endoplasmic reticulum (SER) is a well-documented change seen secondary to administration of xenobiotics that induce cytochrome P450 enzymes (Guengrich, 2007). Hepatocytes have a full complement of cytochrome P450s (CYP) because one of the liver’s main functions is metabolism of foreign substances. Induction most commonly occurs in centrilobular hepatocytes. Not all xenobiotics that induce CYP cause proliferation of SER. Proliferation is dependent on the subset of CYP that are induced, as no single agent induces all CYPs. Cytochrome P450 enzymes are grouped into families (e.g., CYP1, CYP2, CYP3) with capital letters designating subfamilies (e.g., CYP1A, CYP1B, CYP1C) and numerals further identifying individual enzymes (e.g., CYP1A1). Not all CYP inducers cause SER proliferation and liver enlargement (Barka and Popper, 1967); however, SER proliferation is nearly pathognomonic for CYP induction. Classic inducers of SER proliferation are phenobarbital and its analog. Phenobarbital is metabolized by, and induces, cytochrome P4502B, the major membrane protein of SER (2d). Proliferation can be rapid and dramatic, with morphologic changes occurring within 4 days. Liver weight and size are increased. By light microscopy in Hematoxylin and Eosin (HE) sections, centrilobular hepatocytes are hypertrophied because of increased amounts of homogeneous eosinophilic cytoplasm. SER proliferation begins in centrilobular hepatocytes (Fig. 53.5). With increasing magnitude, proliferation will extend into midzonal regions and rarely to periportal hepatocytes. Withdrawal of the inciting xenobiotic is accompanied by a rapid regression of changes (Cheville, 1994). Effete SER forms aggregates that are removed from the cell by autophagocytosis (Feldman et al., 1980). Hepatotoxicity secondary to hepatocellular enzyme induction can occur through increased activation of xenobiotics to hepatotoxins (Zimmerman, 1999). Subsets of CYP inactivate xenobiotics while others can activate creating reactive electrophiles (Greaves, 2007). Aflatoxin B1, a mycotoxin, is activated to a number of metabolites, including exo-8,9-epoxide, a hepatocarcinogen. Acetaminophen, an analgesic, is activated to a reactive iminoquinone. Trichloroethylene (TCE), an industrial toxicant and previously used anesthetic, is activated to TCE oxide, which forms unstable protein adducts. Troglitazone, an antidiabetes drug, is metabolized to electrophilic reactive metabolites.

Inset A

Inset B

Control

P

C FIGURE 53.5 patocytes. Pathology.

Smooth endoplasmic reticulum proliferation in heCourtesy of Dr. V. Meador, Covance Laboratories Global

Peroxisome Proliferator-Activated Receptor Hepatopathy (Characteristic Lesion by Light and Electron Microscopy) in Rodents Peroxisomes are identified primarily in liver and kidneys, and they are located in cytoplasm often adjacent to mitochondria. Drug-induced effects on peroxisomes have been reported to increase numbers and cause qualitative changes in appearance, but decrease in numbers has not been reported. Stimulation of the hepatocytes with peroxisome proliferator-activated receptor (PPAR) a causes proliferation of peroxisomes (Fig. 53.6). Drugs that induce CYP4A, e.g., PPARa, cause proliferation of hepatic peroxisomes. The PPARa receptor is the transcription factor that activates CYP4A. In general, the change is quantitative causing an increase in numbers, which is appreciable in the liver, but has also been reported in the kidney. By light microscopy, liver has centrilobular hepatocellular hypertrophy, with enlarged hepatocytes having a finely stippled eosinophilic granular appearance. Liver weight can be increased. Changes in appearance of peroxisomes have been reported, but apart from disappearance of the crystalline core, qualitative changes are seldom detected. There is species susceptibility to peroxisome proliferation: rodents are most susceptible, with dogs, nonhuman primates, humans, and guinea pigs being poorly susceptible. For the purpose of this discussion, “disorder” is considered as any morphologic change of peroxisomes as evidenced by a quantitative and/or qualitative change compared to control. Compromise of

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HEPATOPATHIES

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Inset B

Inset A

FIGURE 53.6 Peroxisome proliferation in a rat given peroxisome proliferator-activated receptor alpha. Courtesy of Dr. V. Meador, Covance Laboratories Global Pathology.

hepatocellular integrity or function is generally minimal, until the magnitude of peroxisome proliferation change is marked, at which point direct effects of peroxisome proliferation may be related to the massive cellular enlargement physically leading to compromised cellular integrity and/or pressure-induced collapse of vascular and canalicular spaces. Diagnosis of peroxisome proliferation is by electron microscopy and staining of tissues with diaminobenzidine. The demonstration of lysosomal lamellar bodies by TEM might be considered as a pathologic biomarker of phospholipidosis. A readily accessible, reliable, and noninvasive clinical biomarker of phospholipidosis need to be found. Phospholipidosis results from a multiplicity of different molecules involving different tissues. One impediment is that each phospholipidosis-producing chemical must be evaluated independently (Chatman et al., 2009).

Iron Toxicosis Iron-containing products can increase iron pigment in hepatocytes and/or Kupffer cells. Excessive iron supplementation in dogs and cats has resulted in hemochromatosis due to iron overload in the liver. Cases of iron poisoning have occurred in suckling pigs administered iron dextran intramuscularly and in newborn foals administered ferrous fumarate to prevent anemia (Cullen, 2006). In severe toxicosis, there is significant mortality due to massive hepatic necrosis and affected piglets die soon after injection. There is yellowish brown discoloration (icterus) of tissues, notably those near the injection site. Although the yellowish brown

FIGURE 53.7 Icterus, hemolytic anemia, dog. Reproduced from Pathologic Basis of Veterinary Disease (Fig. 1e72), fourth ed. p. 58. Academic Press/Elsevier.

discoloration is considered a gross pathological biomarker in the case of iron dextran poisoning, icterus has been reported in other diseases and conditions with extensive liver damage; see Fig. 53.7.

Copper Poisoning in Sheep In ruminants, especially sheep, accumulation of copper in the liver may occur over time because of poor regulation of copper storage. Copper toxicity in sheep results from: (1) dietary excess from contamination of pastures from sprays or fertilizer or access to coppercontaining mineral blocks intended for cattle; (2) grazing on pasture grasses containing normal copper but inadequate levels of molybdenum, which antagonizes copper uptake by the liver; and (3) grazing on pastures with hepatotoxic plants such as Heliotropium, Crotalaria, and Senecio, which produce pyrrolizidine alkaloids (Cullen, 2006). Sudden release of the copper results in severe hemolytic anemia and extensive hepatic necrosis. The breakdown of erythrocytes results in release of hemoglobin, which stains the plasma pink and discolors the kidney dark blue and the urine dark red. Gross findings are usually characteristic and include generalized icterus, greatly enlarged “gunmetal blue” kidneys with hemorrhagic mottling, mildly enlarged, friable and yellowish liver, distended gall bladder with thick greenish bile, and enlarged brown to black spleen. Microscopically, there are multifocal areas of hepatocellular vacuolation and necrosis in the liver, and the kidney tubules have hyaline and coarsely granular hemoglobin casts resulting from intravascular hemolysis with glomerular filtration of hemoglobin into the uriniferous space.

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NEPHROPATHIES Polycystic Kidney Disease in Rats Dietary administration of diphenylthiazole to rats for 4e8 weeks resulted in three- to fivefold bilateral and symmetrical enlargement of the kidneys due to marked cystic changes (Carone, 1986). The most prominent enlargement occurs in the outer medulla with radially arranged dilated collecting tubules and occasional dilation of cortical collecting and distal tubules. The histologic correlates include focal degeneration and necrosis and mild tubular dilation of collecting ducts at 2 weeks of feeding and marked cystic change at 4e8 weeks. Recovery from the distinctive morphologic changes occurred after diphenylthiazole treatment withdrawal of 4e8 weeks. Urinary neutrophil gelatinase-associated lipocalin (NGAL) and IL-18 excretion have been shown to be slightly elevated in ADPKD. However, the increased level does not correlate with alterations in total kidney volume or kidney function. A potential explanation could be the discontinuity between individual cysts and the urinary collecting system in this pathology (Parikh et al., 2012).

Alpha 2-Microglobulin Nephropathy Abnormal accumulation of alpha 2-microglobulin (alpha-2 mu-globulin) in kidney lysosomes of male rats has been described in nephropathy resulting from exposure to a variety of chemicals, including D-limonene, a constituent of orange juice. The nephropathy is characterized by distinctive cytoplasmic hyaline droplets in epithelial lining cells of proximal convoluted tubules and granular casts at the junction of the inner and outer stripe of the outer medulla and linear mineral deposits in the descending limb of the loop of Henle. Accumulation of hyaline droplets leads to tubular apoptosis followed later by regeneration, exacerbation of spontaneous renal disease, and tumor formation.

Tubulointerstitial Nephritis A considerable number of xenobiotics can induce kidney injury. Most nephrotoxicants damage the renal tubules with many toxicants affecting specific segments. Microscopic findings include interstitial inflammation, edema, fibrosis, and atrophy. Acute kidney injury has been proposed to encompass the full spectrum of renal injury from minor elevations of serum chemistry values to anuric renal failure (Gwaltney-Brant, 2012). In druginduced tubulointerstitial nephritis (DTIN) microscopic findings include interstitial inflammation, edema, fibrosis, and atrophy (Wu et al., 2010). Lack of correlation has limited the usefulness of established noninvasive diagnostic biomarkers in defining severity and

progression of kidney injury. For instance, renal azotemia (increased BUN) occurs only after approximately 75% of the nephrons have lost function (Turk et al., 1997). According to Wu et al. (2010), a renal biopsy is the gold standard for diagnosis, prognosis, and selection of therapies for DTIN. Urinary monocyte chemotactic peptide-1 (MCP-1) levels correlated with the degree of severity of acute lesions in DTIN. The roles of NGAL and a1-microglobulin in chronic lesions need further evaluation (Wu et al., 2010), and to monitor the progression rate of drug-induced chronic DTIN, urinary N-acetyl-b-D-glucosaminidase (NAG) and matrix metalloproteinases (MMPs) 2 and 9 are two possible candidates to consider (Shi et al., 2013).

Ethylene Glycol (Oxalate) Poisoning Toxic amounts of ethylene glycol, which is commonly used as antifreeze, get metabolized in the liver via alcohol dehydrogenase resulting in formation of oxalates and development of hypocalcemia. The latter occur from the formation of insoluble calcium oxalates that are widely deposited but most severely in the kidney, resulting in crystalluria, which is used as a biomarker for this toxicity. The precipitated calcium oxalates, seen better under polarized light as numerous pale yellow, birefringent (rosette-shaped) crystals in proximal convoluted tubules are consistent and characteristic in animals that die from ethylene glycol toxicity (Thrall et al., 1985; Stice et al., 2018). In addition to ethylene glycol, oxalate poisoning can result from ingestion of oxalate salts, plants with toxic levels of oxalates, and plants infected with fungi that produce oxalates. Grossly, the kidneys are pale, swollen, and feel gritty when cut. Microscopically, degeneration and necrosis of proximal convoluted tubules associated with pale yellow variable-shaped crystals forming sheaves, rosettes, or prisms within proximal tubular lumens, and cytoplasm of the lining of epithelial cells and interstitial spaces confirm the diagnosis of oxalate poisoning.

Melamine Poisoning Melamine cyanurate crystals (MCA) precipitate in the kidney of dogs and cats ingesting commercial pet food contaminated with melamine and cyanuric acid (Bischoff, 2018). Grossly, there is hemorrhage and interstitial edema primarily of the medullary region and histologic correlates of tubular degeneration and necrosis of distal straight tubules associated with pale yellow to brown crystals that were fan to starburst to globular in appearance within the lumen of the distal straight and collecting tubules. The more downstream location and different morphology of the crystals is diagnostic of MCA poisoning.

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NEPHROPATHIES

FIGURE 53.8 Lead-acid fast inclusion bodies in rat. Reproduced from Pathologic Basis of Veterinary Disease (Fig. 11e41), fourth ed. p. 654. Academic Press/Elsevier.

Lead Toxicity In the kidney, toxic tubular necrosis is caused by several classes of naturally occurring and synthetic compounds, including heavy metals such as lead, cadmium, thallium, and inorganic mercury. The nephrotoxic heavy metal cannot be identified by renal lesions alone, except for lead. Exposure to old paints and batteries may result in lead toxicity in which renal tubular epithelial cells have irregularly shaped intranuclear inclusion bodies that are acid fast (Myers and McGavin, 2006; Fig. 53.8). The inclusion bodies, composed of lead protein complexes, are considered to be a diagnostic pathological biomarker for lead toxicosis.

Nephrotoxicity and Acute Renal Injury One major factor that explains the distinct susceptibility of the kidney to injury is the exposure to a variety of exogenous compounds (e.g., aminoglycosides, cisplatin, radiocontrast agents) and endogenous compounds (e.g., free hemoglobin after hemolysis, free myoglobin after rhabdomyolysis) via the blood flow. Both kidneys receive roughly 25% of cardiac output. Heavy metals (e.g., chromium, lead, and mercury), analgesics (including acetaminophen and nonsteroidal antiinflammatory drugs), aminoglycoside antibiotics (e.g., neomycin, gentamicin), immunosuppressive agents (e.g., cyclosporine, tacrolimus), and chemotherapeutic agents (e.g., cisplatin, carboplatin) are known common nephrotoxic drugs that cause kidney damage (Schnellman, 2008; Pazhayattil and Shirali, 2014). For druginduced renal dysfunction, the potential mechanisms underlying their toxicity include alterations in renal perfusion and glomerular filtration, tubular cell damage, and tubular obstructions.

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To detect and monitor renal dysfunction, clinical pathology end points such as BUN, serum creatinine (sCr), phosphorus, and urine-specific gravity has been established and used over the past decades. However, these parameters are relatively insensitive indicators in chronic injury and exhibit alteration only when more than two-thirds nephrons have functional deficit (Stockham and Scott, 2008). In addition to BUN and sCr, there are seven renal injury biomarkers with improved sensitivity that was qualified by the FDA to monitor druginduced renal injury in rats (Dieterle et al., 2010a,b). These seven renal injury biomarkers are kidney injury molecule-1, albumin, total protein, b2-microglobulin, cystatin C, clusterin, and trefoil factor-3 (Rosenberg and Paller, 1991; Witzgall et al., 1994; Ichimura et al., 1998; Han et al., 2002; Davis et al., 2004; Filler et al., 2005; Vaidya et al., 2006; Perez-Rojas et al., 2007; Bonventre et al., 2010; Dieterle et al., 2010a,b; Yu et al., 2010; Vlasakova et al., 2014). Clusterin and renal papillary antigen-1 have been used to detect acute drug-induced renal tubule alterations in rats. Several biomarkers such as NGAL (lipocalin-2) have been used in drug development settings (Ennulat and Adler, 2015; Mishra et al., 2003). For several decades, more traditional biomarkers employing enzymes that are released from damaged renal tubular cells such as ALP, lysosomal enzymes N-acetyl-b-glucosaminidase (NAG), and gamma glutamyltransferase (GGT) have been used as markers of renal injury in animal studies (Clemo (1998); Emeigh (2005); D’Amico and Bazzi (2003). It is important to understand the relationship between biomarkers and microscopic pathologic changes on the kidney as assessed by light microscopy. Because of renal cell injury, enzymes such as BUN are elevated prerenal, renal, or post renal; but, BUN can also increase with diet, without renal injury. For the kidneys, histopathologic findings remain the gold standard by which biomarkers are validated for use (Table 53.3).

Polioencephalomalacia Polioencephalomalacia is diagnosed at necropsy of cattle, sheep, and, less commonly, goats observed with clinical signs including depression, stupor, ataxia, head pressing, apparent cortical blindness, opisthotonos, convulsions, and recumbency with paddling of the limbs. Although primarily associated with thiamine deficiency, this may be a multifactorial metabolic disorder. Multiple dietary factors have been proposed, such as feeding high carbohydrate rations with little roughage (Zachary, 2006). In cattle, microcavitation of the deep cortical lamina adjacent to subcortical white matter is characteristic of chronic polioencephalomalacia.

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958 TABLE 53.3

53. PATHOLOGICAL BIOMARKERS IN TOXICOLOGY

Summary of Renal Injury Biomarkers

Biomarkers

Sources

Interpretation

Kidney injury molecule 1 (KIM-1, TIM-1, HAVCR-1)a

Proximal tubules

Toxic, ischemic, septic renal tubular injury

Neutrophil gelatinase-associated lipocalin (NGAL/Lipocalin-2)b

Proximal and distal tubules, neutrophils, bone marrow

Renal tubular injury (urinary), inflammatory (blood)

Clusterin

Proximal and distal tubules

Toxic, ischemic, septic renal tubular injury

Cystatin C

All nucleated cells, renal tubules

Plasma levels inversely related to GFR; renal tubular injury (urine)

Alkaline phosphatase (ALP)

Renal tubules

Renal tubular injury

N-Acetyl-b-glucosaminidase (NAG)

Renal tubules

Renal tubular injury

Gamma glutamyltransferase (GGT)

Renal tubules

Renal tubular injury

Total protein

Plasma protein

Glomerular injury

Albumin (microalbumin)

Plasma protein

Glomerular injury

a1/b2-microglobulin

Plasma protein

Glomerular injury and/or proximal tubular injury

a

May not be useful in canines. False-positive increases can be induced in neutrophils by various stimuli (inflammation, infections, neoplasia). Reproduced from A Comprehensive Guide to Toxicology in Nonclinical Drug Development (Table 17.1), first ed. Academic Press/Elsevier, p. 457.

b

Results from a previous study suggest that gut fill, Cu status, and cytochrome c oxidase activity in brain tissue might be influenced and compromised by wet distillers grains with solubles when fed at 60% of diet dry matter in diets based on steam-flaked corn. It might imply a greater susceptibility to polioencephalomalacia (Ponce et al., 2014).

Sodium Chloride Toxicity

repens) for long periods of time (Cordy, 1978). The gross lesions, which were usually bilateral, correlated histologically with necrosis and loss of neurons as well as necrosis of axons, glia, and blood vessels in the anterior regions of the globus pallidus and substantia nigra. In addition, localized encephalomalacia (necrosis) of the nucleus of the inferior colliculus (Fig. 53.9), the mesencephalic nucleus of the trigeminal nerve, and the dentate nucleus was also reported in horses fed dried Russian knapweed (Young et al., 1960). Morphologic findings

Sodium chloride toxicity or salt poisoning has been reported primarily in pigs, poultry, and occasionally in ruminants, horses, and dogs following increased intake of sodium chloride from feed rations or supplements with limited access to drinking water. Gross lesions include inconsistent meningeal congestion and edema, and transverse slices of fixed brain have shown subgross evidence of laminar necrosis. Histologically, laminar cerebrocortical necrosis accompanied by astrocytic swelling and infiltration of meningeal and cerebral blood vessels by eosinophils were observed in pigs. The latter was considered the microscopic pathology biomarker of salt poisoning.

Nigropallidal Encephalomalacia in Horses Sharply demarcated foci of pale yellowish discoloration to buff-colored foci of softening or cavitation of the substantia nigra and the globus pallidus have been reported in horses grazing on yellow star thistle (Centaurea solstitialis) or Russian knapweed (Acroptilon

FIGURE 53.9 Equine nigropallidal encephalomalacia. Reproduced from Pathologic Basis of Veterinary Disease (Fig. 11e79), fourth ed. p. 921. Academic Press/Elsevier.

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CONCLUDING REMARKS AND FUTURE DIRECTIONS

are distinctive for the neurotoxicity that develops in horses. A potential biomarker of nigropallidal encephalomalacia in horses would be in relation to oxidative stress. Results from a previous study suggest that glutathione (GSH) depletion by the sesquiterpene lactone repin may increase the susceptibility to oxidative damage in equine nigropallidal encephalomalacia (Tukov et al., 2004).

Phospholipidosis Cationic amphophilic drugs (CADs) from different pharmacologic classes are known to induce accumulation of phospholipids and drugs within a variety of cells (Reasor, 1989). When taken up by the lysosomal system the CADs, such as chlorphentermine, amiodarone, and chloroquine, may raise intracellular pH and form complexes with polar lipids or mucopolysaccharides, which interfere with lysosomal enzyme activity, resulting in accumulation of polar lipids (Hein et al., 1990). By light microscopy, affected cells are foamy because of presence of lamellar inclusion bodies. Wu¨nschmann et al. (2006) reported that captive Humboldt penguins found dead after treatment for avian malaria with higher than usual doses of chloroquine had neuronal storage disease. Affected birds were found histologically to have moderate to marked vacuolation of Purkinje cells, neurons of the brainstem, and motor neurons of the spinal cord. By electron microscopy, the cytoplasmic vacuoles were multilayered lamellar structures by electron microscopy, characteristic of drug-induced lysosomal storage disease (Fig. 53.10). For chloroquine toxicity, gangliosides rather than phospholipids are primarily stored (Klinghardt et al., 1981). A tentative

FIGURE 53.10 Chloroquine toxicity in penguin. Electron micrograph of a spinal cord motoneuron with distended perikaryon due to the presence of numerous multilayered concentric lamellar structures. Bar ¼ 300 nm. Courtesy of Dr. A. Wu¨nschmann, College of Veterinary Medicine, University of Minnesota.

959

histopathologic diagnosis may be enhanced by Immunohistochemistry (IHC) with antibodies for autosomal membrane associated protein-2 (LAMP-2) and antiADRP for adipophilin. However, the widely accepted standard for a diagnosis of lysosomal storage disease is electron microscopic identification of concentric lamellar inclusion bodies in secondary lysosomes (Monteith et al., 2006). The central nervous system that contains numerous cell bodies with specific functions is protected from xenobiotics by the bloodebrain barrier (BBB); but, some molecules can cross BBB by nature of their physicochemical properties or design. As of this writing, “there are no routinely used molecular/chemical validated biomarkers to assess xenobiotic related injury to the nervous system.”

CONCLUDING REMARKS AND FUTURE DIRECTIONS This chapter focused on morphologic (gross and microscopic) and clinical pathology findings as pathological biomarkers of adverse effects of drugs, food additives, biopharmaceuticals, medical devices, environmental chemicals, and plants. Introduction of the autopsy/necropsy and the use of the light microscope to investigate diseases were important milestones that provided recognizable postmortem morphologic end pointsdthe gross and microscopic pathological biomarkers. Later, integration of special morphologic pathology techniques resulted in increased accuracy of diagnoses reported accurately and interpreted correctly in a timely manner. Now there are validated biomarkers being used to fine tune diagnosis, prognosis, and therapy with more being discovered through “omics” technologies (Milburn et al., 2013). Because morphologic techniques remain typically the gold standard against which specificity and sensitivity of novel biomarkers are being validated, the toxicologic pathologist should be aware of and adapt to his/her expanding role. As per Best Practices Guideline: Toxicologic Histopathology (Crissman et al., 2004), the toxicologic pathologist must be qualified and possess credentials that document a high level of education, training, experience, and expertise and shall be provided well-prepared slides, laboratory test results, and other study information necessary to conduct an accurate pathologic evaluation. A draft guidance regarding the use of histopathology for biomarker qualification studies has been issued (Food and Drug Administration, 2011). Continued improvements in pathology processes and procedures will come from the alignment with Best Practices Guidelines. Taken together with the personal integrity of the toxicologic pathologist, this assures a high-quality pathology

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report, contributing to the final reports of R&D safety, toxicity, and efficacy studies for the benefit of man and animals.

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54 Oral Pathology Biomarkers Anupama Mukherjee Oral Pathology, Microbiology and Forensic Odontology, Goa Dental College and Hospital, Panaji, India

INTRODUCTION The oral cavity is regarded as a window to the human body. The stomatognathic system plays a critical role in numerous physiological processes such as digestion, respiration, and speech. Subtle and early signs of various diseases often manifest in the oral tissues. This necessitates a detailed study of the oral cavity and its associated structures to identify and understand local and systemic disease processes. Oral and maxillofacial pathology is a specialty of dentistry and pathology, which deals with the nature, identification, and management of diseases affecting the oral and maxillofacial regions. It is a science that investigates the causes, processes, and effects of these diseases. The practice of oral and maxillofacial pathology includes research along with diagnosis of diseases using clinical, radiographic, microscopic, biochemical, or other examinations and the subsequent management of patients. Numerous developmental disturbances/disorders often involve the orofacial regions. These may be as a result of hereditary factors, administration of drugs, or even exposure to radiation during dental development. Additionally, the use of tobacco in a smoked or smokeless form or betel quid chewing in synergy with alcohol consumption, predispose an individual to oral potentially malignant disorders (OPMDs) and subsequently oral cancer (George et al., 2011; Sarode et al., 2012). A continuous and excessive exposure to inorganic substances such as fluoride, silica, asbestos, and heavy metals (lead, mercury, etc.) may manifest in the oral mucosa, as a sign of toxicity. Various biomolecules (growth factors, enzymes, and interleukins) and the presence of toxicants can be assessed in oral samples. Saliva, a biofluid, exclusive to the oral cavity has emerged as a significant diagnostic tool (Langie et al., 2017). The altered concentrations or unusual presence of certain biomolecules in saliva are Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00054-2

now being utilized as biomarkers for diagnosis and prognosis. Biomarkers of oral diseases are more routinely evaluated using tissue biopsies. The application of special techniques such as histochemistry and immunohistochemistry allow a more detailed evaluation of the proteomic and molecular profile of diseases. This chapter describes oral pathology biomarkers of select toxicants and diseases.

ANATOMICAL AND HISTOLOGICAL CONSIDERATIONS OF THE ORAL CAVITY The stomatognathic system displays a harmonious coexistence of functional hard and soft tissues. The hard tissue components include the teeth, temporomandibular joint, mandible, and maxilla, whereas the soft tissue components include the oral mucosa (including tongue) and the salivary glands (Nanci, 2017). (Fig. 54.1).

Teeth The teeth represent the exposed mineralized structures of the oral cavity. They play a vital role in mastication along with aesthetics and provide support to facial tissues such as the lips and cheeks. Teeth are composed of enamel, dentine, and cementum, which are mineralized, whereas the pulp accounts for the vital, soft, connective tissue enclosed within the tooth. The oral mucosa refers to the soft tissue lining the oral cavity. Mucosal organization at the various anatomic sites of the oral cavity is categorized as (1) lining mucosa, (2) masticatory mucosa, and (3) specialized mucosa based on its regional variation in structure and function (Table 54.1). The salivary glands produce a multifunctional fluid, saliva, which moistens the oral cavity. The predominant

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Upper lip (labium) Frenulum of upper lip Gingiva covering the maxillary alveolar process

Superior vestibule

Hard palate Fauces

Soft palate Uvula

Palatine tonsil

Cheek

Tongue

Molars

Frenulum of tongue Openings of submandibular ducts

Premolars

Gingiva covering the mandibular alveolar process

Canine

Frenulum of lower lip

Incisors Inferior vestibule

Lower lip (labium)

FIGURE 54.1 Shows the various anatomical regions of the oral cavity. Image courtesy: Seeley
Stephens
Tate: Anatomy and Physiology, sixth ed., Part 4-Regulations and Maintenance, Chapter: 24 Digestive System, The McGraw
Hill Companies, 2004.

functions of saliva include lubrication, mastication, gustation (taste) and digestion. It also plays an important role in speech, oral pH maintenance, and ion exchange between tooth surfaces, along with providing defense against early microbial invasions. Saliva also acts as a vehicle for numerous substances, such as vitamins, drugs, glucose and salts, facilitating their dispersion and rapid absorption by the oral mucosa.

HISTOLOGY OF ORAL MUCOSA The oral mucosa comprises the oral epithelium and connective tissue. (Fig. 54.2). The epithelium is stratified squamous in nature and may be keratinized or nonkeratinized in different areas. It forms a physical barrier to the external environment. The epithelium is supported by the connective tissue, designated as the lamina propria and submucosa. The connective tissue comprises the vascular, muscular, and neural components. The submucosa in the oral cavity also shows the presence of specialized glandular structures. These are the serous or

seromucous minor salivary gland acini. The organizational relations of the various tissue elements when viewed under a microscope are shown in Fig. 54.3. The basement membrane is a specialized zone that maintains the structural integrity of the epithelium, along with its intimate association with the underlying connective tissue. Histologically, it is observed as an interface between the basal layer of epithelial cells and the lamina propria (Nanci, 2017). Thus the variable histomorphological features confer each oral site with a specialized function. The areas covered by the masticatory mucosa protect the underlying tissues from microtrauma and abrasions during mastication. The specialized mucosa on the dorsum of the tongue allows for taste perception. Based on the variable keratinization of the oral mucosa, some sites (sublingual, floor of the mouth) are more permeable as compared to others (palate). (Fig. 54.4). The presence of minor salivary gland acini, in conjunction with the major salivary glands, maintains a moistened mucosa. The water content of these secretions acts as a vehicle to facilitate intercellular transport and absorption (Nanci, 2017).

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HISTOLOGY OF ORAL MUCOSA

TABLE 54.1

A Comparative Overview of the Numerous Regional Variations of the Oral Mucosa

Region

Overlying Epithelium

Lamina Propria

Submucosa

Soft palate

Thin (150 mm), nonkeratinized stratified squamous epithelium; few taste bud present.

Thick with numerous short papillae; elastic fibers forming an elastic lamina; highly vascular with well-developed capillary network

Diffuse tissue containing numerous minor salivary gland acini

Ventral surface of the tongue

Thin, nonkeratinized, stratified squamous epithelium.

Thin with numerous short papillae and some elastic fibers; a few minor salivary gland acini; capillary network in subpapillary layer.

Thin and irregular, where absent, mucosa is bound to connective tissue surrounding tongue musculature.

Floor of the mouth

Very thin (100 mm), nonkeratinized stratified squamous epithelium.

Short papillae; some elastic fibers; extensive vascular supply with short anastomosing capillary loops.

Loose fibrous connective tissue, containing fat and minor salivary glands.

Alveolar mucosa

Thin, nonkeratinized stratified squamous epithelium.

Short papillae, connective tissue containing many elastic fibers; capillary loops close to the surface.

Loose connective tissue containing thick elastic fibers attaching it to the periosteum of alveolar process; minor salivary gland acini.

Labial and Buccal mucosa

Very thick (500 m), nonkeratinized stratified squamous epithelium.

Long, slender papillae; dense fibrous connective tissue containing collagen and some elastic fibers; rich vascular supply giving off anastomosing capillary loops into papillae.

Mucosa firmly attached to underlying muscle by collagen and elastin; dense collagenous connective tissue with fat, minor salivary glands, some sebaceous glands.

Lips: Vermilion zone

Thin, orthokeratinized, stratified squamous epithelium.

Numerous narrow papillae; capillary loops close to surface in papillary layer.

Mucosa firmly attached to underlying muscle; some sebaceous glands in vermilion border, minor salivary glands and fat in intermediate zone.

Lips: Intermediate zone

Thin, parakeratinized, stratified squamous epithelium.

Long irregular papillae; elastic and collagen fibers in connective tissue.

Gingiva

Thick (250 mm), orthokeratinized or parakeratinized, stratified squamous epithelium often showing stippled surface.

Long, narrow papillae, dense collagenous connective tissue; long capillary loops with numerous anastomoses.

No distinct layer, mucosa firmly attached by collagen fibers to cementum and periosteum of alveolar process.

Hard palate

Thick, orthokeratinized (parakeratinized in some parts), stratified squamous epithelium thrown into transverse palatine ridges (rugae).

Long papillae; thick, dense collagenous tissue, especially under rugae; moderate vascular supply with short capillary loops.

Dense collagenous connective tissue attaching mucosa to periosteum (“mucoperiosteum”), fat and minor salivary gland acini are packed into connective tissue in regions where mucosa overlies lateral palatine neurovascular bundles.

Thick, keratinized, and nonkeratinized stratified squamous epithelium forming various types of papillae, some bearing taste buds.

Long papillae, minor salivary gland acini in posterior region; rich innervation, particularly near taste buds; capillary plexus in papillary layer, large vessels lying deeper.

No distinct layer; mucosa is bound to connective tissue surrounding musculature of tongue.

LINING MUCOSA

MASTICATORY MUCOSA

SPECIALIZED MUCOSA Dorsal surface of the tongue

Courtesy: Antonio Nanci. Ten Cate’s Oral Histology-Development, Structure and Function, ninth ed. Elsevier, Amsterdam.

Cellular permeability is attributed to cell structure and concentrations of substances in the cytoplasm. The lipid bilayer configuration of the cell membrane allows trafficking of lipid soluble substances. Special protein channels and transport proteins help carry

water-soluble substances across the cell membrane. These structural characteristics allow for selective permeability across the membrane. The mechanisms by which substances travel across the cell membrane include (1) active transport, (2) passive diffusion, and

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FIGURE 54.2 Schematic representation of the various components of the oral mucosa. Image courtesy: Antonio Nanci, 2017. Ten Cate’s Oral Histology-Development, Structure and Function, ninth ed.

(3) facilitated diffusion. Occasionally, endocytosis may be demonstrated by a cell. However, this is not common in oral epithelial cells. The stratified squamous epithelium lining the oral mucosa, like all epithelial cells, shows the presence of membrane surface specializations called cell junctions, which allow cell-to-cell communication along with the passage of substances through and between cells. Thus, epithelial cells can allow and promote the diffusion of gases, liquids, and nutrients. Epithelial cells demonstrate four major types of cell-cell junctions. The tight junction (zonula occludens) and the adherent junction (zonula adherens) are typically close together and each forms a continuous ribbon around the cell’s

apical end. Multiple ridges of the tight, occluding junctions prevent passive flow of material between the cells. The adhering junctions, usually located immediately below the tight junctions, serve to stabilize and strengthen circular bands around the cells and help hold the layer of cells together (Fig. 54.5). Both desmosomes and gap junctions make spot-like plaques between two cells. Bound to intermediate filaments inside the cells, desmosomes form very strong attachment points and play a major role in maintaining the integrity of the epithelium. Gap junctions, which are patches of many connexons in the adjacent cell membranes, have little strength but serve as intercellular channels for the flow of molecules. The specific

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BIOMARKERS IN THE ORAL CAVITY

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FIGURE 54.3 A pictomicrograph showing the tissue organization of the oral mucosa. MSG, minor salivary glands; P, connective tissue papillae; R, reticular lamina propria; SM, submucosa. Image courtesy: Ellen Eisenberg, Easwar Natarajan, Bradley K. Formaker, 2018. Oral Mucosa and Mucosal Sensation, Department of Oral Health and Diagnostic Sciences, School of Dental Medicine, University of Connecticut, Storrs, CT, USA.

arrangement of these junctions forms a “junctional complex” and defines the basolateral surface of a cell (Nanci, 2017).

ORAL PHYSIOLOGY AND PHARMACODYNAMICS The oral cavity allows for two major routes of administration of drugs, oral (as when ingested) and mucosal. Saliva constantly bathes the oral mucosa and acts as a vehicle allowing dissolution of substances to pass across. Mucosal absorption bypasses first pass metabolism in the liver and directly delivers the drug/substance into the vascular system. Absorption by the oral mucosa occurs predominantly via passive diffusion into the lipid membrane. Numerous studies have been initiated to understand the process of mucosal absorption, particularly for various drugs. A rapid absorption of drugs/substances via the mouth into the mucosal membranes does not imply that they are immediately transported to the systemic circulation. This is because the rate-limiting step could be the transport of molecules through the mucosal membranes into the systemic circulation. Mucosal absorption is usually rapid and short, depending largely on duration of contact and permeability of the mucosa. The sublingual area/floor of the mouth is most permeable followed by the buccal mucosa and palate. Mucosal permeability is attributed to relative thickness of epithelium, keratinization, and vascularity. The floor of the mouth is mostly vascular and thinly epithelialized. Thus, it demonstrates a rate of absorption about 3e10 times greater than that seen via other routes of administration (Narang and Sharma, 2011).

A key role, in mucosal absorption, is played by the secretory glands of the oral cavitydthe major and minor salivary glands. Saliva comprises water, mucin, enzymes, and salts. The anatomical location of the sublingual salivary glands also contributes to maintaining a moist environment that can act as a vehicle for the dissolution and distribution of substances (Nanci, 2017). Certain factors that influence oral mucosal absorption include lipid solubility and permeability of solution (osmosis), molecular weight of substances, and the ionization state (pH). The cells of the oral epithelium and epidermis are also capable of absorbing by endocytosis. These engulfed particles are usually too large to diffuse through its wall. It is unlikely that this mechanism is used across the entire stratified epithelium. It is also unlikely that active transport processes operate within the oral mucosa. Occasionally large inert substances (heavy metals/pigments) may accumulate in the fine capillaries and tissues present as clinical signs of toxicity or pigmentation.

BIOMARKERS IN THE ORAL CAVITY Biomarkers have been defined as a biological characteristic that is objectively measured and evaluated as an indicator of normal biological/pathological processes or a response to a therapeutic intervention (Mayeux, 2004). The WHO has defined biomarkers as “almost any measurement reflecting an interaction between a biological system and a potential hazard, which may be chemical, physical, or biological. The measured response may be functional and physiological, biochemical at the cellular level, or a molecular interaction.”

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Biomarkers can be broadly categorized as biomarkers of exposure, which are used in risk prediction, and biomarkers of disease, which are used in screening, diagnosis, and monitoring of disease progression (Mayeux, 2004). Although drug and metal toxicity may manifest as clinically and histologically observable features, these are predominantly biomarkers of exposure/risk. For other oral diseases such as periodontitis, oral squamous cell carcinoma, pemphigus, pemphigoid, oral lichen planus (Sultan et al., 2014; Gopalakrishnan et al., 2016), etc., the evaluation of distinct histological features, proteomic profiles, and gene expression have been used as biomarkers for screening to assess disease progression and aggressiveness (Ferna´ndez-Gonza´lez et al., 2011; Mehdipour et al., 2018). To simplify and categorize the various biomarkers observed in the oral cavity, a three-tier approach may be adopted. This would include a) clinical biomarkers, observable clinical features that reflect the risk/presence of a disease either systemically or locally in the oral cavity (brownish mottled teeth suggestive of fluorosis), and b) tissue biomarkers, the histopathological characteristics that are informative of the degree of variation from normal. They aid in providing a definitive diagnosis and help ascertain the severity or aggressiveness of the disease (keratin pearls as dyskeratosis as seen in squamous cell carcinoma (Gopalakrishnan et al., 2016)). c) Molecular biomarkers include molecular profiles or the expression of specific protein(s), the presence of which may be increased/decreased or be abnormal in a diseased state. These markers may also be used to monitor disease progression or for the evaluation of therapy (antibodies directed against DSG-3 and/or DSG-1 in pemphigus).

COMMON DRUGS AND TOXICANTS SHOWING ORAL MANIFESTATIONS

FIGURE 54.4

Schematic representation comparing the variable epithelial and connective tissue thickness at various oral sites. The buccal mucosa and floor of the mouth are richly vascular area. Images courtesy: Niki M. Moutsopoulos, Joanne E. Konkel, 2018. Tissue-specific immunity at the oral mucosal barrier. Trends Immunol. 39 (4), 276e287.

1. Drugs a. Tetracycline/Minocycline b. Bisphosphonates c. Hydantoin 2. Heavy Metals a. Mercury b. Silver c. Lead d. Arsenic e. Bismuth 3. Poisonings a. Cyanide b. Organophosphates c. Aluminum phosphide

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FIGURE 54.5 Schematic diagram showing the various cell junctions in epithelial cells. Image courtesy: Mescher, A.L. Junqueira’s Basic Histology: Text and Atlas, twelfth ed. http://www.accessmedicine.com.

SELECT DISEASES OF THE ORAL CAVITY 1. Periodontal disease 2. Immunologically mediated oral diseases/ Autoimmune diseases a. Pemphigus b. Pemphigoid c. Oral Lichen Planus 3. Oral squamous cell carcinoma

ORAL BIOMARKERS OF EXPOSURE AND EFFECTS OF SELECT DRUGS/TOXICANTS Tetracycline/Minocycline Tetracycline is a broad-spectrum antibiotic introduced in 1948. It was routinely administered to children and adults for the treatment of common infections caused by Gram-positive bacteria. A few Gramnegative bacteria also showed sensitivity to the drug. Minocycline, a semisynthetic derivative of tetracycline, is commonly administered to treat acne. A prominent side effect of long-term administration of tetracycline is its incorporation into various mineralizing sites. This occurs because of the select affinity of tetracycline for calcium ions. The drug forms a chelate with the calcium hydroxyapatite crystals and is deposited into teeth and bones during mineralization. Minocycline causes staining via different mechanisms. The drug assumes a highly protein-bound state and preferentially binds to

collagen-rich tissues such as teeth and bones. Alternatively, the chelation of a minocycline breakdown product similar to hemosiderin forms an insoluble complex with teeth (Raymond and Cook, 2015). The excretion of minocycline in the gingival cervical fluid and its subsequent oxidation when incorporated into enamel could also cause staining, which appears as a blackish discoloration. The administration of tetracycline and its derivatives to pregnant mothers (second and third trimester) or children (up to 7 years of age) results in staining of teeth and bones. The distribution and intensity of color vary based on the specific form of the drug along with the duration of exposure. The occurrence of staining is noted in 3%e6% of minocycline-treated patients. Clinical biomarkers of tetracycline administration include the following: • Yellowish or brownish-gray staining of deciduous and permanent teeth. Discoloration is more intense at the time of tooth eruption and gradually becomes browner. Occasionally a bluish discoloration has also been reported (Johnston, 2013). • Chlortetracycline administration leads to brownishgray color. Oxytetracycline leads to a yellowish color. • Bright yellow fluorescence under ultraviolet light. • Minocycline administration causes “black bones,” “black or green roots,” and a blue-gray to gray hue, darkening the crowns of permanent teeth. Such teeth usually pose an esthetic challenge and are not functionally compromised. Various techniques such

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as composite restorations or dental veneers are often employed to improve patient aesthetics. Mercury and Silver Mercury is a glistening, odorless liquid that becomes a colorless and odorless gas when heated. Mercury is very toxic and exceedingly bioaccumulative. It is a major cause of acute heavy metal poisoning. Because mercury is ubiquitous in the environment, individuals are routinely exposed to it via air, water, and food. According to WHO, the maximum allowable environmental level of mercury is 50 mg/day in the workplace. Three major forms of mercury in descending order of toxicity are organomercury, mercury vapor, and inorganic mercury. Organomercury (methyl-, ethyl-forms) is well absorbed or may even be formed because of the interaction of gut flora with inorganic mercury. The toxic effects of mercury are noted at a cellular level and eventually manifest clinically. Mercury affects cellular integrity and disrupts intracellular calcium homeostasis. Excessive mercury also interferes with transcription and translation, resulting in the disappearance of ribosomes and endoplasmic reticulum activity. These cell organelles are vital for cell functions (Jaishankar et al., 2014). Mercury is largely neurotoxic as it depletes glutathione and thiols. It also leads to the generation of reactive oxygen species (ROS) and oxidative stress. High levels of mercury in the blood can be nephrotoxic and neurotoxic. Severe mercury toxicity, particularly from methyl mercury is termed as the “Minamata disease”. It is a severe neurological syndrome characterized by headache, loss of peripheral vision, numbness in hands and feet, general muscle weakness, and damage to hearing and speech. The disease was first noted in Japanese residents of the city of Minamata in 1956 (Yorifuji et al., 2017). Silver is a heavy metal occurring naturally as a soft, glistening metal. It occurs as a powdery white (silver nitrate and silver chloride) or dark gray to black compound (silver sulfide and silver oxide). Exposure to silver occurs via jewelry, silverware, medication, printed photographic material, electronic equipment, and dental fillings with silver in its metallic form. Excessive exposure to silver results in argyria. A clinical biomarker would be a gray to blue-gray discoloration of the skin and body tissues. This phenomenon is also referred to as “ashen-Gray” discoloration. Dental amalgam is an alloy of silver and mercury. It contains about 50% mercury, along with other toxic metals such as tin, copper, nickel, palladium, etc. Amalgam restorations continuously leak mercury due to its low vapor pressure and loss due to the galvanic action of mercury with dissimilar metals in the mouth. It has been reported that an amalgam filling releases about 0.8e0.10 mg of mercury vapor each day. Often,

residual amalgam from a restoration may be lodged into the gingiva. This can result in a grayish-black discoloration of the localized area referred to as an “Amalgam tattoo.” This poses no significant health hazard (Sivapathasundharam, 2016). A key microscopic biomarker is the appearance of black, granular deposits of silver salt in the submucosa. This is a key feature to rule out localized pigmentation. The measurement of urinary mercury levels in adults and children has been used as a parameter in various studies aimed at understanding the toxic effects of dental amalgam. Results remain elusive because age of the patient, time of sampling, duration since filling, and even masticatory habits have shown to play a role. Dental practitioners and dental assistants are at a high risk to develop mercury toxicity because of the use of amalgam in dental practice. Handling of mercury during restorative procedures, removal of old amalgam restorations, and mercury spills/open disposal jars pose a long-term health risk. Numerous studies have evaluated mercury levels in the blood, urine, hair, and nails of dental practitioners and reported higher values as compared with patients and nondental professionals. Lead Lead is a bluish-gray metallic element that is soft, ductile, durable, and heavy. It is used in a variety of products such as batteries, paint, solders, and ceramics. More recently the use of lead in pipes, gasoline, and cosmetics has been curbed owing to the propensity to cause toxicity. Exposure to lead can occur via ingestion, absorption (from skin), or inhalation. Lead poisoning (plumbism, saturnism, painter’s colic) may manifest as acute or chronic poisoning. The severity of presentation depends on the amount of lead in the blood and tissues, as well as duration and amount of exposure. Organic forms of lead are more toxic than inorganic forms. The excess lead binds to the sulfhydryl groups of proteins. It also causes an inhibition in the synthesis of heme, an essential component of hemoglobin, myoglobin, and various cytochromes. Plumbism can present in an acute or chronic form based on duration of exposure. Nausea, vomiting, diarrhea, muscle pain, abdominal pain, and weakness are noted in clinically acute lead poisoning. Headaches, fatigue, stupor, and anemia are noted due to chronic lead poisoning. Additionally, oral signs would include astringency, metallic taste in the mouth, and the appearance of “lead hue,” a biomarker of chronic lead exposure. It appears as a characteristic bluish-gray line along the free gingiva around the tooth. A tissue biomarker of plumbism is the perivascular deposit of lead sulfide in the submucosa and basement

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IMMUNOLOGICALLY MEDIATED ORAL DISEASES

membrane zone. In tissue samples, lead can be demonstrated histochemically using the rhodizonate method. Blood levels of lead have often been used to aid in the diagnosis of lead toxicity/poisoning, which is a clinicopathological correlation. It is suggested that cognitive impairment occurs at blood lead levels of 5 mg/dL, particularly in children. At highly elevated blood lead levels of 70 mg/dL, encephalopathy, seizures, and even death may occur (Jaishankar et al., 2014; Ihmed, 2016). In a study by Gardner et al. (2016), saliva has been reported as a reliable sample for lead evaluation when screening children for toxicity. Based on their study, saliva was reliable for lead concentrations of less than 5 mg/dL. Salivary samples with a concentration of lead greater than 1.40 mg/dL should be confirmed using blood evaluation. The oral cavity provides subtle indications of toxicities and poisonings. Data regarding the correlation between the clinical biomarkers and serum and/or salivary levels of metals remain unexplored. Often in case of acute toxicity or poisoning, the systemic manifestations are overwhelming, and diagnosis based solely on oral biomarkers is not necessitated. In the case of inert metals, such as silver or exposure to amalgam, oral findings are often suggestive and diagnostic when correlated with history and examination.

BIOMARKERS OF SELECT DISEASES Biomarkers have assumed a forefront in the assessment of numerous oral diseases. This approach allows them to be categorized as screening, diagnostic, and prognostic biomarkers. Biomarkers are evaluated using techniques ranging from routine histopathology to specialized techniques, such as immunohistochemistry, ELISA, immunofluorescence, and even fluorescence in situ hybridization (to detect gene fusions and characterize soft tissue tumors).

PERIODONTAL DISEASE Periodontal disease refers to a microbial infection affecting the supporting structures of the tooth, that is, the gingiva, periodontal ligament, and alveolar bone. It predominantly affects the connective tissue structures and is clinically preceded by gingivitis. If not intercepted, periodontal disease progressively leads to loss of tooth support and eventually tooth loss. Periodontal disease affects almost 50% of the adult population in the United States. The clinically observable biomarkers of gingivitis include bleeding on probing the gingival

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crevice, along with redness and edema of gingival tissues (Taba et al., 2005). The clinically evaluated diagnostic biomarkers for periodontitis include pocket formation due to loss of attachment at the dentogingival unit and gingival recession, which is the apical migration of the gingiva. These features are often accompanied by tooth mobility. A diagnostic radiographic finding of loss of alveolar bone is significant. Aggressive forms of periodontal disease can lead to early tooth loss resulting in masticatory difficulties and poor aesthetics. Over a long period, it may also render the jawbones unsupportive to rehabilitation techniques such as dental implants or prosthesis. Hence, methods for early detection and assessment of disease progression could decrease morbidity. Tools to measure periodontal disease at the clinical, cellular, and molecular level have been devised. Studies evaluating saliva and gingival crevicular fluid (GCF), a fluid found in the space between the tooth and gingiva, for potential biomarkers of periodontal disease have been carried out. Considering that periodontitis is characterized by connective tissue destruction, the levels of matrix metalloproteinases (MMP) in GCF have been evaluated. In particular, MMP-8 has demonstrated an 83% sensitivity and 96% specificity in differentiating periodontitis from gingivitis and other disease sites. A study by Holmlund et al. (2004) reported an increase in IL-1a, IL-1b, and IL-1 receptor antagonist, which are associated with bone resorption, particularly in samples from diseased sites. It has been established that an increase in prostaglandins (PGE2), IL-1b, and TNF-a is associated with the severity of periodontal disease. A review by Loos and Tjoa (2000) identified GCF-related probable diagnostic biomarkers of periodontitis. This included alkaline phosphatase, b glucuronidase, cathepsin B, collagenase-2 matrix metalloproteinase (MMP-8), gelatinase (MMP-9), dipeptidyl peptidase (DPP) II and III, and elastase. Early detection could help address and arrest the disease in its initial stage, using suitable therapeutic regimens of antibiotics, oral prophylaxis, and root planning procedures.

IMMUNOLOGICALLY MEDIATED ORAL DISEASES Autoimmune diseases encompass a group of diseases where an immunological dysfunction results in failure of the host to recognize and distinguish self- from none self-tissues. Some conditions which present as mucocutaneous diseases, may manifest solely in the oral cavity, at a given time and pose a diagnostic challenge. In such a situation, the use of specific biomarkers can help eliminate other differential diagnoses and allow effective

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treatment planning. Some of the immunologically mediated oral diseases are listed below: • Hypersensitive Reaction

• Reiter’s Syndrome

• Pemphigus vulgaris

• Linear IgA

• Paraneoplastic pemphigus

• Epidermolysis bullosa

• Cicatricial or mucocutaneous pemphigoid

• Erythema multiforme

• Cutaneous, bullous pemphigoid

• Lupus erythematosus

• Lichen planus

• Systemic drug reaction

• Scleroderma

• Crest syndrome

• Bechet’s syndrome

PEMPHIGUS Pemphigus is classified as a vesiculobullous lesion with an autoimmune pathogenesis. Pemphigus is a broad term that encompasses a number of mucocutaneous presentations. The term “Pemphigus” is derived from the Greek word “Pemphix” implying blister or bubble (Mignogna et al., 2009). Some cases that involve the skin and mucous membranes extensively may pose to be life threatening (Azizi and Lawaf, 2008).There are various subtypes of pemphigus, each of which are characterized by the loss of normal cell-to-cell adhesion secondary to the binding of autoantibodies. This results in the formation of an intraepithelial vesicle with acantholysis (loss of cellular cohesion) (Mignogna et al., 2009; Zunt, 1996). The three major subsets of pemphigus are (1) pemphigus vulgaris, (2) pemphigus foliaceus, and (3) paraneoplastic pemphigus. Pemphigus vulgaris is a relatively rare disease with an onset around 50e60 years of age. It commonly affects the oral mucosa. Oral lesions precede cutaneous lesions and are often the first sign of disease. They are described as “first to show and last to go” because they are the most challenging to address with therapy. Clinically, lesions are noted on the soft palate, buccal mucosa, lower lip, and ventral surface of the tongue. They appear as oral vesicles and bullae that readily rupture to give rise to multiple eroded ulcers that may bleed. The condition is attributed to the abnormal production of autoantibodies directed toward the specific molecules desmoglein-3 (DSG-3) and desmoglein-1 (DSG-1). Both molecules are structural components of desmosomes which are a type of cell junction. (Azizi and Lawaf, 2008). The disruption of cell adhesion leads to

blister formation. The expression of DSG-3 is seen in the parabasal cells of the oral epithelium and epidermis. DSG-1 is noted in the superficial layers of the epidermis and minimally expressed in the oral epithelium. Thus, patients with autoantibodies directed only toward DSG-1 shall exclusively demonstrate dermal lesions, whereas those with autoantibodies directed toward DSG-3 alone, or DSG-1 and DGS-3, shall demonstrate oral lesions (Mignogna et al., 2009).

PEMPHIGOID Pemphigoid is also classified as a vesiculobullous lesion with an autoimmune pathogenesis. The term pemphigoid represents the similarity in its clinical presentation with pemphigus. Yet its histopathological features, immunological findings, and prognosis differ. Two distinct lesions, mucous membrane pemphigoid, and bullous pemphigoid are observed (Neville et al., 2015; Sivapathasundharam, 2016). Mucous membrane pemphigoid or cicatricial pemphigoid refers to a heterogeneous group of blistering lesions caused by autoantibodies that are directed toward any of the numerous components of the basement membrane (Xu et al., 2013). It is usually seen in females 50e 60 years of age. Intraoral lesions are seen more commonly as compared to bullous pemphigoid. Clinically, the appearance of bullae or vesicles is noted. They rupture to form ulcerated, denuded surfaces on the mucosa and may persist for weeks to months if left untreated. Other mucosal surfaces in addition to the oral cavity, such as the conjunctival, nasal, laryngeal, esophageal, and vaginal mucosa, may also be involved (Petruzzi, 2012; Xu et al., 2013). In some cases, dermal involvement is also seen. The clinical course in patients is usually progressive and protracted as compared to bullous pemphigoid (Zunt, 1996). The appearance of denuded gingiva termed as “desquamated gingivitis” is often used as a clinical biomarker to differentiate this lesion from bullous pemphigoid, though it may also be seen in erosive lichen planus or pemphigus vulgaris. Bullous pemphigoid is described as the most commonly occurring autoimmune condition (Xu et al., 2013). It is characterized by the production of autoantibodies toward the components of the hemidesmosome cell surface specializations that attach the basal cells to the basement membrane. Clinically, it is noted in older adults usually 60e 80 years of age with no gender predilection. Involvement of the oral mucosa is not a very common finding. If present, oral mucosal lesions are usually noted as shallow erythematous ulcers with smooth and distinct margins. This is due to the rupture of the intraoral vesicle/bullae as a result of constant low-grade trauma

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ORAL LICHEN PLANUS

TABLE 54.2

Clinical and Tissue Biomarkers That Aid in Eliminating Some Confounding Vesiculobullous/Autoimmune Lesions

Condition

Mean Age

Sex Prediliction

Clinical Features

Histopathology

Pemphigus vulgaris

40e60 years

Equal

Vesicles, erosions, and ulcerations on any oral mucosal site or skin surface

Intraepithelial clefting

Paraneoplastic pemphigus

60e70 years

Equal

Vesicles, erosions, or ulceration on any mucosa or skin surface

Subepithelial and intraepithelial clefting

Mucous membrane pemphigoid

60e70 years

Female

Primarily mucosal lesions

Subepithelial clefting

Bullous Pemphigoid

70e80 years

Equal

Primarily skin lesions

Subepithelial clefting

Based on Xu, H.-H., Werth, V.P., Parisi, E., et al., 2013. Mucous membrane pemphigoid. Dent. Clin. North Am. 57 (4), 611e630; Neville, B., Damm, D.D., Allen, C. et al., 2015. Oral and Maxillofacial Pathology, fourth ed. Elsevier, Amsterdam; Sivapathasundharam, B., 2016. Shafer’s Textbook of Oral Pathology. Elsevier, India.

within the oral cavity. Even though numerous clinical findings can be enlisted to differentiate pemphigus and pemphigoid, histopathological and immunological evaluations provide a more definitive diagnosis, particularly in cases that do not show a classical presentation. Clinical and tissue biomarkers that distinguish and aid in eliminating some confounding vesiculobullous/autoimmune lesions are summarized in Table 54.2.

ORAL LICHEN PLANUS Lichen planus is a chronic inflammatory mucocutaneous condition. Oral lichen planus (OLP) refers to the oral presentation of the disease, and it may/may not be accompanied by skin lesions. Females in the fifth to sixth decade of life are more commonly affected (Ferna´ndez-Gonza´lez et al., 2011). A vast spectrum of clinical presentations are noted, such as unilateral or bilateral white striations, papules, or plaques on the buccal mucosa, labial mucosa, or tongue. The appearance of “striae of Wickham” is a pathognomic clinical feature of oral lichen planus. Lesions involving the gingiva cause inflammation and denudation of the surface epithelium (Kamath et al., 2015). This phenomenon is also referred to as desquamative gingivitis. Erythema, erosion, and blisters may or may not be present. Numerous theories have been cited to understand the pathogenesis. OLP is regarded as a dysregulated T cell-mediated disorder to exogenous triggers or a dysregulated response to autologous keratinocyte antigens (autoimmune) (Ferna´ndez-Gonza´lez et al., 2011). A number of etiological agents have been proposed, such as: 1. local and systemic inducers of cell-mediated hypersensitivity, 2. stress,

3. autoimmune response to epithelial antigens or a dysregulated response to external antigens, and 4. viral infections. Histopathologically, the pathognomonic features of lichen planus include liquefactive degeneration of basal cell layer, saw tooth rete pegs, a band of chronic inflammation in the sub-basilar region, along with supplementary findings such as hyaline/colloidal bodies and MaxeJoseph cleft (Ferna´ndez-Gonza´lez et al., 2011; Kamath et al., 2015; Mehdipour et al., 2018). Lichen planus is not a life-threatening disease, but it continues to be listed as a potentially malignant disorder (OPMD) (Sarode et al., 2012). This has been attributed to mixed reports of malignant transformation of OLP to oral squamous cell carcinoma (Fitzpatrick et al., 2014). The presence of chronic inflammation, as seen with OLP, has been recognized as a potential cause of malignant transformation. Diagnosing OLP is a clinicopathological correlation and may pose a challenge because similar clinical features may also be noted in oral lichenoid reaction (an allergic response to drugs, restorative materials, betel quid chewing, etc.), pemphigus, pemphigoid, and leukoedema. In such cases, evaluation of biomarkers using immunofluorescence can help eliminate other possibilities. Immunofluorescence is based on the principle of antigeneantibody reaction. The resulting complex is tagged with a fluorescent compound to allow visualization. Two major techniques are utilized, direct immunofluorescence (aims at detecting the presence of the antigen in the patient’s biopsy sample) and indirect immunofluorescence (aims at detecting the presence of antigen-directed antibodies in the patient’s serum). Because autoimmunity is a dysregulation of the immune system, antigen and antibody evaluations, along with the distribution of the immune complex, can be utilized as a specific diagnostic biomarker. The application of immunofluorescence in the diagnosis of immune-related

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Application of Immunofluorescence for the Diagnosis of Immune-Mediated Lesions

Lesion

Direct Immunofluorescence

Indirect Immunofluorescence

Pemphigus vulgaris

Intercellular deposition of IgG (IgG 1 and 4) throughout epidermisdchicken wire/fish net appearance

Intercellular circulating IgG autoantibodies that bind to epidermis in 80%e90% cases

Paraneoplastic pemphigus

IgG with or without C3 in an intercellular pattern. Granular or linear deposition of C3, IgG, and/or IgM along dermaleepidermal junction in minor cases

Both intercellular intraepidermal antibody deposition and along dermaleepidermal junction

Cicatricial pemphigoid

Linear deposits of C3, IgG, IgA at dermal epidermal junctiond“shoreline pattern”

Circulating IgG autoantibodies directed against basement membrane one in 20% cases þ circulating autoantibodies to laminin 5

Bullous pemphigoid

IgG (70%) and C3 (90%e100%) deposition at dermaleepidermal junction

Circulating autoantibodies against basement membrane zone

Erythema multiforme

Granular deposits of IgG, C3, IgM, and fibrinogen around dermal vessels or at dermaleepidermal junction

e

Lichen planus

Shaggy deposit at dermaleepidermal junction of IgM (within scattered cytoid bodies), C3 and IgG with fibrin deposition at basement membrane zone

e

Systemic lupus erythematosus

Deposition of IgG, IgM, or C3 in a shaggy band at basement membrane (positive Lupus band test)

e

conditions has gained universal acceptance (Anuradha et al., 2011; Rastogi et al., 2014) (Table 54.3).

ORAL SQUAMOUS CELL CARCINOMA A tumor is defined as an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after the cessation of the stimuli that evoked the change. Tumors can be benign or malignant based on their proliferation characteristics, local tissue invasion, and behavior. The head and neck region is afflicted by distinct tumor entities that may affect the face, oral cavity, nasal and paranasal structures, pharynx, and salivary glands. Oral squamous cell carcinoma (OSCC) accounts for almost 90% of all oral cancers, with over 300,000 new cases reported annually worldwide. OSCC is a malignant epithelial neoplasm affecting the mucosal lining of the oral cavity and oropharynx. Despite attempts aimed at early diagnosis and improving treatment and prognosis, OSCC continues to have a dismal 5-year survival rate of 55%. OSCC can affect the tongue, buccal mucosa, gingiva, palate, floor of the mouth, and lips in decreasing incidence. Most cases are detected at an advanced stage and therapeutic alternatives are expensive and disfiguring. OSCC was reported to be a “disease of the elderly” because it commonly affected males over the age of 50. More

recently, a bimodal distribution of cases has been reported, some as early as in 27e30 year old individuals. The use of tobacco in a smoked/smokeless form with or without the synergistic effects of alcohol consumption is a major risk factor to develop the disease. The malignant transformation of OPMDs (leukoplakia, oral submucous fibrosis, and oral lichen planus) (George et al., 2011; Sarode et al., 2012) continues to bewilder clinicians and researchers because of the unpredictable course of these diseases culminating into OSCC. Recently, the role of chronic inflammation due to microtrauma, chronic mucosal irritation, sharp tooth, or denture clasps has also been recognized as leading to nonhabitassociated OSCC. The molecular aspects of OSCC reveal the derangement of basic cellular processes that govern DNA repair, cell proliferation, and cell survival. This is led by genetic alterations and mutations that dysregulate normal functions. Mutation in tumor suppressor genes-p53 and/or oncogenes such as epidermal growth factor receptor (EGFR), Bmi (B lymphoma Mo-MLV insertion region 1 homolog), c-myc, RAS result in uncontrolled proliferation and cell survival (Lavanya et al., 2016). The tumor also establishes a microenvironment conducive for its proliferative and metabolic needs. As the tumor thrives, the increase in vascular and lymphatic channels provides routes for metastasis to lymph nodes and distant organs. Thus, OSCC cannot be viewed solely as an epithelial pathology but more like a rouge organ.

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SALIVA: A HIDDEN PLETHORA OF BIOMARKERS

Attempts to grade (degree of disorganization of epithelium) and stage (disease severity) the disease have been made so as to facilitate treatment planning and prognostication. Often, cases within the same stage/grade demonstrate a variable outcome. Hence, the accurate evaluation of tumor aggressiveness and behavior remains a challenge. Biomarkers for tumors include histopathological characteristics (tissue markers) and tumor markers. Routine histopathology remains the gold standard for diagnosis and can be regarded as a definitive diagnostic tissue biomarker. Other specialized techniques such as immunohistochemistry, ELISA, and immunofluorescence have been proposed to actively evaluate the various phases of tumor development from initiation, promotion, and progression (Lavanya et al., 2016). A tumor marker is a substance present in or produced by a tumor or the tumor’s host in response to the presence of the tumor and can be used to differentiate a tumor from normal tissues or to determine the presence of a tumor based on its measurement in blood or secretions (Rivera et al., 2017). Scully and Burkhardt (1993) proposed that tumor markers can be categorized based on (1) tissue and cell site specificity, (2) their interaction with tissues, and (3) their association with tumoral processes such as invasion and metastasis. The role of tumor markers as an aid for early diagnosis, disease prognostication, and for treatment evaluation has steadily evolved. The application of tumor markers for OSCC attempts to address two major challenges: • to predict the transformation of oral potentially malignant disorders (leukoplakia, erythroplakia, discoid lupus erythematosus, oral submucous fibrosis) to frank malignancy (OSCC), and • to predict metastasis and understand tumor behavior and aggressiveness. The evaluation of tumor tissue, patient blood, and salivary samples has been carried out in an attempt to unravel efficient biomarkers. Because a universally accepted panel of markers is yet to be proposed, we regard the listed markers as “potential tumor biomarkers.” An enhanced expression of Bmi and survivin in OPMDs, which transformed to OSCC, has been demonstrated. Increased expression of transforming growth factor alpha (TGF-a), epidermal growth factor receptor (EGFR), and fibroblast growth factor receptor (FGFR) have been suggested as early markers for head and neck carcinogenesis (Feng et al., 2013). The expression of cytokeratin (AE-1/3) can aid in characterizing the epithelial cell of origin in tumors, which are atypical or dedifferentiated. Cytokeratin-19 (CK-19) is associated with high-grade dysplasia and

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early OSCC, whereas cytokeratin-13 (CK-13) has been proposed as a prognostic marker to reveal metastasis and tumor progression (Frohwitter et al., 2016). The decrease in expression of cell adhesion molecule E-cadherin and an increase in N-cadherin is indicative of an invasive aggressive tumor. To understand the stromal response, expression of vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP-2,-9,-13), alpha smooth muscle actin (a-SMA), interleukin-8 (IL-8), CD-163, and CD-44 have been evaluated in addition to over 35 other markers with promising results. Although studies have been carried out on tumor tissues, the salivary biomarkers that are significantly altered in OSCC patients are inhibitors of apoptosis (IAP), squamous cell carcinoma associated antigen (SCCeAg), carcinoembryonic antigen (CEA), carcino-antigen (CA19-9), CA128, serum tumor marker (CA125), tissue polypeptide specific antigen (TPS), reactive nitrogen species (RNS) (Radhika et al., 2016) and 8-Oxo-20 -deoxyguanosine (8-OHdG), which are critical markers of DNA damage, lactate dehydrogenase (LDH), and salivary IgA(s-IgA), to name a few.

SALIVA: A HIDDEN PLETHORA OF BIOMARKERS Saliva is the exclusive omnipresent biofluid of the oral cavity. It is the result of synthesis and secretion of products from the major and minor salivary glands. Saliva is attributed with distinct functions, as previously stated, and contributes toward maintaining oral health. In the event of oral disease infection, inflammatory or neoplastic, alterations in salivary constituents have been demonstrated. Over 100 potential salivary biomarkers have been reported. A variety of techniques such as liquid chromatography, gel and capillary electrophoresis, mass spectrometry, microbial cultures, immunoassays, and magnetic bead immunoprecipitation have been used to evaluate the proteomic, metabolomic, and microbiomic classes of salivary biomarkers (Wang et al., 2016). Saliva is considered as the best diagnostic tool for periodontitis. A salivary sample can help ascertain the number and taxa of bacteria and help categorize the disease (Aimetti et al., 2012). A diagnosis of Leishmaniasis in asymptomatic patients can be established by detecting the Leishmania siamensis DNA in salivary samples via polymerase chain reaction (PCR) even before its appearance in blood (Siriyasatien et al., 2016). The detection of HIV antibodies in saliva has paved the way for rapid diagnostic kits allowing self-testing, prior to

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laboratory confirmation (Wang et al., 2016). OraQuick is an in-home oral swab-based testing kit, approved by the FDA for use in the United States (US Food and Drug Administration, 2017). Dental caries is a microbial disease of the teeth, characterized by the irreversible loss of tooth structure due to demineralization of the inorganic component along with dissolution of the organic matrix constituting a tooth. Salivary samples from patients can be evaluated using specific tests referred to as caries activity tests. The Lactobacillus colony count test provides information regarding the microbial load of the cariogenic bacteria Lactobacillus. The Snyder test evaluates the ability of salivary microbes to form organic acids from a carbohydrate substrate, which eventually leads to demineralization of the tooth. Enamel solubility test and saliva flow rate are a few other saliva-based investigations that are employed as predictive biomarkers for dental caries (Wang et al., 2016). As a result of the evaluation, preventive measures may be adopted for highly susceptible individuals or the caries vaccine may be advised. Antibodies (IgG) directed against dengue virus antigen, Ebola virus antigen, and Plasmodium falciparum antigens have also been detected in saliva. The application of these markers in a clinical setting warrants further investigation and validation. Numerous studies have investigated the correlation of serum and salivary glucose levels in diabetics (Gupta et al., 2015). An ongoing challenge of this correlation is to establish a noninvasive, rapid investigation to monitor the glycemic status of patients. In an attempt to facilitate early diagnosis of OSCC, the evaluation of potential biomarkers such as cytokines, i.e., interleukin-6, interleukin-8, matrix metalloproteinases MMP1, MMP3, MMP9, TNF-a, vascular endothelial growth factor A (VEGF-A), fibroblast growth factors, and transferrin in salivary samples has revealed promising results (Radhika et al., 2016). The assessment of CD-59, profilin, and Mac-2ebinding protein (M2BP) have demonstrated an 83% specificity and 90% sensitivity for OSCC detection (Wang et al., 2016). SaliMark OSCC is a recently developed noninvasive salivabased evaluation to detect OSCC. An introduction of these evaluation techniques, as part of a routine workup, needs further validation. Investigating the role of saliva in the detection of distant malignancies is still under way. A study by Bigler et al. (2002) suggests that the response to chemotherapy in breast cancer could be measured by the expression of an oncogenic protein c-erbB-2 in saliva. It is evident that saliva holds numerous biomarkers that are yet to be assayed and correlated. (Fig. 54.6). The major challenges for investigations and research

using saliva include (1) inherent variability of the sample due to dehydration, use of medication or salivary gland disorders, and (2) inconsistent sample collection techniques along with the confounding effects of diet, oral hygiene status, and contamination with oropharyngeal secretions with the samples collected. Yet, salivary diagnostics and investigations remain a promising avenue to be explored owing to the ease of sample collection, patient compliance, and substantial availability of sample, with minimal risks of collection site complications or infections. Thus, the assay of salivary biomarkers could be applied as predictive and diagnostic biomarkers, contributing toward disease prevention, early diagnosis, disease monitoring, and evaluation of treatment.

CONCLUDING REMARKS AND FUTURE DIRECTIONS The exposure to any substance, organic or inorganic, of intrinsic or extrinsic origin, in an excessive amount, can pose to be a threat to normal vital functions. Often individuals may remain oblivious of the risk factors they are exposed to until the disease manifests. The advancements in laboratory techniques have facilitated the early diagnosis of hereditary and acquired pathologies, which would have otherwise presented only at an advanced stage of the disease. Thus health care services now focus not only on treatment but also on prevention and screening. The use of biomarkers has been the key to detection and diagnosis since early medical practice, wherein clinical features of a disease were the only accessible and assessable biomarkers. Over the years, study of the biological response and involvement of organs and organ systems in various diseases has unfurled countless biomarkers ranging from tissue characteristics to the molecular signature of diseases. The oral cavity provides a general impression of an individual’s health status. This view is substantiated by the early and subtle hints of disease, which are often observable at this site. Although not many studies have focused on the oral signs of toxicity, it remains an intriguing area to be investigated because oral signs may precede the actual systemic manifestation of toxicity, thus allowing early intervention. Oral diseases encompass conditions as basic as dental caries and as lethal as oral cancer. A spectrum of other pathologies ranging from hereditary conditions such as amelogenesis imperfecta and regional odontodysplasia to orofacial cysts such as dentigerous cysts, radicular cysts, odontogenic keratocyst, and even tumors such as

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REFERENCES

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Albumin

Saliva Biomarkers

MIP-1β

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N-acetyl-β-D-hexosaminidase

Irisin

Antioxidants

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FIGURE 54.6 An overview of the various biomarkers that have been detected in saliva along with their association with specific disease processes. Image courtesy: Prasad, S., Tyagi, A.K., Aggarwal, B.B., 2016. Detection of inflammatory biomarkers in saliva and urine: potential in diagnosis, prevention, and treatment for chronic diseases. Exp. Biol. Med. 241, 783e799.

ameloblastoma, pleomorphic adenomas and calcifying epithelial odontogenic tumors are also encountered. The use of specific clinical (radiographic) and tissue biomarkers is often employed for diagnosis. Molecular biomarkers in oral diseases continue to be an evolving arena. The discussion of each pathology and its biomarkers is extensive and external to this chapter. The sublingual route of drug administration has garnered much attention as an efficient means of systemic drug delivery. Various pharmaceuticals have developed drug preparations such as lozenges, oral sprays, gels, and dispersible tablets to best use this route. What remains to be achieved is the proposal and acceptance of a standardized universal panel of biomarkers, which can be used to detect, diagnose, and evaluate efficacy of treatment for a particular disease. As newer facts regarding etiopathogenesis and pathophysiology are unfurled, novel biomarkers continue to emerge while many go on to become obsolete. Further

studies to quantify the various organic and inorganic constituents of saliva, as affected by various diseases, could aid in devising a quick, noninvasive and simple screening test.

References Anuradha, C.H., Malathi, N., Anandan, S., et al., 2011. Current concepts of immunofluorescence in oral mucocutaneous diseases. J. Oral Maxillofac. Pathol. 15 (3), 261e266. Azizi, A., Lawaf, S., 2008. The management of oral pemphigus vulgaris with systemic corticosteroid and dapsone. J. Dent. Res. Dent. Clin. Dent. Prosp. 2 (1), 33e37. Bigler, L.R., Streckfus, C.F., Copeland, L., et al., 2002. The potential use of saliva to detect recurrence of disease in women with breast carcinoma. J. Oral Pathol. Med. 31 (7), 421e431. Eisenberg, E., Natarajan, E., Formaker, B.K., 2018. Oral Mucosa and Mucosal Sensation. Department of Oral Health and Diagnostic Sciences, School of Dental Medicine. University of Connecticut, Storrs, CT, USA. Feng, J.Q., Xu, Z.Y., Shi, L.J., et al., 2013. Expression of cancer stem cell markers ALDH1 and Bmi1 in oral erythroplakia and the risk of oral cancer. J. Oral Pathol. Med. 42 (2), 148e153.

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54. ORAL PATHOLOGY BIOMARKERS

´ lvarez, R., Reboires-Lo´pez, D., Ferna´ndez-Gonza´lez, F., Va´squez-A et al., 2011. Histopathological findings in oral lichen planus and their correlation with the clinical manifestations. Med. Oral Patol. Oral Cir. Bucal 16 (5), e641ee646. Fitzpatrick, S.G., Hirsch, S.A., Gordon, S.C., 2014. The malignant transformation of oral lichen planus and oral lichenoid lesions: a systematic review. J. Am. Dental Ass. 145 (1), 45e56. Frohwitter, G., Buerger, H., Van Diest, P.J., et al., 2016. Cytokeratin and protein expression patterns in squamous cell carcinoma of the oral cavity provide evidence for two distinct pathogenetic pathways. Oncol. Lett. 12 (1), 107e113. Gardner, S.L., Geller, R.J., Hannigan, R., et al., 2016. Evaluating oral fluid as a screening tool for lead posioning. J. Anal. Toxicol. 40 (9), 744e748. George, A., Sreenivasan, B.S., Sunil, s, et al., 2011. Potentially malignant disorders of oral cavity. Oral Maxillofac. Pathol. J. 2 (1), 95e100. Gopalakrishnan, A., Balan, A., Kumar, N.R., et al., 2016. Malignant potential of oral lichen planus an analysis of literature over the past 20 years. Int. J. Appl. Dental Sci. 2 (2), 1e5. Gupta, S., Sandhu, S.V., Bansal, H., et al., 2015. Comparison of salivary and serum glucose levels in diabetic patients. J. Diabetes Sci. Technol. 9 (1), 91e96. Holmlund, A., Ha¨nstro¨m, L., Lerner, U.H., 2004. Bone resorbing activity and cytokine levels in gingival crevicular fluid before and after treatment of periodontal disease. J. Clin. Periodontol. 31 (6), 475e482. Ihmed, M.H.M., 2016. Heavy metal toxicity - metabolism, absorption, distribution, excretion and mechanism of toxicity for each of the metals. World News Nat. Sci. 4, 20e32. Jaishankar, M., Testeen, T., Anbalagan, N., et al., 2014. Toxicity, mechanism and health effects of some heavy metals. Interdiscipl. Toxicol. 7 (2), 60e72. Johnston, S., 2013. Feeling blue? Minocycline-induced staining of the teeth, oral mucosa, sclerae and ears e a case report. Br. Dental J. 215 (2), 71e73. Kamath, V.V., Setlur, K., Yerlagudda, K., 2015. Oral lichenoid lesions - a review and update. Indian J. Dermatol. 60 (1), 102. Langie, S.A.S., Moisse, M., Declerck, K., et al., 2017. Salivary DNA methylation profiling: aspects to consider for biomarker identification. Basic Clin. Pharmacol. Toxicol. 121, 93e101. Lavanya, R., Mamatha, B., Waghray, S., et al., 2016. Role of tumor markers in oral cancer: an overview. Br. J. Med. Med. Res. 15 (7), 1e9. Loos, B.G., Tjoa, S., 2000. Host-derived diagnostic markers for periodontitis: do they exist in gingival crevice fluid? Periodontology 39, 53e72. Mayeux, R., 2004. Biomarkers: potential uses and limitations. J. Am. Soc. Exp. NeuroTher. 1, 182e188. Mehdipour, M., Shahidi, M., Manifar, S., et al., 2018. Diagnostic and prognostic relevance of salivary microRNA-21, -125a,e31and -200a levels in patients with oral lichen planus - a short report. Cell. Oncol. https://doi.org/10.1007/s13402-018-0372-x. Mignogna, M.D., Fortuna, G., Leuci, S., 2009. Oral pemphigus. Minerva Stomatol. 58 (10), 501e518.

Moutsopoulos, N.M., Konkel, J.E., 2018. Tissue-specific immunity at the oral mucosal barrier. Trends Immunol. 39 (4), 276e287. Nanci, A., 2017. Ten Cate’s Oral Histology- Development, Structure and Function, ninth ed. Elsevier, Amsterdam. Narang, N., Sharma, J., 2011. Sublingual mucosa as a route for systemic drug delivery. Int. J. Pharm. Pharmaceut. Sci. 3 (2), 18e22. Neville, B., Damm, D.D., Allen, C., et al., 2015. Oral and Maxillofacial Pathology, fourth ed. Elsevier, Amsterdam. Petruzzi, M., 2012. Mucous membrane pemphigoid affecting the oral cavity: short review on etiopathogenesis, diagnosis and treatment. Immunopharmacol. Immunotoxicol. 34 (3), 363e367. Prasad, S., Tyagi, A.K., Aggarwal, B.B., 2016. Detection of inflammatory biomarkers in saliva and urine: potential in diagnosis, prevention, and treatment for chronic diseases. Exp. Biol. Med. 241, 783e799. Radhika, T., Jeddy, N., Nithya, S., et al., 2016. Salivary biomarkers in oral squamous cell carcinoma e an insight. J. Oral Biol. Craniofac. Res. 6 (1), S51eS54. Rastogi, V., Sharma, R., Misra, S.R., et al., 2014. Diagnostic procedures for autoimmune vesiculobullous diseases: a review. J. Oral Maxillofac. Pathol. 18 (3), 390e397. Raymond, J., Cook, D., 2015. Still leaving stains on teethdthe legacy of minocycline? Austr. Med. J. 8 (4), 139e142. Rivera, C., Oliveira, A.K., Costa, R.A.P., et al., 2017. Prognostic biomarkers in oral squamous cell carcinoma: a systematic review. Oral Oncol. 72, 38e47. Sarode, S.C., Sarode, G.S., Tupkari, J.V., 2012. Oral potentially malignant disorders: precising the definition. Oral Oncol. 48 (9), 759e760. Scully, C., Burkhardt, A., 1993. Tissue markers of potentially malignant human oral epithelium lesions. J. Oral Pathol. Med. 22, 246e256. Siriyasatien, P., Chusri, S., Kraivichian, K., et al., 2016. Early detection of novel Leishmania species DNA in the saliva of two HIV-infected patients. BMC Infect. Dis. 16, 89. Sivapathasundharam, B., 2016. Shafer’s Textbook of Oral Pathology. Elsevier, India. Sultan, M.K., Sadatullah, S., Shaik, M.A., 2014. Have biomarkers made their mark? A brief review of dental biomarkers. J. Dent. Res. Rev. 1 (1), 37e41. Taba Jr., M., Kinney, J., Kim, A.S., et al., 2005. Diagnostic biomarkers for oral and periodontal diseases. Dent. Clin. North Am. 49 (3), 551e571. US Food and Drug Administration, 2017. Available from: https:// www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/ DentalProducts/DentalAmalgam/ucm171094.htm. Wang, A., Wang, C.P., Tu, M., et al., 2016. Oral biofluid biomarker research: current status and emerging frontiers. Diagnostics 6 (4), 1e15. Xu, H.-H., Werth, V.P., Parisi, E., et al., 2013. Mucous membrane pemphigoid. Dent. Clin. North Am. 57 (4), 611e630. Yorifuji, T., Kashima, S., Suryadhi, M.A.H., et al., 2017. Temporal trends of infant and birth outcomes in Minamata after severe methylmercury exposure. Env. Poll. 231 (Pt. 2), 1586e1592. Zunt, S.L., 1996. Vesiculobullous disease of the oral cavity. Dermatol. Clin. 14 (2), 291e302.

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55 Biomarkers of Mitochondrial Dysfunction and Toxicity Carlos M. Palmeira, Joa˜o S. Teodoro, Rui Silva, Anabela P. Rolo Center for Neurosciences and Cell Biology of the University of Coimbra and Department of Life Sciences of the University of Coimbra Largo Marqueˆs de Pombal, Coimbra, Portugal

INTRODUCTION Mitochondria play a central role in the life and death of cells. They are not only the mere center for energy metabolism and ATP generation but are also the prime location for different catabolic and anabolic processes, calcium fluxes, and various signaling pathways, while also playing major role in cell life-defining processes such as apoptosis. Mitochondria maintain cellular homeostasis by interacting with reactive oxygenenitrogen species and responding adequately to different stimuli. In this context, the interaction of pharmacological agents with mitochondria is an aspect of molecular biology that is too often disregarded, not only in terms of toxicology but also from a pharmaceutical point of view, especially when considering the potential therapeutic applications related to the modulation of mitochondrial activity. Numerous works have shown that mitochondria are a major toxicological target, with their dysfunction being a major mechanism of drug-induced injury. The aim of this chapter is to highlight the role of mitochondria and the modulation of mitochondrial activities in pharmacology and toxicology and also to stress some of the potential therapeutic approaches. In recent years, there has been extraordinary progress in mitochondrial science that has further outlined the critical role of these organelles in cell biology, pathophysiology, and the diagnosis and therapeutic treatment of different human diseases, such as ischemic diseases, diabetes, some forms of neurodegeneration, and cancer (Duchen, 2004b; Scatena et al., 2007; Giorgi et al., 2012). Mitochondrial physiology and pathophysiology is notably complex, and the role of mitochondria in bioenergetics is also linked, as mentioned earlier, to other essential functions, such as anabolic metabolism,

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00055-4

the balance of redox potential, cell death and differentiation, and mitosis. In addition to these basic functions, mitochondria are associated with more specialized cell activities, including calcium homeostasis and thermogenesis, reactive oxygen species (ROS) and reactive nitrogen species signaling, maintenance of ion channels, and the transport of metabolites. Consequently, the basis of different congenital mitochondrial diseases on a molecular level is equally complex and heterogeneous, making mitochondrial pathophysiology difficult to investigate (Hamm-Alvarez and Cadenas, 2009; Cardoso et al., 2010). This field is made even more challenging by recent evidence that suggests mitochondrial structure and function is dynamic. Specifically, mitochondria possess many interesting properties, such as the ability to fuse or divide, move along microtubules and microfilaments, or undergo turnover (Westermann, 2010; Michel et al., 2012), and these unique properties are often overlooked in research. Undoubtedly, much is still unknown about the mutual interactions between mitochondrial energetics, biogenesis, dynamics, and degradation (Detmer and Chan, 2007), and the contribution of these interactions to mitochondrial toxicology and pharmacology.

MITOCHONDRIAL FUNCTION: GENERAL OVERVIEW The mitochondrion consists of four main structures or compartments: two membranes, the intermembrane space, and the matrix within the inner membrane. The mitochondrial outer membrane (MOM) separates the cytosol from the intermembrane space. The MOM is responsible for interfacing with the cytosol and its interactions with cytoskeletal elements, which are important for

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the movement of mitochondria within a cell. This mobility is essential for the distribution of mitochondria during cell division and differentiation. The mitochondrial inner membrane (MIM) separates the intermembrane space from the matrix. The foldings of the MIM toward the matrix (cristae) serve to increase the surface area of this membrane. Mitochondria also move along intermediate actin filaments, using kinesin and dynein proteins. The MIM hosts the most important redox reactions converting the energy of nutrients into ATP. These reactions are catalyzed by the mitochondrial electron transport chain (ETC), which transports electrons from several substrates to oxygen, in the complex multistep process termed as mitochondrial respiration. According to the chemiosmotic theory, mitochondrial respiration generates a transmembrane potential (DJm) across the inner membrane, which is used by ATP synthase to phosphorylate ADP. The MIM is normally virtually impermeable to protons and other ions, and this solute barrier function of the MIM is critical for energy transduction. Permeabilization of the MIM dissipates the DJm and thereby uncouples the process of respiration from the ATP synthase, halting mitochondrial ATP production (Kushnareva and Newmeyer, 2010). Hence, the free energy of respiration is used to pump protons from the matrix to the intermembrane space (IMS), establishing an electrochemical gradient. Because the MIM displays an extremely low passive permeability to protons, an electrochemical gradient (DmHþ) is built across the membrane. The electrochemical gradient is the sum of two components: the proton concentration difference and the electrical potential difference across the membrane. The estimated magnitude of the proton electrochemical gradient is about 220 mV (negative inside), and under physiological conditions most of the gradient is in the form of electrical potential difference. The proton gradient is converted in ATP by the F1F0-ATP synthase. F1F0-ATP synthase couples the transport of the protons back to the matrix with the phosphorylation of ADP to ATP. Inefficient electron transfer through complexes IeIV causes human diseases in part not only because of loss of energy generation capacity but also of insults to the various enzymes (particularly complexes I, II, and III) induce production of toxic ROS. Defects of complex Vare also a cause of mitochondrial dysfunction (Schapira, 2006; Wu et al., 2010a; Abramov et al., 2011). It has also been reported that the deterioration of mitochondrial function underlies common metabolic-related diseases (Rolo and Palmeira, 2006; Palmeira et al., 2007; Turner and Heilbronn, 2008), and several studies have identified compromised oxidative metabolism, altered mitochondrial structure and dynamics, and impaired biogenesis and gene expression in insulin resistance or type 2 diabetes (T2DM) models (Cheng et al., 2010; Rolo et al., 2011; Gomes et al., 2012; Dela and Helge, 2013; Teodoro et al., 2013). In addition to the process of ATP formation, mitochondria are highly dynamic organelles that have been

implicated in the regulation of a great and increasing number of physiological processes. Cells need energy not only to support their vital functions but also to die gracefully, through programmed cell death, or apoptosis (Kushnareva and Newmeyer, 2010). Execution of an apoptotic program includes energy-dependent steps, including kinase signaling, formation of the apoptosome, and effector caspase activation. Furthermore, mitochondrial regulation is also present beyond cell death mechanisms. Indeed, besides oxidative ATP production, mitochondria assume other functions such as heme synthesis, b-oxidation of free fatty acids, metabolism of certain amino acids, production of free radical species, formation and export of Fe/S clusters, and iron metabolism and play a crucial role in calcium homeostasis (Duchen, 2004a; Michel et al., 2012). In addition, initially described as a key checkpoint of intrinsic programmed cell death, accumulating data point to mitochondria as a central platform involved in many cellular pathways, such as those recently highlighted participating in the innate immune response (West et al., 2011) or its lipidic contribution to autophagosomal membrane genesis during starvation-induced autophagy (Hailey et al., 2010). Still, the regulatory roles of mitochondria over normal physiology include the transduction pathway that underlies the secretion of insulin in response to glucose by pancreatic b-cells and in the evaluation of oxygen tension necessary for sensing oxygen in the carotid body and the pulmonary vasculature. Mitochondria also house key enzyme systems quite distinct from those required for intermediary metabolismdthe rate-limiting enzymes in steroid biosynthesis and even the carbonic anhydrase required for acid secretion in the stomach (Duchen, 2004b). By accumulating calcium when cytosolic calcium levels are high, mitochondria play subtle roles in coordinating the complexities of intracellular calcium-signaling pathways, at least in some cell types, in which their contribution may be extremely important in the finer aspects of cell regulation. The physiological “uncoupling” of mitochondria plays a central role as a heat-generating mechanism in nonshivering thermogenesis in young and small mammals. It has also been suggested that the production of free radical species by mitochondria might play a key role as a signaling mechanismdfor example, in the regulation of ion-channel activities and also in initiating cytoprotective mechanisms in stressed cells (Michel et al., 2012).

MITOCHONDRIAL TOXICITY Mitochondrial dysfunction is a fundamental mechanism in the pathogenesis of several significant toxic effects in mammals, especially those associated with the liver, skeletal and cardiac muscle, and the central nervous system. These changes can also occur as part

XENOBIOTICS AND MITOCHONDRIAL DYSFUNCTION

of the natural aging process and have been linked to cellular mechanisms in several human disease states including Parkinson’s and Alzheimer’s diseases, as well as ischemic perfusion injury and the effects of hyperglycemia in diabetes mellitus (Amacher, 2005). Knowledge of the effects of xenobiotics on mitochondrial function has expanded to the point that chemical structure and properties can guide the pharmaceutical scientist in anticipating mitochondrial toxicity. Recognition that maintenance of the mitochondrial membrane potential is essential for normal mitochondrial function has resulted in the development of predictive cell-based or isolated mitochondrial assay systems for detecting these effects with new chemical entities. The homeostatic role of some uncoupling proteins, differences in mitochondrial sensitivity to toxicity, and the pivotal role of mitochondrial permeability transition (MPT) as the determinant of apoptotic cell death are factors that underlie the adverse effects of some drugs in mammalian systems. To preserve mitochondrial integrity in potential target organs during therapeutic regimens, a basic understanding of mitochondrial function and its monitoring in the drug development program are essential. At the mitochondrial level, there are several potential drug targets that can lead to toxicity, but a real clinical counterpart has been demonstrated only for a few of them. Recently, antiviral nucleoside analog have shown mitochondrial toxicity through the inhibition of DNA polymerase gamma. Other drugs targeted to different components of the mitochondrial channels can disrupt ion homeostasis or affect the MPT pore. Many molecules are known as inhibitors of the mitochondrial ETC, interfering with one or more of the complexes in the respiratory chain. Some drugs, including nonsteroidal antiinflammatory drugs (NSAIDs), may lead to uncoupling of oxidative phosphorylation, whereas the mitochondrial toxicity of other drugs seems to depend on the production of free radicals, although this mechanism has yet to be clearly defined. Besides toxicity, other drugs have been targeted toward mitochondria to treat mitochondrial dysfunctions. A clear example is the recent development of drugs that target the mitochondria of cancer cells to trigger apoptosis or necrosis, thus promoting cell death and fighting cancer (Rohlena et al., 2011).

XENOBIOTICS AND MITOCHONDRIAL DYSFUNCTION Mitochondria, because of their central role in metabolism and cell function, have been often used to assess chemical-induced toxicity. Organophosphorus (OPs) pesticides are a class of widely used pesticides in agriculture and in domestic uses. Mitochondria as a site of

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cellular oxygen consumption and energy production can be a target for OP poisoning as a noncholinergic mechanism of OPs toxicity (Gupta et al., 2001a,b; Karami-Mohajeri and Abdollahi, 2013). Some toxic effects of OPs arise from the dysfunction of mitochondrial oxidative phosphorylation through alteration of the activities of all respiratory complexes and disruption of the mitochondrial membrane. Reduction of ATP synthesis or induction of its hydrolysis can impair the cellular metabolism. The OPs perturb cellular and mitochondrial antioxidant defenses, ROS generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome c released from mitochondria and consequent activation of caspases. Mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, and electron donors and increasing the cytosolic ATP level. Moreover, other organophosphates have been reported to induce neuron apoptosis in hen spinal cords, which might be mediated by the activation of the mitochondrial apoptotic pathway, causing neuropathy (Zou et al., 2013). Some compounds used as food additives for growth promotion, such as olaquindox, induce DNA damage and oxidative stress, causing apoptosis in liver cells through the mitochondrial pathway (Zou et al., 2011). More recently, there have been reports of an increase in the frequency of mitochondrial DNA (mtDNA) somatic mutations in lung tissues of fruit growers that had been exposed to pesticides multiple times via inhalation (Wang and Zhao, 2012). The mitochondrial genome is particularly prone to DNA damage, because of its limited DNA repair capabilities, lack of protective histone proteins, and the low tolerance of damaged DNA. Moreover, mitochondria are known to be the major source of reactive oxygen in most mammalian cell types, as well as a major target organelle for oxidative damage (Chomyn and Attardi, 2003). Mitochondrial superoxide and H2O2 can cause direct damage to mitochondrial proteins, resulting in nuclear and mitochondrial genotoxicity (Shen et al., 2005), and initiation of apoptosis. It has been reported that dioxins cause sustained oxidative stress and damage in liver mitochondria from mice exposed to TCDD and in hepatocytes (Senft et al., 2002; Aly and Dome`nech, 2009); thus, mitochondria are also a direct target for dioxin-induced toxicity. In both hepatic mitochondria isolated from TCDDtreated mice and mitochondria incubated in vitro with TCDD, a number of functional alterations have been observed, including a defect in ATP synthesis and increased ROS production (Senft et al., 2002; Shen et al., 2005; Shertzer et al., 2006; Kopf and Walker, 2010). TCDD decreases hepatic ATP levels through changes in mitochondrial F0F1-ATP synthase and

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ubiquinone and generates mitochondrial oxidative DNA damage, which is exacerbated by decreasing mitochondrial reduced (active) glutathione and by inner membrane hyperpolarization. These mitochondrial effects of TCDD are also associated with altered expression of nuclearly encoded mitochondrial genes (Forgacs et al., 2010; Dere et al., 2011), as well as apoptosis induction involving calcium/calmodulin signaling (Kobayashi et al., 2009). In primary hepatocytes, TCDD has been shown to induce an oxidative stress response involving mitochondrial dysfunction (Aly and Dome`nech, 2009) and, in mice, exposure to TCDD causes a loss in mitochondrial membrane potential mediated by AhR-dependent production of ROS (Fisher et al., 2005). Furthermore, previous studies have identified mitochondrial targets of environmental pollutants, namely ROS production and decreased ATP content (Shertzer et al., 2006). Consequently, maintenance of cellular function is strictly dependent on the existence of a healthy population of mitochondria, given that alterations of mitochondrial bioenergetic features by toxicants reduce energetic charge and may ultimately result in cell death. Some detrimental effects of dibenzofuran (DBF), a ubiquitous dioxin-like compound considered to be an environmental pollutant, have already been reported in lung mitochondria (Duarte et al., 2011) and lung cells (Duarte et al., 2012); in addition, some previous studies reported that environmental toxicants induce mitochondrial damage (Palmeira and Madeira, 1997), proving that some pollutants injure mitochondria directly. More recently, siRNA-mediated knockdown of the AhR in lung epithelial cells and fibroblasts was shown to increase sensitivity to smoke-induced apoptosis (Souza et al., 2013), and these effects involved mitochondrial dysfunction, decreased antioxidant enzymes, and oxidative stress.

MITOCHONDRIA AND DISEASE In a clinical setting, research has shown a significant relationship between mitochondrial metabolic abnormalities and tumors found in renal carcinomas, glioblastomas, paragangliomas, or skin leiomyoma, which has led to the discovery of new genes, oncogenes, and oncometabolites involved in the regulation of cellular and mitochondrial energy production with a particular focus on reevaluating the Warburg effect (Fulda et al., 2010; Ralph et al., 2010; Solaini et al., 2011). Furthermore, the examination of rare neurological diseases, such as Charcot-Marie Tooth type 2a, autosomal dominant optic 53 atrophy, lethal mitochondrial and peroxisomal fission, and spastic paraplegia, has suggested the involvement of MFN2, OPA1, DRP1, or paraplegin in

the auxiliary control of mitochondrial energy production (Benard et al., 2010; Du and Yan, 2010; Zhu, 2010). Advances in the understanding of mitochondrial apoptosis have suggested a supplementary role for Bcl-2 or Bax in the regulation of mitochondrial respiration and dynamics, which has led to the investigation of alternative mechanisms of energy regulation (Benard et al., 2010). In addition, different metabolic diseases, such as diabetes, obesity, and nonalcoholic fatty liver disease (NAFLD), and the more general metabolic syndrome underline the role of dysfunctional mitochondria in pathogenesis (Dalgaard, 2011; Rolo et al., 2011).

MITOCHONDRIAL DYSFUNCTION IN DIABETES In a situation of excess nutrients, mitochondrial membrane potential (DJm) can rise to abnormally high levels, with concomitant excessive reduction of the mitochondrial respiratory chain complexes. This occurs because of elevated levels of ATP and low levels of ADP, meaning that the membrane potential generated by the oxidation of substrates is not totally utilized and begins to build up. Although there is a normal buildup of membrane potential, when it reaches high enough values it can lead to extremely dangerous situations. Overreduction means that the electrons obtained from substrate oxidation can no longer reach molecular O2 at complex IV or cytochrome c oxidase to generate H2O. As the vectorial ejection of protons against their gradient is a requirement for electronic transport across the respiratory chain, given a high enough membrane potential, the electronic leap between each complex no longer carries enough energy to transport protons against their enlarged gradient, and for that reason electrons get “stuck” inside the respiratory chain. This is most dangerous, for these proteic complexes are in an altered, unstable conformational state, which they must abandon by getting rid of the electrons to anything that will take them. That turns out to be molecular O2, resulting in the heightened generation of ROS. Given enough time, the abnormal ROS generation overwhelms natural antioxidant defenses and creates mitochondrial damage, further increasing their generation and leading ultimately to cellular and tissue dysfunctions (Fig. 55.1). The increase in nutrients offered without elevated demand for ATP leads to the abnormal augment of membrane potential (DJm). This, in turn, leads to increased ROS generation that, if prolonged enough, causes cellular and mitochondrial damage. By activating PGC-1a (by phosphorylationdAMPK and deacetylationdsirtuins), one can induce the activation of the mitochondrial biogenic program, leading to the generation of more mitochondria. More mitochondria allows for better

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ATP

Sirtuins

AMPK

A c

A c

Cellular and mitochondrial damage

ADP PGC1α

P

PGC1α

P

Transcrip on Factors

Oxida ve damage

ROS

More mitochondria

Reduced ROS genera on

FIGURE 55.1 Role of mitochondria in diabetes and obesity. AMPK, AMP-activated protein kinase; PGC1a, peroxisome proliferator-activated receptor g coactivator 1a; ROS,./ reactive oxygen species; UCP1, uncoupling protein 1.

handling of the excess nutrients, thus reducing ROS generation. Another way to reduce DJm is by mildly uncoupling the oxidative phosphorylation. UCP1 (and other members of the uncoupling protein family) accomplishes this by reducing DJm and generating heat, thus preventing ROS generation. It was found that mitochondria from high-fat fed (HFD) rats were morphologically and structurally altered (Lieber et al., 2004; Kim et al., 2008). These studies demonstrate that there appears to exist a direct correlation between altered mitochondrial functionality and insulin resistance, obesity, and diabetes (Vial et al., 2010). As such, correct mitochondrial structure and function correction could lead to the unveiling of therapeutic strategies to treat obesity and diabetes. The master regulator of mitochondrial biogenesis, the peroxisome proliferatoreactivated receptor g (PPARg) coactivator 1a (PGC1a), is of great necessity for the correct number, structure, and function of mitochondria. The regulation of PGC1a can occur by several means: its expression, phosphorylation, and acetylation status (Fernandez-Marcos and Auwerx, 2011), for example. The ones highlighted are particularly important, for they appear to be dependent on the cell’s energetic status. In fact, it has been shown that sirtuin 1 (SirT1) regulates PGC1a. Sirtuins are a class of NADþ-dependent deacetylases and, as such, their activity on gene

transcription can be classified as a nutrient-sensitive action. Sirtuins’ activity as gene transcription modulators has been explored in various fields of investigation, from aging to obesity, diabetes, and Alzheimer’s, to name a few (Yamamoto et al., 2007). SirT1 effects on metabolic regulation were found on SirT1-null mice, which have decreased insulin release, whereas overexpression of SirT1 has increased insulin response to glucose (Moynihan et al., 2005). Also, SirT1 deacetylates and thus activates PGC1a, correlating directly to improved metabolic status (Nemoto et al., 2005). Curiously, SirT1 decreases uncoupling protein 2 (UCP2) expression, resulting in increased mitochondrial coupling and thus reducing substrate utilization (Moynihan et al., 2005), which makes some sense because elevated NADþ levels activate SirT1 and, as such, the cell has energetic needs and should not waste DJm. Conversely, PPARg’s expression is downregulated by SirT1, resulting in decreased adipogenesis and increased lipolysis (Picard and Auwerx, 2002). SirT1 activation of PGC1a in brown adipocytes leads to increased mitochondrial biogenesis and thus increased thermogenic dissipation of excess nutrients (Lagouge et al., 2006). For further reading on SirT1, readers are encouraged to read the excellent work by Yamamoto et al. (2007). These works are sometimes conflicting and make it difficult to understand the effects of SirT1

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on metabolism. In fact, until very recently, the role of resveratrol (the most famous natural SirT1 activator) was still questioned and not fully understood (Hubbard et al., 2013). SirT1 is the most famous and most studied sirtuin, but it is not the only one. Another extremely important sirtuin is the normally mitochondrial native SirT3. Expression of SirT3, in white and brown adipose tissue (WAT and BAT, respectively), is induced by calorie restriction and cold exposure. One of the most interesting facts about SirT3 is that its constitutive expression causes increased levels of PGC1a and UCP1 expression, and as such it is a very attractive target for obesity reduction. Because it is a mitochondrial sirtuin, SirT3’s activity leads to decreased acetylation of mitochondrial proteins, which invariably results in increased activity (Shi et al., 2005). We have recently shown that the isoquinoline alkaloid berberine is a potent inducer of SirT3’s activity in high fatefed rats, which at least partially explains this compound’s potent anti-obesity effects (Teodoro et al., 2013). Because SirT1 is a NADþ-dependent deacetylase, its activity is dependent on the cell’s reductive status, and a high nutrient ambient leads to decreased SirT1 activity, where PGC1a remains acetylated and its activity diminished (Canto´ and Auwerx, 2009). Another metabolic sensor, the AMP-activated protein kinase (AMPK), is activated in the presence of low energy levels, i.e., when the ATP levels are low (or, more appropriately, when AMP levels are high). It phosphorylates PGC1a, activating it to produce more mitochondria to try to elevate ATP levels (Canto´ and Auwerx, 2009). As such, these sensors’ activities on PGC1a were designed to counter situations of low energy stress and are completely shut down in obesity and diabetes. Consequently, the artificial induction of their activation can be considered a potential and extremely attractive therapeutic strategy, especially considering that oxidative phosphorylation inhibition is a hallmark of HFD and diabetic animals. AMPK is an important metabolic sensor and regulator, being involved in, among other effects, glucose uptake, lipidic b-oxidation, and mitochondrial biogenesis. Its effects are present on several organs, from the liver to the brain, from WAT and BAT to skeletal muscle, i.e., all metabolic-relevant tissues (Winder et al., 2000). Because AMP activates AMPK, a rise in this adenosine nucleotide (with concomitant decrease in ATP) signals the cell to begin substrate oxidation processes to generate ATP. As paralleled by NADþ and SirT1, this (and subsequent downstream effects) can be explored (and has been extensively studied) for obesity management. Although SirT1 deacetylates proteins and histones, AMPK phosphorylates and alters proteins’ activity (either increasing or decreasing) (Hardie et al., 2012). AMPK induces GluT1 activation and GluT4 migration to the cellular membrane and thus increased

glucose uptake and oxidation (Barnes et al., 2002; Pehmøller et al., 2009). AMPK also mediates fatty acid uptake in cardiac cells (Habets et al., 2009), while improving their uptake and oxidation mainly by the inhibition of acetyl-CoA carboxylase and thus increasing mitochondrial import of fatty acids (Merrill et al., 1997) and by increasing the glycolytic rate (Marsin et al., 2002). Another key effect of AMPK is on mitochondrial biogenesis for, unsurprisingly, AMPK phosphorylates and activates PGC1a, thus increasing mitochondrial content, particularly in skeletal muscle (Winder et al., 2000). Finally, AMPK can also activate (and be activated by) SirT1, by increasing cellular NADþ levels (Canto´ et al., 2010). As with sirtuins, these are just some effects of AMPK on metabolism, for it is also involved in many other biological functions. For further reading, we refer the reader to Hardie et al. (2012). Oxidative stress also plays a major role in mitochondrial dysfunction in high-energy situations. In fact, as noted before, increased ROS generation is common in high-energy situations. The increased ROS generation, along with mitochondrial damage, also causes the activation of inflammatory pathways (as the c-Jun N-terminal kinase [JNK] and mitogen-activated protein kinase [MAPK]). These cause the inactivation of the insulin signaling pathway and loss of GluT4 (the insulin-sensitive glucose transporter) translocation to the cellular membrane (Qatanani and Lazar, 2007), increasing insulin resistance and thus exacerbating the problem. Also, because of the high energy levels, it comes as no surprise that the expression of proteins involved in lipid handling and mitochondrial lipid b-oxidation is diminished (Schreurs et al., 2010). Furthermore, ROS contribute to diminish glycolytic rates, as it is known that ROS inhibit the key glycolytic enzyme GAPDH (Du et al., 2000), which appears to be a self-defense mechanism against glucose damage (Rolo and Palmeira, 2006). Also, the persistent excess of nutrients leads to the maintenance of said inhibition and worsening of the situation. All of this contributes to increased lipid deposition inside cells, which affects not just mitochondrial function, but also the entire cell. Increasing the number of mitochondria is an attractive strategy for it leads to more units to carry the load of more nutrients, because not only would ROS generation be attenuated but also more antioxidant defenses would also be present. On the other hand, mildly uncoupling mitochondria leads to decreased ROS generation by the caloric dissipation of the electrochemical protonic gradient (Korshunov et al., 1997; Skulachev, 1998). There is already much work being conducted on both perspectives, both yielding very promising results (for further reading, please refer to Ren et al., 2010; Wu et al., 2013).

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In terms of increasing mitochondrial numbers, there has been an enormous push toward research on brown adipose tissue since its recent discovery in human adults. Brown adipose tissue (BAT) evolved into the main source of heat generation in small mammals by having a high content of UCP1-expressing mitochondria (UCP1 can be and is considered a hallmark of BAT) (Cannon and Nedergaard, 2004). When activated, BAT generates a high metabolic rate, sustained by a rather large rate of substrate oxidation, which is obtained both from its lipid droplets and from circulation (Bartelt et al., 2011). As such, overactivation of BAT is, theoretically, a highly effective therapeutic strategy for obesity. In fact, we have recently shown just that, with the use of the bile acid chenodeoxycholic acid (CDCA) an obese phenotype can be normalized by elevating BAT UCP1 activity (Teodoro et al., 2014). It was thought that BAT was not present in adult humans, voiding such therapeutic approaches, but, as noticed before, it has been shown otherwise (Whittle, 2012). However, Vosselman et al. (2012) demonstrated that overstimulation of BAT in adult humans was hardly a valid strategy. As such, thermogenic therapy for obesity in adult humans could only be a failed idea, if not for the fact that adipocytes are highly plastic cells. This means that, given the right stimuli, white adipocytes can be, to some extent, converted into brown-like cells, the so-called “brite” or beige adipocytes (the opposite, i.e., the conversion of brown into white is also possible). As such, the next “big thing” in metabolic research is the conversion of white into brown adipocytes thus creating elevated basal metabolic rates, burning more fuel, and decreasing obesity. Most therapeutic strategies already studied and reported, which reduce adiposity in white adipocytes, produce metabolic alterations that are common to what is described to happen in “brite” inductiondi.e., the activation of PPARa and induction of lipolysis, increased leptin release, and induction of mitochondrial biogenesis (Flachs et al., 2013). These alterations are usually associated with UCP1 induction and nonshivering thermogenesis. Curiously, UCP1-null mice are obesityresistant when exposed to cold, but not at thermoneutrality (Anunciado-Koza et al., 2008). To make matters worse, the work by Nedergaard and Cannon (2013) skillfully argues that, despite the huge increase in UCP1 expression in WAT (arising from virtually zero), the overall contribution of these newly formed “brite” cells to the body’s basal metabolic rate is negligible at best. As such, the authors propose that some other mechanism is responsible for the effects demonstrated in other works. Flachs et al. (2011) suggest that n-3 PUFA antiobesogenic effects are not UCP1-dependent, which is also the case when combined with calorie restriction, but they are caused by increased cycling of triglyceride/free fatty acid cycle (TG/FFA cycle), a so-called

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metabolic futile cycle, for it consumes energy while not causing the generation of products. This cycle could be behind many anti-obesogenic effects of countless compounds, whose effects are clear, but whose mechanisms are not. Research into these metabolic pathways will probably become a hot topic for obesity research in the near future. We have shed some light into this matter, by demonstrating once more using CDCA that there is more than just thermogenic dissipation behind bile acids’ anti-obesity effects, in particular, an acceleration of metabolic functions (Teodoro et al., 2016).

MITOCHONDRIAL DYSFUNCTION IN ISCHEMIA/REPERFUSION Ischemia/reperfusion (I/R) injury is a phenomenon whereby damage to a hypoxic organ is accentuated following the return of the oxygen supply, and it has been recognized as a clinically important pathological disorder. I/R may occur in many clinical situations such as transplantation, resection, trauma, shock, hemorrhage, and thermal injury. The mitochondrial function is impaired in I/R settings, leading to an alteration of energy metabolism. Ischemia leads to the cessation of oxidative phosphorylation, which causes tissue ATP and creatine phosphate concentrations to decrease with a simultaneous increase in ADP, AMP, and inorganic phosphate (Pi) concentrations. During ischemia, anaerobic glycolysis and ATP degradation produce Hþ-maintaining mitochondrial membrane potential. As maintenance of ion gradients across the plasma membrane and between cellular compartments depends on ATP-driven reactions, metabolic disruption by injurious stresses may rapidly perturb cellular ion homeostasis. During oxygen deprivation the intracellular Hþ, Naþ, and Ca2þ levels are elevated, inducing osmotic stress and causing mitochondrial damage. Intracellular Hþ accumulation activates the Naþ/Hþ exchanger, leading to Naþ influx. Naþ efflux is attenuated because the Naþ/Kþ-ATPase is inhibited during ischemia. Therefore, Naþ/Hþ exchange activity leads to increasing intracellular Naþ (Inserte et al., 2002, 2006; Murphy and Steenbergen, 2008). This augmentation of intracellular Naþ during ischemia is accompanied by an increase in intracellular Ca2þ through reverse mode of the Naþ/Ca2þ exchanger. Although Naþ overload stimulates Ca2þ influx by the Naþ/Ca2þ exchanger and depletion of ATP reduces Ca2þ uptake by the endoplasmic reticulum, the Ca2þ level is maintained as modest during ischemia because acidosis inhibits the Naþ/Ca2þ exchanger, and cytosolic Ca2þ is taken up by the mitochondria as long as its membrane potential is maintained. Influx of extracellular Ca2þ is responsible

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for irreversible cell injury as shown by studies in which removal of extracellular Ca2þ protects against various hepatotoxicants (Schanne et al., 1979; Farber, 1982). ROS generation plays a major role in damaging the organ during ischemia and sensitizing it to reperfusion. The source of the ROS is uncertain, perhaps involving complexes I and III of the ETC of mitochondria, or perhaps xanthine/xanthine oxidase (X/XOD) acting on xanthine formed from the degradation of adenosine (AMP is slowly converted into adenosine and then inosine and xanthine through a purine degradation pathway). There is a gradual decline in cellular integrity as a consequence of combinative action of ATP depletion, elevated intracellular Ca2þ, and ROS. Thus, the ATP-dependent repair processes are incapable of operating. Maintenance of mitochondrial integrity is a critical determinant of cell outcome. As such, if mitochondria remain sufficiently intact to generate ATP after short periods of ischemia, tissue damage is reversible and can be repaired. But if the ischemia is more aggressive then recovery is not possible. The ATP restored during reperfusion will exacerbate the damage to the organ due to metabolic disorders that accumulate during ischemia leading to cell death. The increased Ca2þ and bursts of ROS generation are characteristics of reperfusion. Probably the majority of ROS is formed by uncoupled mitochondria, mainly from mitochondrial complexes I and III of the ETC (Jaeschke and Mitchell, 1989; Turrens, 2003). When the respiratory chain is inhibited by absence of oxygen and then reexposed to oxygen, ubiquinone can become partially reduced to ubisemiquinone. It can then react with oxygen to generate superoxide that is reduced to hydrogen peroxide by superoxide dismutase. Hydrogen peroxide is removed by glutathione peroxidase or catalase, but if ferrous ions (or other transition metals such as copper) are present it will form the highly reactive hydroxyl radical through a Fenton reaction (Becker, 2004). In fact, mitochondrial lipids and proteins that are damaged during ischemia favor ROS generation during reperfusion (Inserte et al., 2002). ROS cause peroxidation of cardiolipin of the inner mitochondrial membrane, impairing electron flow through the ETC (Petrosillo et al., 2003; Paradies et al., 2004). Moreover, lipid peroxidation causes the release of reactive aldehydes such as 4-hydroxynonenal that alters membrane proteins (Echtay et al., 2003). ROS also have direct effects on several respiratory chain components and can cause inhibition of the ATP synthase and ANT (adenine nucleotide translocase). There is depletion in superoxide dismutase, glutathione peroxidase, and glutathione during reperfusion that enhances oxidative stress. Mitochondria are the major target of ROS and Ca2þ overload. These agents are potent inducers of the MPT,

resulting in mitochondrial-initiated cell death. A major consequence of MPT induction is inhibition of oxidative phosphorylation, which when unrestrained will lead to necrotic cell death. The permeability transition has also been pointed to as being involved in apoptosis, through the release of proapoptotic factors, such as cytochrome c, and other apoptosis-inducing factors into the cytosol (Forbes et al., 2001; Murata et al., 2001). In response to proapoptotic signals, Bax, a proapoptotic member of the Bcl-2 family, is translocated to the mitochondria and can form channels that allow the release of cytochrome c from the mitochondrial intermembrane space (Borutaite and Brown, 2003). In conditions of ATP depletion, apoptosis can deviate to necrosis (necroapoptosis). Changes in mitochondrial morphology achieved by fission and fusion may play an important role as a determinant of cell viability. It is important to understand the molecular mechanisms of mitochondrial dynamics and their relationship with ischemia-reperfusion injury. Given the role of mitochondria in ischemia/reperfusion injury, strategies have been developed that focus on maintaining mitochondrial function and consequently reducing the damage. Perfusion with GSK-3b inhibitors reduces cell death induced by I/R (Tong et al., 2002; Gross et al., 2004; Pagel et al., 2006; Gomez et al., 2008). It is thought that the mechanism of protection elicited by GSK-3b inhibition is related to modulation of MPT, by interaction between GSK-3b and components of the MPT process (Juhaszova et al., 2008). Phospho-GSK-3b can bind to the ANT, voltagedependent anion channel (VDAC), or Cyclophilin D (CypD) (Pastorino et al., 2005; Nishihara et al., 2007). Pretreatment with indirubin-30 -oxime (an inhibitor of GSK-3b) in conditions of hepatic I/R protects the liver by maintaining mitochondrial calcium homeostasis, thus preserving mitochondrial function and hepatic energetic balance (Varela et al., 2010). GSK-3b inactivation by indirubin-30 -oxime acts as pharmacological preconditioning, modulating the susceptibility to MPT induction and preserving mitochondrial function after I/R. The suppression of the ANT-CypD interaction may contribute to the elevation of the threshold for MPT induction. CypD null mice mitochondria have been demonstrated to have higher Ca2þ buffering capacity, demonstrating a desensitization of these mitochondria to Ca2þ-induced MPT (Baines et al., 2005). Recently, a relationship was established between SirT3 and CypD: SirT3 deacetylates and inactivates CypD causing its dissociation from the ANT (Shulga et al., 2010). The decrease in SirT3 activity leads to increased activation of the MPT in response to Ca2þ increases, cardiac stress, and aging, resulting in a decline in cardiac function (Hafner et al., 2010). This ability to suppress MPT formation indicates SirT3 as a potential target for new drugs that protect against I/R.

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Mitochondrial K-ATP channels are normally closed in vivo because of the inhibitory concentrations of ATP and ADP. Mitochondrial K-ATPedependent matrix alkalization, preservation of mitochondrial volume, and the structure of the intermembrane space, as well as MPT inhibition, are involved in a preconditioning protective action (Andrukhiv et al., 2006; Costa et al., 2006; Costa and Garlid, 2008). Diazoxide is a selective mitochondrial K-ATP channel agonist that was previously shown to decrease I/R injury induced by orthotopic liver transplantation (Huet et al., 2004). The protective effects of diazoxide against hepatic I/R were dependent on Bcl-2 expression and also with the inhibition of mitochondrial cytochrome c release, being abolished by siRNA knockdown of Bcl-2 (Wu et al., 2010b). We have recently contributed to the demonstration of the essential role of mitochondrial function preservation in an I/R setting (Alexandrino et al., 2016) (Fig. 55.2). The events of ischemia/reperfusion lead to a number of cellular alterations, with particular relevance for mitochondrial function. During ischemia, restriction of blood flow limits access to nutrients, ions and, most relevantly, oxygen. Because ATP requirements are maintained (and, in some cases, elevated), ATP generation drains the mitochondrial membrane potential. Eventually, the cell has to resort to anaerobic generation

[O 2]

of ATP through glycolysis, which results in the accumulation of lactate and concomitant decrease in pH. Furthermore, ion exchanges are altered leading to intracellular Naþ accumulation and Ca2þ overload (see full text for further details). Accompanying the decrease in mitochondrial function, there is a mild increase in ROS generation. If the ischemic event is prolonged and if the cell was not prepared for it (for example, by preconditioning it with pharmacological agents or with short, repetitive ischemia/reperfusion events), during reperfusion, the restoration of blood flow and particularly of oxygen restores mitochondrial activity but in a totally different setting. Mitochondrial environment and function are compromised because of alterations suffered during ischemia, and ROS generation is highly exacerbated, with resulting damage to mitochondrial components such as the members of the respiratory chain and cardiolipin, heightening the problem. All this leads to the induction of the mitochondrial permeability pore, with concomitant release of cytochrome c and other proapoptotic factors, which might lead to cell death. Various therapeutic agents have already been shown to be modulators of mitochondrial function and normalizers during ischemia/reperfusion. Of note, berberine leads to an overactivation of SirT3 and GSK-3b inhibitors such as indirubin-30 -oxime (see text for further details).

[O2] Berberine

Anaerobic glycolysis

ATP degrada on H+

GSK-3β inhibitors

Re-energiza on of mitochondria

pH restora on

Lowering pH Cell Death

Na+

GSK-3β Sirt3

Na+

ETC

Ca2+

ETC

ROS

Mild ROS amount

GPx Catalase

H+ Oxidazed CL

CL

Na+ ROS & Ca2+ overload

Na+ Ca2+

∆Ψm

∆Ψm

FIGURE 55.2 Role of mitochondria in Ischemia/Reperfusion injury. DJm, mitochondrial membrane potential; CL, cardiolipin; ETC, electronic transport chain; GPx, glutathione peroxidase; MPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species.

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MITOCHONDRIAL DYSFUNCTION IN CANCER As noted before, mitochondria are responsible for a wide array of reactions and phenomena in the cell. Energy (ATP) production, redox status maintenance, ROS generation, Ca2þ storage, and apoptosis control, to name a few, are all dependent on and/or take place in mitochondria. As such, it comes as no surprise to find that mitochondrial alterations and/or deregulation are involved in cancer development. In fact, alterations in biosynthetic pathways, cell signaling, or DNA replication can shift a cell from a quiescent to a proliferative status (Wallace, 2012). The idea of mitochondrial dysfunction in cancer is rather ancient, dating back to the description of the Warburg effect, meaning the realization that cancerous cells have increased lactic acid production in the presence of oxygen, the so-called aerobic glycolysis. This led to the idea that cancer cells have damaged mitochondria because pyruvate is converted into lactate, and glycolysis is the main source of ATP for the cancerous cell. This is further supported by the notion that the interior of tumors is a highly anoxic area, elevating the need for glycolytic-derived ATP and downplaying the need for a functional mitochondrial population. All these factors would come into play in creating a highly proliferative, malignant cell. As discussed below, a healthy mitochondrial population is as vital to a normal cell as it is to a cancerous one. It is noteworthy, however, that it is true that mtDNA is highly mutated in cancer cells and these cells have a higher glycolytic rate than do normal cells. It is the fact that mitochondria are not essential to the cancerous cell that is in dispute. One way by which we can demonstrate that mitochondria are essential to cancerous cells comes from studies where the mtDNA was removed by ethidium bromide exposure, generating the so-called r0 cells. These cells present a lower metabolic rate, resulting in decreased growth and tumor formation (Cavalli et al., 1997; Weinberg et al., 2010). In his seminal review, Wallace (2012) points to Tasmanian devils’ and dogs’ transmissible tumors, which cause mtDNA decay and which would have disappeared long ago, if not for periodic uptake of normal mtDNA from host cells. Both somatic and germline mtDNA mutations have been reported in almost all cases of tumors and cancers. Despite the fact that some of these alterations could be considered normal heterogeny of the population, there are no doubts about the direct correlation of some of these mutations with a cancerous phenotype. Consequently, almost all cancerous cells demonstrate impaired OXPHOS activity, when compared with normal cells. As such, these alterations can be beneficial to the cancer cell,

by promoting neoplastic alterations and allowing the cancer cell to adapt to a wide array of metabolic environments (Brandon et al., 2006). In fact, several mtDNA mutations have been demonstrated to be positively selected in cancer cells, in contrast to normal cells (Gasparre et al., 2008). But the presence of a mutation that will affect mtDNA and thus lead to a cancerous phenotype appears to not even be necessary, for it has been shown that the nuclear-encoded SUV3 RNA helicase is required for correct mitochondrial function and biogenesis. In fact, SUV3þ/þ can present the same altered mitochondrial phenotype of a heterozygous or double recessive individual, because of maternal inheritance of altered mitochondria (Chen et al., 2012). But if cancerous cells do indeed present mitochondrial alterations and impaired OXPHOS activity, then how is it possible that functional mitochondria are needed for the viability of said cells? For one, mitochondrial activity can be altered by more than just mtDNA mutations. In fact, various nDNA mutations for mitochondrial proteins have been reported in various types of cancers. For example, mutations in the complex II proteins have been shown to lead to increased ROS generation, decreased succinate consumption, and decreased respiratory rates and thus a shift toward glycolysis, in a mechanism that appears to involve the stabilization of the hypoxia-inducible factor 1a, HIF1a (Wallace and Fan, 2010; Kurelac et al., 2011). In the same fashion, fumarate hydratase, an enzyme of the Krebs cycle, has also been shown to be mutated in various cancers, leading to increased succinate levels and thus the shift toward increased glycolytic metabolism but, apparently, without the involvement of HIF4a (Adam et al., 2011; Frezza et al., 2011). There are many other reported mutations in other mitochondrial proteins, for example isocitrate dehydrogenase (Ward et al., 2012), and complexes I, III, IV, and V of the respiratory chain (Wallace, 2012). Mitochondria produce ROS as a by-product of their activity. It is estimated that roughly 5% of all oxygen consumed in a cell is not converted into water but rather into a reactive species. Although a by-product, it has been recently shown that ROS have a signaling activity (Lander, 1997; Devasagayam et al., 2004), resulting in their production being not only required but also needed (within certain limits). As such, a complex (and, for most cases, effective) antioxidant system is in place to reduce them and thus neutralize their activity. However, mitochondrial defects, metabolic imbalances, mutations, and lack of mtDNA histones all lead to increased mtDNA mutations and thus increased cancerous potential. ROS can be one of the most important causal factors for said mutations. In fact, it has been demonstrated that ROS generation is augmented by

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inhibition of the OXPHOS, in particular of the ATP synthase (Sa´nchez-Cenizo et al., 2010). In addition to contributing to increased ROS generation, this blockade of OXPHOS also leads to the already discussed metabolic shift toward glycolysis. We have previously mentioned that the shift toward a greater dependence on glycolytic-derived ATP is a standard condition in the progression from a normal to a cancerous cell. This shift typically involves the overactivation of the PI3KeAkt signaling pathway (Jones and Thompson, 2009), which leads to increased expression of glucose transporters, glycolytic and lipogenic enzymes, and activation of the mechanistic target of rapamycin, mTOR (DeBerardinis et al., 2008), resulting in elevated rates of glycolysis and lactate generation. This pathway also inhibits fatty acid oxidation and prevents carbon flow toward the Krebs cycle (Gatenby and Gillies, 2004; DeBerardinis et al., 2008). Furthermore, by inhibition of the master regulator of mitochondrial biogenesis, PGC1a, this pathway leads to decreased mitochondrial respiratory activity and content, of both respiratory chain units and antioxidant defenses (Daitoku et al., 2003). As mentioned before, HIF1a stabilization is a recurring phenomenon in cancer cells, so it comes as no surprise to realize that HIF1a can impair mitochondrial biogenesis (Zhang et al., 2007). But why does the cancerous cell go to such an extent as to possibly limit its own ATP production? The answer is rather simple: the downplay of mitochondrial bioenergetics is a side effect of the need for the activation of glycolysis-parallel anabolic pathways. By increasing glycolysis, more carbon is shunted toward, for example, the pentose phosphate pathway for nucleotide synthesis and NADPH generation to combat oxidative stress (Gru¨ning et al., 2011). Also, the increased generation of glycerol-3-phosphate in glycolysis leads to heightened lipogenesis (Esechie and Du, 2009), which requires mitochondrial-derived acetyl-CoA, supplied by a complex process that involves the aggressive oncogene Myc, in an anaplerotic refill of Krebs cycle intermediaries (DeBerardinis et al., 2007), making cancer cells dependent on glutamine for their survival (Wise et al., 2008). The antitumor p53 protein provides another proof of mitochondrial dependence of cancer cells. p53 inhibits glycolysis and diverts carbon toward the pentose phosphate pathway, leading to increased NADPH generation; in addition, its activation by telomere shortening prevents PGC1a-mediated mitochondrial biogenesis, thus leading to increased ROS generation and cellular senescence (Sahin and Depinho, 2010). As such, it is clear that, for a cancerous cell, a viable, efficient mitochondrial population is a vital requirement (Vander Heiden et al., 2009). There are many other ways by which mitochondrial function is linked with cancer (masterfully reviewed in

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Wallace, 2012). If mitochondria are so vital for the cancer cell, then it is only reasonable to assume that targeting mitochondria to try to treat cancer is a viable therapeutic strategy. In fact, various works (Fulda et al., 2010; Hockenbery, 2010; Wenner, 2012) have focused on the subject. Therefore, we will briefly approach the thematic in these works. Cancer trademarks, such as immortal potential, unresponsiveness to growth arrest signaling, increased anabolic metabolism, and decreased apoptosis and autophagy, have already been linked to mitochondria (Galluzzi et al., 2010). Despite the already discussed dependence on mitochondrial activity, cancer cells have structurally and functionally different mitochondria when compared with normal cells (ModicaNapolitano and Singh, 2004). One way to target mitochondria is by targeting its membrane potential (DJm). Specific ANT ligands can lead to the induction of the MPT (Lehenkari et al., 2002; Don et al., 2003; Oudard et al., 2003) and could be a valid strategy because cancer cells should be more susceptible owing to having higher metabolic rates and higher Ca2þ loads. Also, the peripheral benzodiazepine receptor (PBR) is thought to be a component of the MPT (as well as ANT) and binds to the voltage-dependent anion channel (VDAC) and prevents MPT induction. Thus, it comes as no surprise that PBR is typically overexpressed in various cancers, as it blocks the antiapoptotic effect of the Bcl-2 protein family. As such, PBR ligands have shown antitumor activity (Decaudin et al., 2002). These are but a few examples of how reducing DJm and inducing apoptosis can be considered a valid and promising anticancer strategy. In fact, various compounds are already in test that target these described mechanisms. It is evident that a compound or therapeutic strategy that would increase ROS generation or inhibit antioxidant defenses in cancer cells could be immensely helpful. Along these lines, there are already some promising results with ROS inducers (Sarin et al., 2006; Bey et al., 2007; Mehta et al., 2009) and antioxidant defense compounds (Alexandre et al., 2006; Trachootham et al., 2006; Dragovich et al., 2007), to name a few. In the mitochondrial intermembrane space reside various proapoptotic factors (e.g., cytochrome c, cyt c), and their release is a common phenomenon in mitochondrial-dependent apoptosis, a phenomenon that usually involves the proapoptotic proteins BAX and BAK (Chipuk et al., 2006). As such, the modulation of the activities of the pro- and antiapoptotic family of proteins (for example, the Bcl-2 family) can provide interesting therapeutic approaches. The compounds ABT-737 (Oltersdorf et al., 2005) and ABT-263 (Tse et al., 2008) are just two examples. Of course, targeting mitochondrial metabolic activity is also a strong possibility. Inhibition of the pyruvate

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dehydrogenase kinase (which blocks PD activity of converting pyruvate into acetyl-CoA) by dichloroacetate leads to increased ROS generation in cancerous, but not in normal cells (Bonnet et al., 2007). Also, inhibition of the expression of lactate dehydrogenase A has shown promising results, by impeding the conversion of pyruvate into lactate (Fantin et al., 2006). Pyruvate is also actively sent toward lipid synthesis, so the block of the key enzyme ATP citrate lyase has also shown promising results (Hatzivassiliou et al., 2005). These are only a few examples of how targeting mitochondrial metabolism is a valid therapeutic strategy to combat cancer. There are many other compounds and therapeutic strategies and targets being currently tested against cancer, many of them also involving mitochondria. Therefore, it would be no surprise if the cure for cancer came from a strategy that involved mitochondria.

CONCLUDING REMARKS AND FUTURE DIRECTIONS Considering that mitochondria play an essential role in cellular homeostasis and signaling processes, identifying biomarkers of mitochondrial dysfunction and toxicity is of fundamental relevance. Research into these mitochondrial targets is at present a hot topic in several diseases (diabetes, obesity, cancer, etc., to name just a few examples) and drug-induced toxicity. An improved understanding of the changes that occur at the mitochondrial level is essential to discover new therapeutic targets for mitochondria-related diseases and toxicity exposure.

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C H A P T E R

56 Biomarkers of BloodeBrain Barrier Dysfunction Rekha K. Gupta1, Ramesh C. Gupta2 1

School of Medicine, University of Louisville, Louisville, KY, United States; 2Toxicology Department, Breathitt Veterinary Center, Murray State University, Hopkinsville, Kentucky, United States

INTRODUCTION Out of all vital organs in the body, the central nervous system (CNS) is the most complex organ in terms of its structure and function. It is composed of certain interfaces, such as the bloodebrain barrier (BBB), the bloodecerebrospinal fluid barrier (BCSFB), and the bloodespinal cord barrier. These can collectively be called bloodeCNS barriers or brain barriers. Evidence for the existence of the BBB came from the observations (based on staining technique) of Nobel Laureate Paul Ehrlich in the early 20th century. In 1921, the findings of a Russian neurophysiologist, Lina Stern, further supported the existence of BBB and designated it as the “hematoencephalic” barrier. Finally, electron microscopic observations confirmed the presence of the BBB (tight junctions between the endothelial cells that form brain capillaries) (Abbot et al., 2006, 2010; Palmer, 2010). Deli et al. (2005) defined the BBB as a dynamic interaction between cerebral and endothelial cells constituting the anatomical basis of the BBB and other neighboring cells, such as astroglia, pericytes, perivascular microglia, and neurons. The cross talk between these cells endows endothelial cells with a unique BBB phenotype comprising not only the morphological barrier of endothelial tight junctions but also the biochemical (enzymatic and metabolic) barriers, as well as the uptake and efflux transport systems (Abbott et al., 2010). The BBB is formed of extremely tight junctions offering a wide range of functions, such as transendothelial transport systems, enzymes, and regulations of leukocyte permeation, which thereby generates the transport, enzymatic, and immune regulatory functions of the BBB (Abbott and Friedman, 2012). Studies have also revealed important stages, cell types, and signaling pathways involved in BBB development.

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00056-6

Under physiological conditions, the BBB regulates the exchange of nutrients, waste, and immune cells between the blood and the nervous tissue of the CNS and is the most important component preserving CNS homeostasis and neuronal function (Abbott et al., 2010). The BBB also protects the brain from xenobiotics, pharmaceuticals, nutraceuticals, pathogens, and various cells, proteins, and neurotransmitters present in the blood. Dysfunctional bloodeCNS barrier mechanisms contribute to the pathology of neurological conditions, ranging from trauma to neurodegenerative diseases (Erickson and Banks, 2013). The brain barriers, particularly BBB, are very sensitive to the toxic insult of chemicals and biotoxins, and these barriers play pivotal roles in the initiation and progression of neurodegenerative diseases. It is noteworthy that unlike toxicants that compromise the integrity of BBB, compounds such as melatonin can promote BBB integrity via multiple molecular pathways (Jumnongprakhon et al., 2016). This chapter describes in brief the structure and function of brain barriers and the biomarkers of toxic effects of metals, pesticides, mycotoxins, drugs of abuse, and some diseases involving the CNS.

STRUCTURE AND FUNCTION OF BRAIN BARRIERS The structure and function of brain barriers are very complex and maintain the neuronal microenvironment, playing pivotal roles in CNS homeostasis, fibrinolysis and coagulation, vasotonus regulation, and blood cell activation, and migration during physiological and pathological processes (Zheng et al., 2003). The BBB is also vital for protecting the CNS from systemic

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56. BIOMARKERS OF BLOOD-BRAIN BARRIER DYSFUNCTION

perturbations and from elements of the peripheral immune system, so that any impairment of its integrity could have far-reaching consequences on the health of the CNS (Drouin-Ouellet et al., 2015). The BBB is formed by a complex cellular system of endothelial cells, astroglia, pericytes, perivascular macrophages, and a basal lamina (Bradbury, 1985; Abbott et al., 2006; Gupta et al., 2015). Endothelial cells regulate the selective transport and metabolism of substances from blood to brain as well as in the opposite direction from the parenchyma back to the systemic circulation. Astrocytes project their end feet tightly to the cerebral endothelial cells (CEC), influencing and conserving the barrier function of these cells (Abbott et al., 2006). The prominent role of astrocytes in supporting a healthy BBB may be the result of trophic factors secreted by astrocytes that nourish and regulate brain microvascular endothelial cells (BMVEC) (Ivey et al., 2009). CEC are embedded in the basal lamina together with pericytes and perivascular macrophages (De Vries et al., 1997; Abbott et al., 2006). Pericytes are characterized as contractile cells that surround the brain capillaries with long processes, and they play a role in controlling the growth of endothelial cells. Pericytes may influence the integrity of the capillaries and conserve the barrier function. The lumen of the cerebral capillaries is covered by CEC in which the functional and morphological basis of the BBB resides. The BBB acts as a physical and metabolic barrier because a complex tight junction system between adjacent endothelial cells restricts most paracellular movement of ions and solutes across the brain endothelium (Partridge, 2002). Tight junctions of the BBB are composed of an intricate combination of at least three integral transmembrane proteins (claudins, junctional adhesion molecules (JAMs), and occludin), and cytoplasmic accessary proteins, such as zonula occludens (ZO-1, ZO-2, and AF6). Daneman et al. (2010) reported that a large number of transcripts are expressed and enriched at the BBB. These transcripts encode tight junction molecules, transporters, metabolic enzymes, signaling cascades, and proteins of unknown function. The best-characterized molecules at the BBB and tight junctions include claudin-5 and claudin-12, occludin, ZO-1 and ZO-2, marveld2, cingulin-like 1, jam4, and pard3. Also at the BBB, several signaling pathways have been identified and characterized, including serotonin receptor signaling, clathrin-mediated endocytosis signaling, dopamine receptor signaling, wnt/betacatenin signaling, and several others. In more than 99% of the brain capillaries, a BBB is present, but in some areas of the brain, a BCSFB can be found. This barrier is present in the circumventricular areas, such as the median eminence, pituitary, choroid plexus, subfornical organ, organum vasculosum of the lamina terminalis, and the area postrema. The BCSFB

is not as strict as the BBB, but it does prevent the entrance of blood-borne compounds into the brain. Because the surface of the BBB is 5000-fold greater than that of the BCSFB, the main route of entry for compounds from plasma into the brain is via the brain capillaries (Partridge, 1986). Major tight junction proteins include occludin and claudin. Claudin-5 is a major functional constituent and a critical determinant of BBB paracellular permeability and charge-selective hydrophilic paracellular pores, whereas occludin enhances tight junction tightness. Recently, it has been reported that endophilin-1 regulates the expression of ZO-1 and occludin via the EGFR-JNK signaling pathway (Chen et al., 2015). Several enzymes (monoamine oxidase A and B, catechol O-methyltransferase, epoxy hydrolase, endopeptidases, acetylcholinesterase (AChE), pseudocholinesterase, dopa decarboxylase, g-glutamyl transpeptidase, etc.) present in endothelial cells are important elements of the BBB, constituting the socalled metabolic barrier. The role of the BBB as a metabolic barrier was further substantiated by the presence of mitochondria in CECs (Fenstermacher, 1989). Thus, the BBB is considered a physical as well as a metabolic barrier. The brain barriers have been assigned many vital functions, and one of them is to provide required nutrients to the CNS. These essential nutrients are transported into the brain by means of selective/carrier mechanisms. Several transport systems have been characterized varying from passive transport (such as diffusion) to active and energy requiring processes (De Vries et al., 1997). The diffusion of compounds across the plasma membranes of the endothelial cells of the BBB is dependent on the physicochemical properties, such as lipophilicity, molecular weight, electrical charge, and extent of ionization. Lipid-soluble compounds penetrate the brain barriers readily and equilibrate easily between blood and brain tissue. Many transporters are expressed at the BBB (Garrick et al., 2003; Zlokovic, 2008) and BCSFB (Herbert et al., 1986; Ghersi-Egea and Strazielle, 2002). Compounds that are a substrate for P-glycoprotein (Pgp) are less efficiently transported across the BBB. Specific carrier systems have been characterized: glucose transporter system (GLUT-1) for sugars and large neutral amino acids system for amino acids, in addition to carrier system for purine, nucleoside, thiamine, monocarboxylic acid, and thyroid hormone. The other two carrier transporters in the human BBB are ATP-binding cassette (ABC) transporters and solute carrier transporters (Geier et al., 2013). Some transporters that are involved in drug distribution in the brain are also involved in drug efflux. The ABC efflux transporter Pgp has been demonstrated as

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STRUCTURE AND FUNCTION OF BRAIN BARRIERS

a key element of the BBB that can actively transport a huge variety of lipophilic drugs out of the brain capillary endothelial cells (Lo¨scher and Potschka, 2005). Drees et al. (2005) and Lo¨scher and Potschka (2005) reported that the multidrug resistance proteins BMDP/ABCG2 and BCRP are also involved in efflux pump at the BBB.

In Vivo and In Vitro Models to Study the BloodeBrain Barrier The movement of compounds from the circulating blood into the brain is strictly regulated by the brain capillary endothelial cells, which constitute the BBB. The importance of the BBB is not only in the passage of nutrients and toxicants but therapeutic drugs and antidotes as well. So, to understand the crossing of compounds of various classes, drugs, and antidotes, various in vivo and in vitro models have been developed (reviewed in Gupta et al., 2015). In Vivo Model Birngruber et al. (2013) reported a cerebral open flow microperfusion as a new membrane-free technique for measuring substance transport across the intact BBB in SpragueeDawley rats. This in vivo technique is based on a probe that is inserted into the brain, thereby rupturing the BBB. The BBB is usually reestablished within 15 days, which then allows sampling of interstitial brain fluid under physiological conditions. This technique also allows monitoring of BBB permeability, which can be useful for measuring pharmacokinetics across the BBB and pharmacodynamics in the brain. Using tracers, such as Evans blue (EB), horseradish peroxidase, and [131I]albumin, breakdown of the BBB in humans and experimental animals has been studied under many conditions, such as hypoglycemia, hypertension, seizures/convulsions, inflammation, etc. ¨ ztaș, 1996). (O In Vitro Models In vitro reconstituted models of the BBB from different mammalian species have been employed since the late 1970s. Bowman et al. (1983) introduced the first in vitro BBB filter model. An insert was made of nylon mesh and polycarbonate tubing, and bovine brain endothelial cells were seated on it for studying the effect of calcium-free medium and osmotic shock on sucrose flux. Since then, a variety of chambers and inserts from different materials and with diverse pore size have become commercially available. Garberg et al. (2005) used an in vitro model for BBB permeability based on the use of a continuous cell line and to investigate the

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specificity of this model. These authors developed a coculture procedure that mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. Under these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the BBB, including tight junctions and enzymes (such as g-glutamyl transpeptidase and monoamine oxidase (MAO)) activities. Deli et al. (2005) presented permeability data from various in vitro BBB models by measuring transendothelial electrical resistance (TEER) and by calculation of permeability coefficients for paracellular or transendothelial tracers. These authors summarized the results of primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin. They also described the effect of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as the physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the BBB function. The strong correlation between the in vivo (Oldenhorf method) and in vitro (coculture) drug transport, the relative ease with which such cocultures can be produced in large quantities, and the reproducibility of the system, provide evidence for an efficient system for the screening of drugs that are active in the CNS (Dehouck et al., 1997). These authors suggested that the coculture method is a useful system for investigating passive diffusion, carrier-mediated transport, and P-glycoprotein (Pgp)edependent drug transport. The in vitro permeabilities of propranolol and cyclosporine A were parallel with indications from in vivo extraction, showing that transporters and Pgp are expressed in the coculture system. For further details, readers are referred to Deli et al. (2005) who reviewed various in vitro models covering bovine, human, porcine, and rodent (murine and rat) brain endothelial cellebased systems. Bovine systems provide a high yield of brain endothelial cells sufficient for pharmacological screening, and they are widely used in basic as well as in applied research. Mouse brain yields the least endothelial cells compared to other species. Some examples of the modulators of BBB permeability in in vitro models are (1) both cAMP elevator peptide hormone adrenomedulin and calcitonin generelated peptide decrease paracellular permeability, (2) a glucocorticoid hormone, hydrocortisone, improves the barrier properties, (3) insulin exerts a tightening effect on tight junctions, and (4) catecholamines (adrenaline and noradrenaline) increase the sodium fluorescein flux. In essence, in vitro models have been widely used in pharmacological research for screening drugs and drug

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candidate molecules for either modifying BBB permeability or investigating brain penetration (Deli et al., 2005). This area of research is very important for permeability screening during drug development in the pharmaceutical industry (Gerlach et al., 2018).

Toxicants Affecting the Central Nervous System Barriers Metals A number of metals (aluminum, copper, iron, lead, manganese, and mercury) are known to modulate directly or indirectly the structure, function, or permeability of the BBB and/or BCSFB. Effects of these metals on brain barriers and the brain are described here in brief (Table 56.1). Aluminum Aluminum (Al)-induced neurotoxicity has been known for more than a century. Al has also been involved in neurodegenerative diseases, such as Alzheimer’s (AD), encephalopathy, and amyotrophic lateral sclerosis (ALS) (Delonche and Pages, 1997; Savory et al., 2006; Bondy, 2010; Garcia et al., 2010). In a pharmacokinetic study, Yokel (2012) mentioned that Al can enter the brain from blood by two routes: (1) through the BBB and (2) through the choroid plexus into the CSF of the ventricles within the brain and then into the brain. Al appears to bind with transferrin and citrate. Al concentration in serum can be 25-fold higher than in CSF. The glutamate transporter system Xc
has been suggested to mediate brain Al citrate uptake. Al mainly deposits in the hippocampus, cortex, and amygdala, which are the areas of brain that are also rich in glutamatergic neurons and transferrin receptors. Al is known to associate with many epithelia and endothelia in the BBB and may be responsible for compromising the properties and integrity of these membranes, thereby altering the barrier function (Vorbrodt et al., 1994). Al-related toxicological effects are observed on both sides of the BBB (Delonche and Pages, 1997). Al is known to cause development of neurofibrillary tangles and degeneration of cerebral neurons in laboratory animals (Zheng, 2001). Al directly affects neuronal pathways and appears to act as a direct BBB toxicant. Accumulation of Al in lysosomes (protease-rich vacuoles) and the hyperphosphorylation of neurofilaments are involved in the molecular mechanism of Al-induced neurotoxicity and neurodegenerative disease, such as AD. Copper and Iron Copper (Cu) is an essential element and involved in many vital biochemical reactions and physiological

functions, such as in Cu-containing enzymes, angiogenesis, nerve myelination, and endorphin action (Choi and Zheng, 2009; Pal, 2014). Cu access in the body, due to mutation in the Cu-transporter ATP7B gene located on the long arm (q) of chromosome 13 (13Q 14.3), may lead to the neurologic disorder known as Wilson’s disease. In contrast, Cu deficiency in liver and brain, due to a genetic disorder in the expression of the Cutransporter ATP7A, may lead to Menkes disease. An imbalance in Cu homeostasis due to defective Cu transporters or any other reasons at the BBB may play a role in the pathogenesis of neurodegenerative diseases, such as AD, Parkinson’s (PD), spongiform encephalopathies, and ALS. In serum, about 65%e90% of Cu tightly binds with ceruloplasmin, and the rest loosely binds with albumin, transcuprein, and amino acids. It appears that ceruloplasmin sequesters Cu and thus tightly regulates the movement of Cu into the CSF (Choi and Zheng, 2009). Free Cu ions are the main species for Cu transport into the brain, and uptake and distribution of Cu varies between different brain regions. The BBB appears to serve as the main entrance for Cu to get access to brain parenchyma, whereas the BCSFB is more likely involved in the regulation of Cu homeostasis in the CSF. Uptake of Cu into the cells is mediated by two transporter proteins (Cu transporter 1 (Ctr1) and divalent metal transporter 1 (DMT1)). Inside the cells, ATP7A and ATP7B are Cu transport proteins that participate in Cu efflux (Choi and Zheng, 2009; Pal, 2014). Monnot et al. (2011) reported that Cu appears to enter the brain primarily via the BBB and is subsequently removed from the CSF by the BCSFB. Iron (Fe) is a trace mineral that acts as both an electron acceptor and electron donor, and thereby it is essential to life (McCarthy and Kosman, 2015). Fe enters the brain by crossing the BBB and BCSFB. Fe is essential for normal brain function, as it is involved as a cofactor in myelination, mitochondrial energy generation, neurotransmission, oxygen transport, and cellular division. Fe is also involved in several neurological diseases, such as AD, PD, and aceruloplasminemia. There are two possible mechanisms for Fe uptake into BMVEC: one, which is referred to as transferrin-bound iron uptake involving transferrin (Tf) endocytosis, and the other is uptake of Fe from nontransferrin-bound iron. The latter process involves an Fe transporter, which is a divalent cation transporter such as DMT1. High intracellular Fe concentration is toxic to cells. The endothelial cells of the BBB sequester Fe from the blood at their apical surface and release Fe into the brain at their basolateral surface. Ferroportin is the only known mammalian cellular Fe exporter. Monnot et al. (2011) investigated how Fe levels regulated brain Cu levels transport through BBB and BCSFB

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TABLE 56.1

Toxicants That Can Cross the Brain Barriers May Cause Dysfunction/Damage to Barriers

Toxicants Metals

Pesticides

Biomarkers

References

Aluminum (Al)

Decreased serum inorganic phosphorus, neurofibrillary tangles development, cerebral neurons degeneration, neuropeptide delta sleepinducing peptide (DSIP), transferrin, disruption of BBB, and increased BBB permeability

Vorbrodt et al. (1994), Zheng (2001), and Yokel RA (2012)

Copper (Cu)

High Cu in Serum/plasma and urine, ceruloplasmin, transporters in BBB and BCSFB, and Cu chaperone for SOD protein

Zheng et al. (2003), Choi and Zheng (2009), and Pal (2014)

Iron (Fe)

Ferritin, transferrin, amyloid-b precursor protein, Zheng et al. (2003) and McCarthy and increased amyloid-b aggregates, ferroportin, Kosman (2015) ceruloplasmin, zyklopen, and hephaestin

Lead (Pb)

Increased BBB permeability, increased Ab1-40 in choroid plexus, protein kinase C (PKC-zeta), reduction of tight junction protein (occludin), and tyrosine kinase Src, and reduction of transthyretin in CSF

Laterra et al. (1992), Bradbury and Deane (1993), Zhao et al. (1998), Zheng et al. (2003), Behl et al. (2009), Song et al. (2014), and Gupta et al. (2015)

Manganese (Mn)

Blood and saliva Mn levels, and transferrin receptor in BBB and BCSFB

Aschner and Gannon (1994), Zheng (2001), Crossgove et al. (2003), Zheng et al. (2003), Milatovic et al. (2011), and Ge et al. (2018)

Mercury (Hg)

Hg concentration, and BBB disruption

Chang and Hartman (1972), Aschner and Clarkson (1988), and Zheng et al. (2003)

Zinc (Zn)

Increased BBB permeability, BBB breakdown, increased Zn and Na concentrations, Cu, Fe, and Mg deficiency in brain, inhibition of Na/K-ATPase

Yorulmaz et al. (2013) and Giacconi et al. (2017)

Organophosphate pesticides and nerve agents

OP residue, increased BBB permeability, AChE inhibition, BuChE inhibition, increased ACh level, seizures, BBB dysfunction, edema, and neuronal damage/loss

Ashani and Catravas (1981), Drewes and Singh (1985), Petrali et al. (1985), Carpentier et al. (1990), Sinha and Shukla (2003), Bhavari and Reddy (2005), Zaja-Milatovic et al. (2009), Mercey et al. (2012), and Martin-Reina et al. (2017)

Carbamates(including pyridostigmine bromide)

Carbamate residue, AChE inhibition, BuChE inhibition, increased ACh level, seizures, and increased BBB permeability, and neuronal damage/loss

Ropp et al. (2008), Gupta et al. (2007), Amourette et al. (2009), and MartinReina et al. (2017)

Chlorinated hydrocarbons

Increased BBB permeability

Sinha et al. (2003) and Malik et al. (2017)

Pyrethrins/pyrethroids

Increased BBB permeability, BBB dysfunction, and pyrethrin/pyrethroid residue

Sinha et al. (2004), Amaraneni et al. (2016), and Martin-Reina et al. (2017)

Neonicotinoids (Imidacloprid)

Imidacloprid residue

Rose (2012)

Nicotine

Nicotine residue

Rose (2012)

Rotenone

Mitochondrial complex I inhibition, autoradiographic findings, mitochondrial dysfunction, decreased ATP level, degeneration of dopaminergic neurons, and Parkinsonian motor deficits

Higgins and Greenamyre (1996), Gupta (2012), Heinz et al. (2017), and Terron et al. (2018)

Herbicides (paraquat)

Paraquat level, reduced tyrosine hydroxylase, and dopamine level

Shimizu et al. (2001) and Prasad et al. (2009)

PCBs/Dioxin/TCDD

Eum et al. (2008) Continued IX. SPECIAL TOPICS

1002 TABLE 56.1

56. BIOMARKERS OF BLOOD-BRAIN BARRIER DYSFUNCTION

Toxicants That Can Cross the Brain Barriers May Cause Dysfunction/Damage to Barriersdcont’d

Toxicants

Biomarkers

Mycotoxins/ Aflatoxins Biotoxins/Endotoxin

Reduced tight junction proteins (ZO-1, ZO-2, and AF6) AFB1 concentration in brain, AFB1-DNA (AFB1-N7-guanine) adducts, and HBMEC death

Experimental chemicals/drugs

References

Qureshi et al. (2015)

Deoxynivalenol (vomitoxin)

BBB and BCSFB impairment, reduced BBB integrity, increased BBB permeability, reduced TEER, increased MAPK, decreased claudin-3 and claudin-4, and increased lactate dehydrogenase release

Prelusky et al. (1990), Maresca (2013), and Behrens et al. (2015)

T-2 and HT-2 toxins

Increased BBB permeability, reduced protein synthesis, decreased MAO activity, and enhanced MMP-9

Wang et al. (1998), Ravindran et al. (2011), and Weidner et al. (2013)

Fumonisins/moniliformin

Increased BBB permeability, fumonisin B1 level, sphingosine: sphingosine ratio, cerebral edema, and increased albumin and IgG in CSF

Kwon et al. (1997), Foreman et al. (2004), Osuchowski et al. (2005), Behrens et al. (2015), and Smith (2018)

Ergot (ergotamine, ergocristinine, and others)

Ergot residue in CSF and reduced BBB integrity

Hovdal et al. (1982), Mulac et al. (2012), and Behrens et al. (2015)

Pertussis toxin

Increased BBB permeability

Bru¨ckener et al. (2003)

Endotoxin

Increased BBB permeability

Osuchowski et al. (2005) and De Vries et al. (1997)

Lipopolysaccharide (LPS)

Increased BBB permeability, increased nitric oxide and prostaglandins, and increased a-synuclein expression

Wispelwey et al. (1988), Minami et al. (1998), Osuchowski et al. (2005), and Jangula and Murphy (2013)

Monosodium glutamate

Increased BBB permeability

 ´ tyova´ et al. (1998) Skulte

N-Methyl D-aspartate (NMDA)

BBB breakdown

Zheng (2001)

Kainic acid (Kainate)

Kainic acid and dihydrokainic acid residue in brain, BBB dysfunction, increased BBB permeability, and brain edema

Gynther et al. (2015) and Han et al. (2015)

Pentylenetetrazole

BBB breakdown and increased BBB permeability Sahin et al. (2003) and Oztas et al. (2007)

Pilocarpine

Brain and plasma concentrations of pilocarpine and increased BBB permeability

Uva et al. (2008) and Ro¨mermann et al. (2015)

AFB1, aflatoxin B1; HBMVEC, human brain microvascular endothelial cells; MAO, monoamine oxidase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; TEER, transendothelial electrical resistance; ZO, zonula occludens.

and Cu homeostasis. Fe deficiency has a more profound effect on brain Cu levels than Fe overload. Fe deficiency increases Cu transport at the brain barriers and prompts Cu overload in the CNS. The BCSFB plays a key role in removing excess Cu from the CSF. The potential biomarkers of Fe exposure and effects on brain, such a ferritin, transferrin, amyloid-b precursor protein, increased amyloid-b aggregates, ferroportin, ceruloplasmin, zyklopen, hephaestin, are listed in Table 56.1. Lead Lead (Pb), a well-known neurotoxicant, can enter the CNS either as free Pb2þ or via the exchange of PbCO3 with an anion, or passively in the form of an inorganic complex, such as PbOHþ. Kinetic studies with 203 Pb continuously infused intravenously into adult

rats revealed that 203Pb uptake into different brain regions was linear with time up to 4 h after infusion (Bradbury and Deane, 1993). Pb accumulates in the choroid plexus of humans as well as animals, and the choroid plexus may be the primary target for Pb-induced neurotoxicity. This effect may alter the function of BCSFB. Behl et al. (2009) demonstrated that Pb significantly increased accumulation of intracellular amyloid-b (Ab1-40) in rat choroid plexus tissues in vivo and in immortalized choroidal epithelial Z310 cells in vitro. Mechanisms involved in leadinduced increase in Ab level at the BCSFB may include (1) a diminished expulsion of Ab molecules from the plexus cells to the extracellular milieu, (2) an increased uptake of Ab from the CSF, blood, or both, (3) an increased synthesis of Ab, and (4) a reduced

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metabolism or degradation of Ab. Pb-induced inhibition of the production of LRP1 (a key intracellular Ab transport protein in the choroid plexus) may be responsible for the accumulation of Ab, a major risk factor for AD (reviewed in Gupta et al., 2015). Pb exposure has also been shown to cause abnormal protein kinase C (PKC-zeta) activity in both the BBB (Laterra et al., 1992) and the BCSFB (Zhao et al., 1998). In addition, Pb appears to accumulate in the same intramitochondrial compartment as Ca2þ, thereby disrupting intracellular Ca2þ metabolism as well as altering transepithelial transport processes (Zheng et al., 2003). Manganese Manganese (Mn) binds readily to transferrin without displacing iron (Fe) in plasma. Brain areas (pallidum, thalamic nuclei, and substantia nigra) having high Mn levels differ from those having high levels of transferrin receptors (nucleus accumbens and caudate putamen), suggesting that perhaps these sites may accumulate Mn through neuronal transport. Like Fe, Mn-loaded transferrin is taken up by receptor-mediated endocytosis at the luminal membrane of brain capillaries. Mn levels, transferrin in BBB and BCSFB, and neurodegeneration and dendritic damage may serve as biomarkers (Aschner and Gannon, 1994; Crossgove et al., 2003; Zheng et al., 2003; Milatovic et al., 2011). Mercury Mercury (Hg) exists in several forms, such as elemental (metallic), inorganic (e.g., mercurous chloride and mercuric chloride), and organic (e.g., methylmercury, ethylmercury, and phenylmercury). In the environment and mammalian systems, various forms of mercury are interchangeable. For example, inorganic mercury can be methylated to methylmercury (MeHg) and MeHg can change to inorganic or elemental Hg. Animals at the top of the food chain tend to bioaccumulated MeHg in their bodies. Therefore, poisoning by Hg is due to consumption of meat or grain contaminated with Hg. The US Food and Drug Administration estimates that, on an average, most people are exposed to about 50 ng Hg/ kg body wt/day in the food they eat. Metallic Hg can stay in the body for weeks to months. Because of its high lipophilicity, it can readily cross the BBB. When metallic Hg enters the brain, it is readily converted to an inorganic divalent Hg and is trapped there for an extended period. The inorganic divalent cation can, in turn, be reduced to metallic Hg. Inorganic Hg compounds do not readily cross the BBB. Yet, compounds such as mercuric chloride can act as direct BBB toxicants (Chang and Hartman, 1972; Zheng et al., 2003). The distribution of MeHg is similar to that of metallic Hg, i.e., a relatively large amount of Hg can accumulate in the brain because of its ability to penetrate

1003

the BBB either by diffusion or by utilizing a transport system. MeHg reacts with sulfhydryl groups of cysteine. The MeHgecysteine complex acts as an amino acid analog, similar in structure to methionine, and is transported by the L system carrier across the BBB (Aschner and Clarkson, 1988). Developing fetuses and neonates are most sensitive to MeHg-induced neurotoxicity because MeHg is more readily transported across the immature BBB as well as because of its inhibitory effects on cell division. Microtubules are essential for cell division (main component of the mitotic spindle), and MeHg reacts with the SH groups of tubulin monomers and thereby disrupts the assembly process. The dissociation process continues, and this leads to depolymerization of the tubule. It is noteworthy that Hg can be involved in the development of AD, multiple sclerosis (MS), and ALS (Mutter et al., 2007). Zinc Zn is a component of more than 300 different enzymes that function in many aspects of cellular metabolism, including metabolism of proteins, lipids, and carbohydrates. The BBB is important for zinc (Zn) homeostasis in the brain (Yorulmaz et al., 2013). Zn serves as a mediator of cellecell signaling and functioning of channels and receptors in the CNS. The transport of Zn into the brain parenchyma occurs via the brain barrier system. Its deficiency or excess can contribute to alterations in behavior, abnormal CNS development, and neurological diseases. In a recent study, circulating Zn has been linked to pathophysiological changes occurring with aging rather than to its nutritional intake (Giacconi et al., 2017). Increased Zn and Na concentrations, inhibition of Na/K-ATPase, increased BBB permeability, and BBB breakdown may serve as biomarkers.

Pesticides Organophosphates Several organophosphate (OP) compounds have been reported to cross the BBB, but very little is known about alterations in the BBB structure or function, except that these compounds can increase its permeability. OP nerve agents, which are irreversible AChE inhibitors, have been studied to a greater extent than pesticides with regard to changes in the BBB. Drewes and Singh (1985) reported that the cerebral transendothelial carrier-mediated transport of glucose and amino acids was not affected in mongrel dogs poisoned by soman. Carpentier et al. (1990) investigated acute changes in BBB permeability to proteins, using EB-labeled serum albumin and plasmatic gamma-immunoglobulin G (IgG) as indicators in rats. An increased BBB permeability to the EBealbumin

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56. BIOMARKERS OF BLOOD-BRAIN BARRIER DYSFUNCTION

complex was macroscopically observed in two-thirds of the convulsive rats exposed to soman (85 mg/kg). Soman produced seizures and reversible BBB opening to the greatest extent after 30e60 min of paroxysmal electroencephalographic (EEG) discharges when signs of cerebral hyperactivity (epileptic EEG pattern, and hyperoxia) were also at their peak. Topographically, the protein leakage was bilateral and restricted to anatomically defined brain structures, some of which were thereafter sites of parenchymal edema and neuronal damage. Interestingly, the first signs of increased BBB permeability were shown to precede the onset of edema. OP nerve agents, being small lipophilic molecules, can easily penetrate the BBB by free diffusion and thereby inhibit AChE in the CNS (Mercey et al., 2012). Increased BBB permeability by OP nerve agents or other ChE inhibitors may lead to their enhanced entry into the brain resulting in greater AChE inhibition and possibly in subsequent maintenance of seizures and aggravation of their pathological consequences, such as edema and neuronal loss in certain brain structures. Carpentier et al. (1990) detected the BBB opening in the amygdaloid complex and in some cortical regions (cingulum, entorhinal, and piriform complex), and that the thalamus was the most frequently and intensely affected structure. Observations from various studies suggest that soman-induced brain alterations are predominantly related to seizures or brain hyperactivity, or to a direct cytotoxic action of soman or acetylcholine (ACh) itself or due to ChE inhibition (McDonough et al., 1987). Domer at al. (1983) found increased permeability of the BBB by systemic administration of ACh. Of course brain hyperactivity alone appears inadequate to be responsible for the BBB opening. Obviously, the short duration of the transient protein leakage (Carpentier et al., 1990) contrasted with the well-known longlasting brain AChE inhibition induced by soman (Petrali et al., 1985). Several other anti-ChE compounds, such as physostigmine and paraoxon, are also known to cause the BBB opening for macromolecules that were seizure-dependent and reversible and unrelated to brain ChE inhibition. Ashani and Catravas (1981) observed that in soman-intoxicated rats, induced damage to BBB integrity was significantly reduced, despite a high degree of AChE and BChE inhibition, and protected from seizures by Nembutal or atropine. In essence, endothelial AChE or BChE play no role in BBB opening, although they may function as an “enzymatic barrier” to ACh. Additional factors, such as increased electrical activity, oxidative/nitrosative stress, decreased energy supply and store, deleterious action of excitatory amino acids, enhanced calcium intrusion, and brain edema,

seemed to play significant roles in the brain damage (Misulis et al., 1987; Carpentier et al., 1990; Solberg and Belkin, 1997; Gupta et al., 2001a,b; Zaja-Milatovic et al., 2009; Prager et al., 2013). Other mechanisms in soman-induced damage to BBB integrity may be related to vasoactive substances (ACh, amines, amino acids, peptides, free radicals, and steroid hormones of the pituitary adrenal axis) and vasogenic events (acidosis, increased blood flow, and hypertension). It is noteworthy that increased BBB permeability may facilitate the entry of an antidote (oxime class) to the brain, which otherwise has limited access because of the BBB (Gupta et al., 2015). Organochlorines and Pyrethrins/Pyrethroids In a number of studies, increased BBB permeability has been reported for organochlorine (Sinha and Shukla, 2003; Malik et al., 2017) and pyrethrin/pyrethroid (Sinha et al., 2004; Amaraneni et al., 2016; Martin-Reina et al., 2017) insecticides. Rotenone Rotenone is one of the naturally occurring insecticides obtained from plants of the “Derris” species (Gupta, 2012). Rotenone causes inhibition of mitochondrial respiratory chain complex I, (NADH-ubiquinone oxidoreductase) and cell death by apoptosis due to excess generation of free radicals. Rotenone has received enormous attention from neuroscientists because it causes inhibition of mitochondrial respiratory chain complex I, followed by mitochondrial dysfunction, impaired proteostasis, degeneration of dopaminergic neurons, neuroinflammation, and finally Parkinsonian motor deficits (Higgins and Greenamyre, 1996; Heinz et al., 2017; Terron et al., 2018). Mitochondrial complex I inhibition, mitochondrial dysfunction, reduced ATP level, degeneration of dopaminergic neurons, and Parkinsonian motor deficits may serve as biomarkers of rotenone’s neurotoxicity.

Herbicides A bipyridyl herbicide paraquat resembles the structure of N-methyl-4-phenyl pyridinium (MPPþ). MPPþ is an experimental agent, which causes PD. Paraquat reaches the brain by crossing the BBB possibly by the neutral amino acid transport system (Shimizu et al., 2001; Prasad et al., 2009). Further, paraquat is taken up into striatal neurons by a Naþ-dependent mechanism. Paraquat inhibits the activity of tyrosine hydroxylase and decreases striatal dopamine levels, thereby inducing Parkinson-like dopaminergic toxicity in the brain.

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STRUCTURE AND FUNCTION OF BRAIN BARRIERS

Mycotoxins In general, the effect of mycotoxins on the BBB is to increase its permeability. Aflatoxin B1 (AFB1) has been reported to form AFB1-DNA (AFB1-N7-guanine) adducts and kill human BMVECs (Qureshi et al., 2015). One of the trichothecenes deoxynivalenol is reported to cause BBB and BCSFB impairment, reduced BBB integrity, increased BBB permeability, reduced TEER, increased MAPK, decreased claudin-3 and claudin-4 (proteins involved in tight junctions), and increased lactate dehydrogenase release (Prelusky et al., 1990; Maresca, 2013; Behrens et al., 2015). T-2 and HT-2 toxins are known to cause increased BBB permeability, reduced protein synthesis, enhanced matrix metalloproteinase-9 (MMP-9), and alterations in neurochemicals by inhibiting MAO activity (Wang et al., 1998; Ravindran et al., 2011; Weidner et al., 2013). Fumonisin B1 commonly affects the equine species causing equine leukoencephalomalacia, cerebral edema, and an elevation of the albumin and IgG levels in CSF (Foreman et al., 2004; Smith, 2018). Ergot alkaloids (such as ergotamine, ergocristinine, and others) are known to cross the BBB by BCRP/ABCG2 transporter system, accumulate in BBB cells, and impair its integrity (Hovdal et al., 1982; Mulac et al., 2012; Behrens et al., 2015).

Drugs of Abuse and Therapeutic Drugs Methamphetamine The BBB has been identified as a target of methamphetamine neurotoxicity (Northrop and Yamamoto, 2015). Methamphetamine causes damage to monoamine

TABLE 56.2

terminals and BBB structural proteins, the tight junction proteins, and BBB function. In experimental animals, methamphetamine has been shown to cause decreased expression of occludin, claudin-5, and zona occludens, as well as decreased TEER, indicative of increased paracellular permeability (Mahajan et al., 2008). These alterations in endothelial structures and function are associated with an increase in ROS. In addition, stress appears to exacerbate methamphetamine-induced increases in ROS, induced BBB disruption by potentiating methamphetamine-induced increases in ROS, inflammatory mediators, mitochondrial dysfunction, and extracellular glutamate. The consequences of methamphetamine-induced BBB disruption are significantly enhanced vulnerability to other disease states, such as stress, HIV infection, Hepatitis C, cognitive decline and depression, etc. (Northrop and Yamamoto, 2015).

Cocaine Depending on the dose and route of administration, cocaine-induced profound hyperthermia and increased plasma and brain serotonin levels lead to BBB breakdown and brain edema (Sharma et al., 2009; Multani et al., 2014). Cocaine also induces cellular stress as seen by upregulation of heat shock protein (HSP 72 kD) expression and results in marked neuronal and glial cell damages at the time of the BBB dysfunction. Increased serotonin levels, hyperthermia, HSP 72 kD expression, and BBB dysfunction can be used as biomarkers of cocaine-induced damage to the BBB (Table 56.2).

Drugs of Abuse That Can Cross the Barriers and May Cause Dysfunction/Damage to the Barriers

Drugs of Abuse

Biomarkers

References

Amphetamine and methamphetamine

Disruption of BBB function, increased BBB permeability, decreased BBB structural proteins (occludin, claudin-5, and zona occludens-1), increased matrix metalloproteinase-9 and nitric oxide, decreased TEER, and increased ammonia and glutamate levels

Mahajan et al. (2008), Silva et al. (2010), Martins et al. (2013, 2017), Multani et al. (2014), Northrop and Yamamoto (2015), and Jumnongprakhon et al. (2016)

MPTP/MPP

Mitochondrial complex I inhibition, mitochondrial dysfunction, reduced ATP level, nigrostriatal degeneration, and Parkinsonian motor deficits

Schildknecht et al. (2017) and Terron et al. (2018)

Cocaine

Increased plasma/serum and brain serotonin levels, increased HSP 72 kD, hyperthermia, increased BBB permeability, BBB dysfunction, and brain edema

Sharma et al. (2009) and Multani et al. (2014)

Alcohol/Ethanol

Myosin light chain kinase activation, decreased occludin, claudin-5, and zona occludens-1, and increased BBR permeability

Haorah et al. (2005), Multani et al. (2014), and Northrop and Yamamoto (2015)

Antipsychotics (chlorpromazine and clozapine)

Increased electrical resistance, apoptosis of BBB endothelia, and impaired BBB permeability

Elmorsy et al. (2014)

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56. BIOMARKERS OF BLOOD-BRAIN BARRIER DYSFUNCTION

Alcohol/Ethanol Ethanol produces activation of myosin light chain kinase and consequent phosphorylation and degradation of tight junction proteins, such as occludin, claudin-5, and ZO-1 (Northrop and Yamamoto, 2015). A decrease in BBB function evidenced by a decrease in TEER and an increase in monocyte migration are also observed in brain microvascular endothelial cells. It has been suggested that the effects of ethanol on tight junction proteins and barrier function are associated with increases in ROS, resulting from ethanol metabolism and increased calcium concentrations. Ethanol in combination with methamphetamine could potentiate methamphetamine-induced BBB disruption through its proinflammatory and prooxidant effects. Biomarkers of ethanol-induced effects on BBB are summarized in Table 56.2. For details on biomarkers of drugs of abuse, readers are referred to Chapter 52 of this book.

Neurodegenerative Diseases and Other Conditions Neurodegenerative Diseases In a number of CNS diseases, including neurodegenerative diseases (AD, PD, MS, ALS, and Huntington), the structure and function of BBB and/or BCSFB are compromised. The BBB itself may play an active role in the mediation of the neuroimmune response either by production of inflammatory mediators or by the expression of adhesion molecules. In neurological diseases, various inflammatory mediators, such as cytokines (TNF, IL-1b, and IL-6), prostaglandins (PGD2, PGE2, PGF1a, and PGF2a), thromboxane A2, and leukotrienes, can be used as biomarkers of inflammation. The common adhesion molecules include (1) intercellular adhesion molecule-1, (2) intercellular adhesion molecule-2, and (3) vascular cell adhesion molecule-1; alterations in these parameters can be used as biomarkers. Brain barriers possibly contribute to the etiology of AD by three aspects: (1) the aging of cerebral vascular structure in the overall aging process of the brain, (2) as the site of transport of extracerebral Ab into the brain, and (3) the ability to prevent aggregation by producing transthyretin (Zheng, 2001). The neuropathological hallmark of AD is the aggregation of Ab peptide in senile plaques and neurofibrillary tangles and is often used as a biomarker of AD. Detailed biomarkers of AD are summarized in Table 56.3 and described in Chapter 50 of this book. The quantification of a-synuclein in CSF has been proposed as a diagnostic biomarker for PD and other a-synucleinerelated diseases, such as multiple system

atrophy and dementia with Lewy bodies (Mollenhauer, 2014). Biomarkers of PD are described in detail in Chapter-51 of this book. BBB disruption is one of the hallmarks of MS. Development of sensitive and specific biomarkers for MS has been a challenge as they could (1) improve the monitoring of disease activity, (2) improve the monitoring of response to MS therapies that target BBB disruption, and (3) advance our understanding of dynamic MS processes participating in BBB disruption (Waubant, 2006). The presence of gadolinium-enhancing lesions on serial brain MRI scans is frequently used to evaluate BBB disruption. In MS, cerebral infections, hypertension, or seizures, enhanced permeability of BBB and is considered to be the result of either opening of tight junctions or of enhanced pinocytotic activity and the formation of transendothelial channels (De Vries et al., 1997). Several studies for ALS biomarkers have been conducted, while others are ongoing (Su et al., 2013). It has been demonstrated that protein biomarkers in both plasma and CSF may aid the diagnosis and prognosis of patients with ALS (Mitchell et al., 2009, 2010). In CSF, various classes of biomarkers have been investigated with special attention to neurofilament protein, tau protein, S100-b, and cystatin C. Cytoskeletal protein tau can be measured in CSF and blood and used as a prognostic biomarker of ALS. Su et al. (2013) measured plasma and CSF levels of 35 biomarkers using multiplex and immunoassay analysis for prognosis of ALS. In a recent study, Gendron et al. (2017) identified the use of phosphorylated neurofilament heavy chain as a prognostic biomarker for clinical trials that may likely be useful in developing a successful treatment for C9ALS. Currently, none of the biomarkers is specific to BBB damage or dysfunction induced by ALS. Epilepsy Biomarkers in epilepsy play multiple roles because they may (1) predict the development of an epilepsy condition, (2) identify the presence and severity of tissue capable of generating spontaneous seizures, (3) measure progression after the condition is established, (4) be used to create animal models for more cost-effective screening of potential antiepileptic and antiseizure drugs and devices, and (5) reduce the cost of clinical trials of potential antiepileptogenic interventions (Engel et al., 2013). The mechanisms involved in the development of epilepsies and the generation of spontaneous recurrent seizures are multifactorial. Biomarkers linked to a precipitating factor could be useful for seizure prediction and the possible development of interventions. In epilepsy, biomarkers can vary from imaging (MRI, PET, or SPECT) and electrophysiological measurements to changes in gene expression and metabolites in blood or tissues (Engel et al., 2013; Obenaus, 2013). Alterations

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TABLE 56.3

Biomarkers of Brain Barriers in Neurodegenerative Diseases and Other Neurological Conditions

Disease

Biomarkers

References

Alzheimer’s disease

b-amyloid (Ab peptide) in senile plaques and neurofibrillary tangles, increased BBB permeability, aberrant protein kinase C (PKC-zeta), dysfunctional BCSFB, decreased transthyretin, haptoglobulin in CSF, IL-1, ICAM-1, increased albumin in CNS, CSF/Serum ratio of albumin, and altered occludin and claudins

Moore et al. (1998), De Vries et al. (1997), Zisper et al. (2007), Abdel-Rehman et al. (2004), Agyare et al. (2013), Erickson and Banks (2013), and Srinivasan et al. (2016)

Parkinson’s disease

Intracellular a-synuclein deposition, CSF a-synuclein and b-amyloid, and a-synuclein/total protein ratio

Shaltiel-Karyo et al. (2013), Mollenhauer (2014), Stav et al. (2015), and Wang et al. (2015)

Multiple sclerosis (MS)

BBB disruption, increased CSF osteopontin, pathological legions, gliosis, gadolinium enhancement on MRI scans, and changes in MMPs

Kermode et al. (1990), De Vries et al. (1997), Minagar and Alexander (2003), Palmer (2010), Waubant (2006), and Szalardy et al. (2013)

Amyotrophic lateral sclerosis (ALS)

Increased phosphorylated neurofilament heavy chain in CSF, tau protein in CSF and blood, cytokines (IL-1b, IL1RA, IL-5, IL-9, IL-8, IL-10, IL-12, IL-15), RANTES, high granzyme B, eotaxin, growth factors, plasma L-ferritin, plasma-CSF interferon-g ratio, high Nogo-A, low cystatin C, genetic biomarkers, and many others

Mitchell et al. (2009, 2010), Su et al. (2013), Bakkar et al. (2015), and Gendron et al. (2017)

Huntington’s disease

Mutant Huntingtin protein (mHtt) aggregates, increased transcytosis, increased blood vessel density, reduced blood vessel diameter, reduced occludin-5 and claudin, BBB disruption, and increased permeability

Drouin-Ouellet et al. (2015)

Schizophrenia

Impaired choroid plexus and BCSFB

Rudin (1979)

Allergic encephalomyelitis

BBB disruption, decrease in number of mitochondria, and increased permeability

Claudio et al. (1989) and Hawkins et al. (1991)

HIV-1 and AIDS dementia

Detection of HIV-1 in bloodeCNS barriers, increased cytokines (TNF, IL-1b, and IL-6) in CSF, HIV-1 gp-120 proteins, fibrinogen, IgG, increased BBB permeability, and BBB breakdown

Harouse et al. (1989), Petito and Cash (1992), De Vries et al. (1997), Kanmogne et al. (2005), Banks et al. (2006), Ivey et al. (2009), and Northrop and Yamamoto (2015)

Gulf war illness

Increased BBB permeability

Gupta et al. (2015)

Traumatic brain injury (TBI)

Brain edema, BBB disruption, BBB breakdown, loss of tight junction proteins (occludin, claudin-5, ZO-1), reduced expression of PDGFR-b, increased BBB permeability, decreased TEER, increased glutamate, aII-spectrin, GFAP, and serum microRNA let-7i

Ling et al. (2009), Shlosberg et al. (2010), Chodobski et al. (2011), Balakathiresan et al. (2012), Hue et al. (2013), Tate et al. (2013), Tomkins et al. (2013), Shetty et al. (2014), and Dobbins and Pan (2015)

Chronic traumatic encephalopathy (CTE)

Tau protein aggregation, tauopathy, and myelinated axonopathy

Goldstein et al. (2014)

Convulsions, seizures, and epilepsy

Increased BBB permeability, BBB dysfunction, electrophysiologic and imaging biomarkers, TGF-b, and GLUT1 deficiency

Ashani and Catravas (1981), Oztas and Kaya (2003), Oby and Janigro (2006), Persidsky et al. (2006), Remy and Beck (2006), Cacheaux et al. (2009), Staba and Bragin (2011), Engel et al. (2013), Obenaus (2013), and Salar et al. (2014)

Reversible cerebral vasoconstriction syndrome (RCVS) (Thunderclap headache)

BBB breakdown, arterial vasoconstriction, and hyper intense vessels

Chen and Wang (2014) and Lee et al. (2017)

Brain edema

Increased BBB permeability

Jangula and Murphy (2013)

Stress (from heat, combat, chemicals, etc.)

Increased serotonin, and increased BBB permeability

 ´ tyova´ et al. Sharma and Dey (1986), Skulte (1998), and Amourette et al. (2009)

Hypoxia

Claudin-5

Koto et al. (2007)

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in RNA, protein, and metabolites are biomarkers of loss of neurons, gliosis, inflammation, changes in BBB, angiogenesis, neurogenesis, etc. A growing body of evidence has shown that inflammatory mechanisms may participate in the pathological changes observed in epileptic brain, with increasing awareness that blood-borne cells or signals may participate in epileptogenesis by virtue of a leaky BBB (Oby and Janigro, 2006). In epilepsy, pharmacoresistance to antiepileptic drugs is a common therapeutic challenge, as it occurs in about 30%e70% patients (Salar et al., 2014). These authors and others demonstrated that a dysfunctional BBB with acute extravasation of serum albumin into the brain’s interstitial space could contribute to pharmacoresistance (Remy and Beck, 2006). BBB dysfunction per se could serve as a biomarker to direct clinical decisions for alternative drugs with high efficiency and lower affinity or no affinity to albumin. Monitoring BBB integrity may therefore also be important in the management of epilepsy. In addition, modulation of drug transporters such as Pgp, MRP2, and BCRP at the BBB could help in easy entry of antiepileptic drugs (Lo¨scher and Potschka, 2005). HIV Infection The BBB can be affected by HIV infection and is also involved in the progression of disease (Banks et al., 2006; Northrop and Yamamoto, 2015). HIV can increase the BBB permeability and infiltration of virus and immune cells. HIV proteins (Tat and gp-120) act on BMECs and result in increased neuroinflammation. HIV proteins cause BBB disruption via oxidative stress and inflammatory mechanisms, decreases in tight junction proteins (claudin-1, claudin-5, and ZO-2) in BMVECs, and cytotoxicity of BMVECs. These alterations are associated with increased expression of MMP-2 and MMP-9. Biomarkers of HIV-induced alterations in BBB are summarized in Table 56.3.

CONCLUDING REMARKS AND FUTURE DIRECTIONS The CNS barriers, such as BBB and BCSFB, play significant roles in protecting the brain from endogenous and exogenous substances thereby maintaining brain homeostasis. These barriers, of course, allow required nutrients to reach the CNS. The structure of the BBB is very complex as it contains many molecules, enzymes, receptors, transporters, and proteins of known and unknown functions. The structure and functions of these barriers are known to be altered by a variety of toxicants (metals, pesticides, and mycotoxins), drugs of abuse and therapeutic drugs in higher doses, neurodegenerative diseases, and conditions of the CNS. These biomarkers

may aid in diagnosis, prognosis, and disease interventions. The future of biomarkers of CNS barriers seems bright, and with newer technologies more specific and sensitive biomarkers will be developed that could detect chemical toxicosis and CNS disease very early and allow timely interventions.

Acknowledgments The authors would like to thank Ms. Robin B. Doss for her technical assistance in preparation of this chapter.

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permeability in developing brain. Int. J. Dev. Neurosci. 22 (1), 31e37.  ´ tyova´, I., Tokarev, D., Jezova´, D., 1998. Stress-induced increase in Skulte blood-brain barrier permeability in control and monosodium glutamate-treated rats. Brain Res. Bull. 45, 175e178. Smith, G., 2018. Fumonisins. In: Gupta, R.C. (Ed.), Veterinary Toxicology: Basic and Clinical Principles. Academic Press/Elsevier, Amsterdam, pp. 1003e1018. Solberg, Y., Belkin, M., 1997. The role of excitotoxicity in organophosphorus nerve agents central poisoning. Trends Pharmacol. Sci. 18, 183e185. Song, H., Zheng, G., Shen, X.-F., et al., 2014. Reduction of brain barrier tight junctional proteins by lead exposure: role of activation of nonreceptor tyrosine kinase Src via chaperone GRP78. Toxicol. Sci. 138 (2), 393e402. Srinivasan, V., Braidy, N., Chan, E.K.W., et al., 2016. Genetic and environmental factors in vascular dementia: an update of blood brain barrier dysfunction. Clin. Exp. Pharmacol. Physiol. 43, 515e521. Staba, R.J., Bragin, A., 2011. High-frequency oscillations and other electrophysiological biomarkers of epilepsy: underlying mechanisms. Biomarkers Med. 5, 545e556. Stav, A.L., Aarsland, D., Johansen, K.K., et al., 2015. Amyloid-b and a-synuclein cerebrospinal fluid biomarkers and cognition in early Parkinson’s disease. Park. Relat. Disord. 21 (7), 758e764. Su, X.W., Simmons, Z., Mitchell, R.M., et al., 2013. Biomarker-based predictive models for prognosis in Amyotrophic lateral sclerosis. JAMA Neurol. 70 (12), 1505e1511. Szalardy, L., Zadori, D., Simu, M., et al., 2013. Evaluating biomarkers of neuronal degeneration and neuroinflammation in CSF of patients with multiple sclerosis-osteopontin as a potential marker of clinical severity. J. Neurol. Sci. 331, 38e42. Tate, C.M., Wang, K.K.W., Eonta, S., et al., 2013. Serum brain biomarker level, neurocognitive performance, and self-reported symptoms changes in soldiers repeatedly exposed to low-level blast: a breacher pilot study. J. Neurotrauma 30, 1e11. Terron, A., Bal-Price, A., Paini, A., et al., 2018. An adverse outcome pathway for parkinsonian motor deficits associated with mitochondrial complex I inhibition. Arch. Toxicol. 92, 41e82. Tomkins, O., Feintuch, A., Benifla, M., et al., 2013. Blood-brain barrier breakdown following traumatic brain injury: a possible role in pasttraumatic epilepsy. Cardiovasc. Psychiatr. Neurol. 2011, 765923. Uva, L., Librizzi, L., Marchi, N., et al., 2008. Acute induction of epileptiform discharges by pilocarpine in the in vitro isolated Guinea-pig

brain requires enhancement of blood-brain barrier permeability. Neuroscience 151 (1), 303e312. Vorbrodt, A.W., Dobrogowska, D.H., Lossinsky, A.S., 1994. Ultracytochemical studies of the effects of aluminum on the blood-brain barrier of mice. J. Histochem. Cytochem. 42, 203e212. Wang, J., Fitzpatrick, D.W., Wilson, J.R., 1998. Effect of T-2 toxin on blood-brain barrier permeability monoamine oxidase activity and protein synthesis in rats. Food Chem. Toxicol. 36 (11), 955e961. Wang, X., Yu, S., Li, F., et al., 2015. Detection of a-synuclein oligomers in red blood cells as a potential biomarker of Parkinson’s disease. Neurosci. Lett. 599, 115e119. Waubant, E., 2006. Biomarkers indicative of blood-brain barrier disruption in multiple sclerosis. Dis. Markers 22 (4), 235e244. Weidner, M., Hu¨wel, S., Ebert, F., et al., 2013. Influence of T-2 and HT-2 toxin on the blood-brain barrier in vitro: new experimental hints for neurotoxic effects. PLoS One 8 (3), 1e10. Wispelwey, B., Lesse, A.J., Hansen, E.J., et al., 1988. Haemophilus influenzae lipopolysaccharide-induced blood-brain barrier permeability during experimental meningitis in the rat. J. Clin. Invest. 82, 1339e1346. Yokel, R.A., 2012. The pharmacokinetics and toxicology of aluminum in the brain. Curr. Inorg. Chem. 2, 54e63. Yorulmaz, H., Seker, F.B., Demir, G., et al., 2013. The effect of zinc treatment on the blood-brain barrier permeability and brain element levels during convulsions. Biol. Trace Elem. Res. 151, 256e262. Zaja-Milatovic, S., Gupta, R.C., Aschner, M., Milatovic, D., 2009. Protection of DFP-induced oxidative damage and neurodegeneration by antioxidants and NMDA receptor antagonists. Toxicol. Appl. Pharmacol. 240, 124e131. Zhao, Q., Slavkovich, V., Zheng, W., 1998. Lead exposure promotes translocation of protein kinase C activity in rat choroid plexus in vitro, not in vivo. Toxicol. Appl. Pharmacol. 149, 99e106. Zheng, W., 2001. Neurotoxicology of the brain barrier system: new implications. Clin. Toxicol. 39 (7), 711e719. Zheng, W., Aschner, M., Ghersi-Egea, J.-F., 2003. Brain barrier systems: a new frontier in metal neurotoxicological research. Toxicol. Appl. Pharmacol. 192, 1e11. Zisper, B.D., Johanson, C.E., Gonzalez, L., et al., 2007. Microvascular injury and blood-brain barrier leakage in Alzheimer’s disease. Neurobiol. Aging 28, 977e986. Zlocovic, B.V., 2008. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178e201.

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57 Biomarkers of Oxidative/Nitrosative Stress and Neurotoxicity Dejan Milatovic1, Snjezana Zaja-Milatovic2, Ramesh C. Gupta3 1

Charlottesville, VA, United States; 2PAREXEL International, Alexandria, VA, United States; 3Toxicology Department, Breathitt Veterinary Center, Murray State University, Hopkinsville, Kentucky, United States

INTRODUCTION Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are molecules or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals, which characterize free radicals with high reactivity. Exogenous agents (such as pesticides, metals, xenobiotics, and ionizing radiation) and a variety of endogenous processes (cellular respiration, antibacterial defense, phagocytic oxidative bursts, and others) can generate significant amounts of ROS and RNS in the human body (Chakravarti and Chakravarti, 2007; Mangialasche et al., 2009; Il’yasova et al., 2012). Both species of free radicals are also products of normal cellular metabolism. Mitochondrial oxidative phosphorylation generates the majority of free radicals in the cell (Federico et al., 2012; Mora´n et al., 2012; Quinlan et al., 2012; Trewin et al., 2018; Sas et al., 2018). ROS are produced mainly as superoxide, when electrons leak onto molecular oxygen in side reactions from prosthetic groups or coenzymes involved in these redox reactions. Superoxide further serves as a precursor to the formation of the hydroxyl radical (HO
), the most reactive radical. The hydroxyl radical is produced by the Fenton and Habere Weiss reactions from hydrogen peroxide (H2O2) and metal species (iron, copper) (Dix and Aikens, 1993; Gue´raud et al., 2010). Superoxide easily reacts with nitric oxide (NO•) and forms peroxynitrite (ONOO), an RNS with very high reactivity (Vatassery, 2004). Peroxynitrite is a powerful oxidant exhibiting a wide array of tissuedamaging effects ranging from lipid peroxidation and inactivation of enzymes and ion channels via protein oxidation and nitration to inhibition of mitochondrial respiration (Virag et al., 2003). Peroxynitrite, which

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00057-8

dissipates during oxidation (Wang et al., 2003), has also been found to nitrate and oxidize adenine, guanine, and xanthine nucleosides (Sodum and Fiala, 2001). Low concentrations of peroxynitrite trigger apoptotic cell death, whereas higher concentrations induce necrosis with cellular energetics (ATP and NAD) serving as a switch between the models of cell death. Superoxide is also involved in the production of nitrogen dioxide radical and hydrogen peroxide, which can be further transformed into lipid peroxidationeinitiating species, namely peroxyl and alkoxyl radicals (ROO
and RO
) (Dix and Aikens, 1993; Gue´raud et al., 2010). Furthermore, ROS and RNS are also normally generated by tightly regulated enzymes or enzyme systems located in or associated with cellular membranes or organelles, in both phagocytic and nonphagocytic cells. Nitric oxide synthases (NOSs) and nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase isoformeproduced ROS/RNS are also involved in cellular signaling, synaptic plasticity in the central nervous system (CNS), neuronal transmission, reactions to stress and various agents, and the induction of mitogenic and apoptotic responses (Beal, 2000; Valko et al., 2007; Mangialasche et al., 2009). ROS and RNS, produced under various mechanisms, can react with lipids, sugars, proteins, and nucleic acids and inhibit normal cell functions (Deavall et al., 2012; Cheignon et al., 2018). This damage can compromise cell viability or induce cellular response leading to cell death by necrosis or apoptosis (Valko et al., 2007; Mangialasche et al., 2009; Du et al., 2018; Fang et al., 2017). Importantly, living systems have developed several mechanisms to control these harmful effects of ROS and RNS. These mechanisms are mainly based on the presence of antioxidants (enzymatic and

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nonenzymatic) and the repair or removal of the injured molecules/systems. Antioxidants are natural or synthetic molecules preventing excessive formation of ROS or inhibiting their reaction with biological structures and/or molecules. Antioxidant defense involves a variety of strategies, both enzymatic, such as superoxide dismutase, catalase, thioredoxin reductase, and glutathione peroxidase (GPx), and nonenzymatic, involving tocopherols, carotenes, ascorbate, glutathione (GSH), ubiquinols, and flavonoids (Halliwell, 2007; Espinosa-Diez et al., 2015; Radomska-Lesniewska et al., 2016, 2017; Valko et al., 2007; Vallejo et al., 2017). Different systems/mechanisms cooperate in the regulation of ROS/RNS production and neutralization, which is essential for avoiding their detrimental effects and preserving equilibrium. This is termed redox homeostasis (Droge, 2002).

LIPID PEROXIDATION AND MARKERS OF OXIDATIVE STRESS Because of the high concentration of substrate polyunsaturated fatty acids (PUFA) in cells, lipid peroxidation is a major outcome of free radicalemediated injury (Montine et al., 2002). Lipid peroxidation is the mechanism by which lipids are attacked by chemical species that have sufficient reactivity to abstract a hydrogen atom from a methylene carbon in their chain. Lipid peroxidation in vivo, through a free radical pathway, requires a PUFA and a reactant oxidant inducer that together form a free radical intermediate. The free radical intermediate subsequently reacts with oxygen to generate a peroxyl radical, which with unpaired electrons may additionally abstract a hydrogen atom from another PUFA. The greater number of double bonds in the molecule and the higher instability of the hydrogen atom adjacent to the double bond explain why unsaturated lipids are particularly susceptible to peroxidation (Pratico et al., 2004; Gao et al., 2006). A critically important aspect of lipid peroxidation is that it will proceed until the oxidizable substrate is consumed or termination occurs, making this fundamentally different from many other forms of free radical injury in that the self-sustaining nature of the process may entail extensive tissue damage (Porter et al., 1995). Decreased membrane fluidity following lipid peroxidation makes it easier for phospholipids to exchange between the two halves of the bilayer, increase the leakiness of the membrane to substances that do not normally cross it other than through specific channels (e.g., Kþ, Ca2þ), and damage membrane proteins, inactivating receptors, enzymes, and ion channels (Halliwell, 2007). Increases in Ca2þ induced by oxidative stress can activate phospholipase A2, which then releases arachidonic acid

(AA) from membrane phospholipids. The free AA can then both undergo lipid peroxidation (Farooqui et al., 2001) and act as a substrate for eicosanoid synthesis. Increased prostaglandin synthesis is immediately linked to lipid peroxidation because low levels of peroxides accelerate cyclooxygenase action on polyunsaturated fatty acids (Smith, 2005). Phospholipase A2 can also cleave oxidized AA residues from membranes. The use of reactive products of lipid peroxidation as in vivo biomarkers is limited because of their chemical instability and rapid and extensive metabolism (Gutteridge and Halliwell, 1990; Moore and Roberts, 1998). For these reasons, other more stable lipid products of oxidative damage have generated intense interest in recent years as in vivo markers of oxidative damage (De Zwart et al., 1999). These compounds include the F2-isoprostanes (F2-IsoPs), F3-Isops, isofurans (IsoFs), and F4-neuroprostanes (F4-NeuroPs) (Morrow et al., 1990; Fessel et al., 2002; Yin et al., 2007; Janicka et al., 2010).

PROSTAGLANDIN-LIKE COMPOUNDS AS IN VIVO MARKERS OF OXIDATIVE STRESS F2-IsoPs are prostaglandin-like compounds that are produced by a noncyclooxygenase free radicale catalyzed mechanism involving the peroxidation of the PUFA, arachidonic acid (AA, C20:4, u-6). Formation of these compounds initially involves the generation of four positional peroxyl radical isomers of arachidonate, which undergo endocyclization to PGG2-like compounds. These intermediates are reduced to form four F2-IsoP regioisomers, each of which can consist of eight racemic diastereomers (Morrow et al., 1990). In contrast to cyclooxygenase (COX)-derived prostaglandins (PGs), nonenzymatic generation of F2-IsoPs favors the formation of compounds in which the stereochemistry of the side chains is cis oriented in relation to the prostane ring. A second important difference between F2-IsoPs and PGs is that F2-IsoPs are formed primarily in situ, esterified to phospholipids, and subsequently released by phospholipases (Famm and Morrow, 2003; Gao et al., 2006), whereas PGs are generated only from free arachidonic acid (Morrow et al., 1990). The basic formation process is depicted in Fig. 57.1. F2-IsoPs analog may be formed by peroxidation of docosahexaenoic acid (DHA, C22:6, u-3), which generates F4-IsoPs. These compounds are also termed neuroprostanes (F4-NeuroPs), because of the high levels of their precursor in the brain (Roberts et al., 1998). In contrast to AA, which is evenly distributed in all cell types in all tissues, DHA is highly concentrated in neuronal membranes (Salem et al., 1986). DHA is obtained mainly through dietary means as the human body can only

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Lipid peroxidation

Chemically reactive products

Endoperoxide intermediate of AA and DHA

Cell dysfunction and death

Increased O2

IsoFs and NeuroFs

Reduction

F2-IsoPs and F4-NeuroPs

Isomerization

D2/E2-IsoPs and D4/E4-NeuroPs

FIGURE 57.1 Schematic of the compound-forming process.

minimally synthesize this fatty acid. DHA deficiency has been linked to slow mental development (Connor et al., 1992), whereas DHA supplementation has been linked to a variety of health benefits including decreased rates of neurodegenerative diseases (Schaefer et al., 2006). DHA can undergo oxidation both in vitro and in vivo, and increased units of unsaturation suggest its higher susceptibility to lipid peroxidation than AA. Thus, although the measurement of F2-IsoPs provides an index of global oxidative damage in the brain, integrating data from both glial and neuronal cells, determination of F4-NeuroPs permits the specific quantification of oxidative damage to neuronal membranes in vivo. In fact, to our knowledge, F4-NeuroPs are the only quantitative in vivo marker of oxidative damage that is selective for neurons. This is particularly important because of the implication of oxidative damage and lipid peroxidation being causative factors in numerous neurodegenerative diseases (Montine et al., 2004; Milatovic et al., 2005b). Another F2-IsoPs analog may be formed by peroxidation of eicosapentaenoic acid (EPA, C20:5, u-3) that leads to the production of F3-IsoPs. Levels of F3-IsoPs can significantly exceed those of F2-IsoPs generated from AA, perhaps because EPA contains more double bonds and is therefore more easily oxidizable (Gao et al., 2006). It has also been shown that in the presence of increased oxygen tension in the microenvironment in which lipid peroxidation occurs, an additional oxygen insertion step may take place (Fessel et al., 2002). This step diverts the IsoP pathway to form tetrahydrofuran ringecontaining compounds termed isofurans (IsoFs), which are functional markers of lipid peroxidation under conditions of increased oxygen tension. Thus, measurements of IsoFs represent a much more robust indicator of hyperoxia-induced lung injury than measurements of F2-IsoPs. Similar to IsoPs, the IsoFs are chemically and metabolically stable, so they are well suited to act as in vivo biomarkers of oxidative damage. Stable isotope dilution, negative ion chemical ionization (NICI) gas chromatography/mass spectrometry (GC/MS) with select ion monitoring (SIM) was the first technique used in early discovery and quantification of isoprostanes by investigators at Vanderbilt University (Morrow et al., 1990). This methodology allows the

lower limit of detection of the F2-IsoPs to be in the low picogram range. Quantification of the F2-IsoPs levels is achieved by comparing the height/areas of the peak containing derivatized F2-IsoPs (m/z 569) with the height of the deuterated internal standard peak (m/z 573) (Fig. 57.2). These properties, along with the assay’s high sensitivity and specificity, allow the F2-IsoPs to be excellent biomarkers of and the most robust and sensitive measure of oxidative stress in vivo. However, mass spectrometryebased methods necessitate a skilled technical staff and are laborious to execute, because of extensive purification and appropriate derivatization procedures. In addition, over the past 20 years other methods such as enzyme immunoassays, radioimmunoassays, liquid chromatography (LC)emass spectrometry, LC-MS-MS, and GC-MS-MS (Wang et al., 1995; Basu, 1998; Liang et al., 2003) have been developed and used to exploit the impact of this biomarker on human health and disease (Morrow et al., 1990; Wang et al., 1995; Pratico et al., 1998; Basu, 2008). Immunoassays, although less specific and quantitative than GC-MS methods, have been found to be helpful tools for new discoveries in medical and pharmaceutical sciences. Immunoassays have a huge sample-analyzing capacity with fairly low expense. Thus, a well-validated technique could be a significant tool for evaluating free radicalemediated reactions in clinical research, where a large number of samples must be analyzed at an affordable cost. Specific antibodies against isoprostanes can also be used for in situ localization by immunostaining in oxidative stresse injured tissues. Immunostaining with specific antibodies opens possibilities for therapeutic application of various radical scavengers in disease-related damage (Basu, 2008).

ALDEHYDES AS LIPID PEROXIDATION PRODUCTS The enzymatic and free radical peroxidation of PUFAs, which contain at least three double bonds, such as AA and DHA, could lead to malondialdehyde (MDA). This product can be generated by thromboxane synthase, but a report from the Biomarkers of Oxidative

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(A)

log 569.50 (569.00 to 570.00): 003.D

Time–> Abundance

40,000 35,000 30,000 25,000 20,000 15,000 10,000 5000 0

6.00

6.50

(B)

Time–> Abundance

7.00

7.50

8.00

8.50

9.00

8.50

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log 573.20 (572.70 to 573.70): 003.D 40,000 35,000 30,000 25,000 20,000 15,000 10,000 5000 0

6.00

6.50

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log 593.00 (592.50 to 593.50): 042.D

(C) 25,000 20,000 15,000 10,000 5000 Time–> Abundance

0

6.00

6.50

7.00

7.50

8.00

8.50

9.00

FIGURE 57.2 Chromatograms for F2-IsoPs and F4-NeuroPs from the mouse cerebrum. All chromatograms plot abundance versus time (min). (A) m/z 569 chromatogram showing F2-IsoPs, (B) m/z 573 chromatogram showing internal standard, (C) m/z 593 chromatogram showing F4NeuroPs.

Stress Study showed that peripheral levels of MDA derive primarily from nonenzymatic peroxidative degradation of unsaturated lipids (Kadiiska et al., 2005). Because MDA reacts with thiobarbituric acid (TBA), MDAeTBA adducts are used to spectrophotometrically measure the levels of oxidative stress and consequent lipid peroxidation (Spickett et al., 2010; Fang et al., 2017). MDA and MDAeTBA complex assessment can be also measured by high-performance liquid chromatography (HPLC) or GC-MS (Maboudou et al., 2002). Although more specific, this approach does not deduce all the limitations of this biomarker (Devasagayam et al., 2003; Sultana et al., 2006). 4-Hydroxy-2-nonenal (HNE) is a reactive aldehyde arising from peroxidation of u6 fatty acid (Uchida, 2003; Gue´raud et al., 2010; Fang et al., 2017). HNE is formed under various conditions such as autooxidation and stimulated microsomal lipid peroxidation (Neely et al., 2005). HNE is found in food (Gasc et al.,

2007) and detected in vivo (Neely et al., 2005). MDA and HNE are able to covalently modify proteins and alter their functions (Butterfield et al., 2006). In addition to protein modification, these lipid peroxidation products can also interfere with synthesis of DNA and RNA, alter cell metabolism and signaling, and mediate brain-induced oxidative damage. Several studies suggest MDA and HNE can promote the degeneration of cholinergic neurons, Ab aggregation, and amyloidogenesis (Mark et al., 1995; Pedersen et al., 1999; Butterfield et al., 2006). In addition, increased levels of HNE and HNE-protein adducts (Montine et al., 1997; Markesbery and Lovell, 1998) have been described in the ventricular CSF and brain of AD patients compared with control subjects (Montine et al., 1997; Markesbery and Lovell, 1998). Another highly reactive aldehydic product, acrolein, derived from the metal-catalyzed oxidation of polyunsaturated fatty acid, including AA and DHA, can also

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EXCITOTOXICITY AND OXIDATIVE DAMAGE

promote the formation of protein adducts and thus promote neuronal damage (LoPachin et al., 2008). Studies suggest that acrolein can modify DNA, promote lipid peroxidation, induce tau oligomerization, and promote neurofibrillary tangles formation (Uchida et al., 1998; Lovell et al., 2000; Kuhla et al., 2007).

REACTIVITY OF LIPID PEROXIDATION PRODUCTS Metabolization of secondary lipid peroxidation products such as HNE in most cells and tissues is rapid and complete. GSH conjugation seems to be the primary and major step. GSH maintains protein sulfhydryl groups in the reduced form and has been implicated in the regulation of cytoskeletal function. SH groups of cystein constitute a main protein-associated target of HNE (Esterbauer et al., 1991). Indeed, cellular detoxification of HNE is mainly achieved through conjugation to the cystein GSH. This reaction is catalyzed by GSH Stransferase and leads to a transient decrease in cellular GSH (Johnson et al., 1993; Hayes and Pulford, 1995; Kinter and Roberts, 1996; Cheng et al., 1999; Radomska-Lesniewska et al., 2017). If GSH is depleted by buthionine sulfoximine, for instance, or by a concomitant oxidative insult, there is a reduction in GSH-HNE conjugate together with an increase in unmetabolized HNE and HNE toxicity (Picklo et al., 2002). On the other hand, when the oxidative insult occurs some time before treatment of cells by HNE, preconditioned cells acquire resistance to HNE-induced apoptosis by metabolizing and excluding HNE at a higher rate when compared with nonpreconditional cells (Rice et al., 1986; Rabinovitch et al., 1993). We previously demonstrated that another main cellular target to HNE is tubulin, the core protein of microtubules containing abundant cystein (Neely et al., 2005). Our studies demonstrated that the exposure of Neuro2A cells to HNE results in the inhibition of cytosolic taxolinduced tubulin polymerization and thus supported the hypothesis that HNE adduction to tubulin is the primary mechanism involved in the HNE-induced loss of the highly dynamic neuronal microtubule network (Neely et al., 2005). ROS and RON can react with the DNA molecule and induce purine or pyrimidine base or sugar lesions, nitration and deaminations of purines, DNAeDNA, or DNAeprotein cross-links (Dizdaroglu et al., 2002). These processes lead to mutations and impaired transcriptional and posttranscriptional processes and compromise protein synthesis (Colurso et al., 2003). In addition, DNA damage, oxidative phosphorylation, and altered cell metabolism may lead to apoptosis and promote neuronal death (Becker and Bonni, 2004; Fishel

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et al., 2007). The most investigated DNA adduct, 8hydroxy-20 -deoxyguanosine (8-OHdG), can be evaluated by multiple techniques including immunoassay, HPLC, GC-MS, capillary electrophoresis, and LC-MS. However, LC-MS techniques for DNA damage evaluation permit the identification of a wide range of base adducts and offer a more complete picture of DNA oxidation (Lovell and Markesbery, 2007; Mangialasche et al., 2009; Fang et al., 2017). Although ROS and RNS can attack any amino acid, sulfur-containing and aromatic amino acids are the most susceptible (Stadtman and Levine, 2003). The oxidation of amino acids leads to the formation of carbonyl derivatives, produced by fragmentation, due to an attack on several amino acid side chains, and by formation of adducts between some amino acids residues and products of lipid peroxidation. Protein carbonyls can be detected with 2,4-dinitrophenylhydrazine (DNPH) and used as biomarkers of oxidative stress (Dalle-Donne et al., 2003). Oxidation of protein also leads to protein fragmentation and protein crosslinking. In addition, peroxynitrite and a hydroxyl radical can react with tyrosine and form other indexes of protein oxidation, 3-nitrotyrosine and orthotyrosine, respectively. These products of oxidative/ nitrosative modification of proteins are relatively stable with sensitive assays available for their detection (Chakravarti and Chakravarti, 2007).

EXCITOTOXICITY AND OXIDATIVE DAMAGE The brain is especially susceptible to oxidative damage. The brain is relatively deficient in antioxidant systems, with a lower activity of GPx and catalase compared to other organs. It has high metabolic activity that requires large amounts of oxygen and contains redox-active metals (iron, copper) that can promote the production of free radicals. During normal physiological conditions, ROS are generated at a low rate in brain and are efficiently removed by scavenger and antioxidant systems. However, in pathophysiological conditions, such as seizures and acetylcholinesterase inhibitors (organophosphates and carbamates) toxicity, a high rate of ATP consumption is accompanied by increased generation of ROS. Previous studies have supported the role for oxidative stress and excessive generation of ROS and RNS in anticholinesterase-induced neurotoxicity (Dettbarn et al., 2001; Gupta et al., 2001a,b, 2007; Milatovic et al., 2005a). During sustained seizures, the flow of oxygen to the brain is greatly increased at a time when the use of ATP is greater than the rate of its generation. This metabolic stress results in a markedly increased ROS generation. A greatly increased rate of

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57. BIOMARKERS OF OXIDATIVE/NITROSATIVE STRESS AND NEUROTOXICITY

ROS production, overwhelming the capacity of inherent cellular defense systems, results in an attack on the mitochondria and cell membranes, leading to peroxidation of lipids, cell lesions, and, in turn, cell death (Sjodin et al., 1990). Processes such as excitotoxicity and inhibition of acetylcholinesterase (AChE) lead to unremitting stimulation of nervous tissue and muscle, which, in turn, causes depletion of high-energy phosphates (HEP), ATP, and phosphocreatine (PCr) and ROS generation (Dettbarn et al., 2001, 2006). If this stimulation is sufficiently low in intensity or brief in duration, cellular recovery will ensue without lasting consequences. If, however, intense cholinergic stimulation is allowed to persist, a self-reinforcing cycle of cellular damage is set in motion. ATP depletion for several hours to approximately 30%e40% of normal levels leads to a fall in the mitochondrial membrane potential that is associated with (1) reduced energy production (because of decrease in complex I and complex IV activities), (2) impaired cellular calcium sequestration, (3) activation of protease/caspases, (4) activation of phospholipases, (5) activation of nitric oxide synthase (NOS), and (6) excessive generation of ROS (Milatovic et al., 2006). Several of these steps are associated with exacerbation and propagation of the initial depletion of ATP; most notable are the decreases in complex I and IV activities, the impairment of mitochondrial calcium metabolism that regulates ATP production even in the face of a constant supply of substrates, and the generation of nitric oxide, which binds reversibly to cytochrome c oxidase (COx) in competition with oxygen, with subsequent sensitization to hypoxia. COx is the terminal complex in the mitochondrial respiratory chain, which generates ATP by oxidative phosphorylation, involving the reduction of O2 to H2O by the sequential addition of four electrons and four Hþ. Electron leakage occurs from the electron transport chain, which produces the superoxide anion radical and H2O2. Under normal conditions, COx catalyzes more than 90% of the oxygen consumption in the cells. The chance of intermediate products, such as superoxide anion radical and H2O2 and hydroxyl radical, escaping is less under conditions where COx remains active. During the hyperactivity of brain or muscle, the activity of COx is reduced (Milatovic et al., 2001), leading to an increased electron flow within the electron transport chain, thereby increasing ROS generation, oxidative damage to mitochondrial membranes, and vulnerability to excitotoxic impairment (Soussi et al., 1989; Gollnick et al., 1990; Bose et al., 1992; Bondy and Lee, 1993; Yang and Dettbarn, 1998). Excitotoxicity-induced cell damage is also thought to result from an intense transient influx of calcium leading to mitochondrial functional impairments characterized by activation of the permeability transition pores in the

inner mitochondrial membrane, cytochrome c release, depletion of ATP, and simultaneous formation of ROS (Cadenas and Davies, 2000; Nicholls and Ward, 2000; Heinemann et al., 2002; Patel, 2002; Nicholls et al., 2003). In addition, increase in cytoplasmic calcium ions triggers intracellular cascades through stimulation of enzymes, including proteases, phospholipase A2, and nitric oxide synthase, which also leads to increased levels of free radical species and oxidative stress (Lafon-Cazal et al., 1993; Farooqui et al., 2001). Because free radicals are direct inhibitors of the mitochondrial respiratory chain, ROS generation perpetuates a reinforcing cycle, leading to extensive lipid peroxidation and oxidative cell damage (Cadenas and Davies, 2000; Cock et al., 2002). Our studies have shown that kainic acid (KA)e induced excitotoxicity and anticholinesterases, such as diisopropylphosphorofluoridate (DFP) and carbofuran (CF), cause neuronal injury by excessive formation of ROS (Yang and Dettbarn, 1998; Gupta et al., 2001a,b; Milatovic et al., 2000a,b, 2001, 2005a; Zaja-Milatovic et al., 2008). Exposure to DFP or carbofuran significantly suppressed AChE activity, induced severe seizure activity, and significantly increased biomarkers of global free radical damage (F2-IsoPs) and the selective peroxidation biomarker of neuronal membranes (F4-NeuroPs) (Gupta et al., 2007; Zaja-Milatovic et al., 2009). Although twofold elevations are seen in F2-IsoPs levels, F4-NeuroPs levels are fivefold higher compared to controls (Fig. 57.3). The selective increase in F4-NeuroPs indicates that neurons are specifically targeted by this mechanism. DFP or CF exposures also caused marked elevation in brain citrulline levels (an indicator of NO/NOS activity) (Gupta et al., 2001b, 2007). Many reports provide evidence that NO impairs mitochondrial/cellular respiration and other functions by inhibiting the activities of several key enzymes, particularly COx, and thereby causing ATP depletion (Yang and Dettbarn, 1998; Dettbarn et al., 2001; Gupta et al., 2001a; b; Milatovic et al., 2001). Results from our experiments also showed that 1 hour after DFP (1.5 mg/kg, s.c.) or CF (1.5 mg/ kg, s.c.) treatment, the levels of ATP and PCr were significantly reduced in the cortex, hippocampus, and amygdala (Gupta et al., 2001a,b). The rapid decrease in energy metabolites at the onset of seizures indicates early onset of mitochondrial dysfunction, in turn further increasing ROS production and neuronal injury. The most consistent pathological findings in acute experiments with anticholinesterases include degeneration and cell death in the pyriform cortex, amygdala, hippocampus (where the CA1 region is preferentially destroyed), dorsal thalamus, and cerebral cortex. The early morphological changes in AChEI-induced status epilepticus (SE) include dendritic swelling of pyramidal

IX. SPECIAL TOPICS

1019

EXCITOTOXICITY AND OXIDATIVE DAMAGE

(B)

(A) a

6

a F4-NeuroPs (ng/g)

5 F2-IsoPs (ng/g)

a

75

4 3 2 1

50

25

0

0 Control

DFP

CF

a

Control

DFP

CF

FIGURE 57.3 Effect of DFP (1.5 mg/kg, s.c.) and carbofuran (CF, 1.5 mg/kg, s.c.) on F2-IsoPs (A) and F4-NeuroPs (B) levels in rat brain. Rats were sacrificed 1 h after DFP or CF injection. Values are mean  SEM (n ¼ 4e6). aSignificant difference between control and DFP- or CF-treated rats (P < .05).

pyramidal neuron from the hippocampal CA1 area of control rats (Fig. 57.4). Taken together, our results revealed that anticholinesterase exposure is associated with oxidative and nitrosative stresses, alteration in energy metabolism, and consequent degeneration of pyramidal neurons from the CA1 hippocampal region of rat brain. Ultimately, the additive or synergistic mechanisms of cellular disruption caused by anticholinesterase agents lead to cellular dysfunction and neurodegeneration. Similar studies investigated the role of glutamatergic excitation, oxidative injury, and neurodegeneration in the model of KA excitotoxicity. We have used intracerebroventricular (icv) injection of KA and investigated whether F2-IsoPs and F4-NeuroPs formations correlated with the vulnerability of pyramidal neurons in the CA1 hippocampal area following KA-induced excitotoxicity.

(A)

Dendritic length (um)

1800 1500 1200 900

300 0

Control

a

600

Control

DFP

Spine density/ µm dendrite

neurons in the CA1 region of the hippocampus (Carpentier et al., 1991). Therefore, we have investigated whether seizure-induced cerebral oxidative damage in adult rats is accompanied by alterations in the integrity of the hippocampal CA1 dendritic system. Our results showed that anticholinesterase induced early increases in biomarkers of global free radical damage (F2-IsoPs), and the selective peroxidation biomarker of neuronal membranes (F4-NeuroPs) was accompanied by dendritic degeneration of pyramidal neurons in the CA1 hippocampal area. Anticholinesterase-induced brain hyperactivity targeted the dendritic system with profound degeneration of spines and regression of dendrites, as evaluated by Golgi impregnation and Neurolucida-assisted morphometry (Fig. 57.4). Rats injected with DFP show a significant decrease in total dendritic length and spine density compared to

0.3

(B)

0.2

a

0.1

0.0

Control

DFP

DFP

FIGURE 57.4 Morphology and quantitative determination of dendritic length (A) and spine density (B) of hippocampal pyramidal neurons from CA1 sector of rats treated with saline (control) or DFP (1.5 mg/kg, s.c.) and sacrificed 1 h after the treatment. Four to six Golgi-impregnated dorsal hippocampal CA1 neurons were selected and spines were counted using Neurolucida system. aSignificant difference between control and DFP-treated rats (P < .05). Treatment with DFP induced degeneration of hippocampal dendritic system and a decrease in the total length of dendrite and spine density of hippocampal pyramidal neurons. Tracing and counting were done using a Neurolucida system at 100 under oil immersion (MicroBrightField, VT). Colors indicate degree of dendritic branching (blue ¼ 1 degree, yellow ¼ 2 degrees, green ¼ 3 degrees, magenta ¼ 4 degrees, orange ¼ 5 degrees).

IX. SPECIAL TOPICS

1020

57. BIOMARKERS OF OXIDATIVE/NITROSATIVE STRESS AND NEUROTOXICITY

Our results showed that icv KA induced an early increase in biomarkers of oxidative damage, F2-IsoPs and F4-NeuroPs. Elevated levels of these in vivo markers of oxidative damage are in agreement with our previous findings (Montine et al., 2002; Milatovic et al., 2005b; Gupta et al., 2007), as well as those of others (Patel et al., 2001; Kiasalari et al., 2013, 2016) and indicate that KA injection leads to profound cerebral and neuronal oxidative damage in mice. Our results also showed that the transient rise in F2IsoPs and F4-NeuroPs is accompanied by rapid evolution of dendritic abnormalities, which is apparent in the significant decrease in dendritic length and spine density of pyramidal neurons as early as 30 min post KA injection. However, the recovery in oxidative damage biomarkers at 60 min following the injection was not paralleled by the rescue of damaged neurons from the CA1 hippocampal area. Extended seizure activity (60 min) induced the same level of dendritic length and spine density decrease when compared to 30 min following KA injection (Table 57.1). Together, these data suggest that both oxidative stress and neurodegeneration occur as an early response to seizures, but they do not establish whether oxidative stress is a cause or effect of seizure-induced CA1 cell damage. Neuronal damage processes triggered by sustained seizure activity may occur as a continuum, last longer than the formation of oxidative lipids and although not evident by the markers, and may already be in progress when the peak increases in F2-IsoPs and F4-NeuroPs occur. Thus, we investigated dynamic changes in lipid peroxidation and dendritic structures immediately after seizures, but future studies over the longer period should be able to determine the long-term time course of these spine and dendritic changes. It is very likely that the spine loss seen in our study is the initial phase of more chronic spine loss and progressive neurodegeneration reported in other studies (Isokawa and Levesque, 1991; Muller et al., 1993; Multani et al., 1994; Jiang et al., 1998; Zeng et al., 2007; Fang et al., 2017; Sabens Liedhegner et al., 2012).

TABLE 57.1

NEUROINFLAMMATION AND OXIDATIVE INJURY Neuroinflammation is a complex response to brain toxicity involving the activation of glia, release of inflammatory mediators, such as cytokines and chemokines, and the generation of ROS and RNS. The links among risk factors and the development of neuroinflammation are numerous and involve many complex interactions that contribute to vascular compromise, oxidative stress, and, ultimately, brain damage (Montine et al., 2002; Milatovic et al., 2003, 2004; Salinaro et al., 2018; Calabrese et al., 2016; Fang et al., 2017; Guangpin and Ping, 2016). Once this cascade of events is initiated, the process of neuroinflammation can become overactivated, resulting in further cellular damage and loss of neuronal functions. Acute neuroinflammatory response resulting in phagocytic phenotype and the release of inflammatory mediators such as free radicals, cytokines, and chemokines (Tansey et al., 2007; Frank-Cannon et al., 2009) may be generally beneficial to the CNS because it tends to minimize further injury and contributes to repair of damaged tissue. In contrast, chronic neuroinflammation is a long-standing and often self-perpetuating neuroinflammatory response that persists long after an initial injury or insult. Sustained release of inflammatory mediators and increased oxidative and nitrosative stress activate additional microglia, promoting their proliferation and resulting in further release in inflammatory factors. Owing to this sustained nature of inflammation, the bloodebrain barrier (BBB) may be compromised, thus increasing infiltration of peripheral macrophages into the brain parenchyma and perpetuating the inflammatory process further (Rivest, 2009). Rather than serving in a protective role, as does acute neuroinflammation, chronic neuroinflammation is most often detrimental and damaging to nervous tissue. Thus, whether neuroinflammation has beneficial or harmful outcomes in the brain may critically depend on the duration of the inflammatory response.

Cerebral Concentrations of F2-IsoPs and F4-NeuroPs and Dendritic Degeneration of Hippocampal Pyramidal Neurons Following KA-Induced Seizures in Mice

F2-IsoPs (ng/g)

F4-NeuroPs (ng/g)

Dendritic Length (mm)

Spine Density (Number of Spinal/ 100 mm Dendrite)

Control

3.07  0.05

13.89  0.58

1032.10  61.41

16.45  0.55

KA 30 min

4.81  0.19*

34.27  2.71*

363.44  20.78*

8.81  0.55*

KA 60 min

3.40  0.18

18.55  1.26

425.71  23.04*

7.44  0.56*

Data from KA-exposed mice were collected 30 min or 60 min postinjection. *One-way ANOVA showed P < .0001 for each end point. Bonferroni’s multiple comparison test showed significant difference (P < .001) compared to vehicle-injected control.

IX. SPECIAL TOPICS

1021

METAL TOXICITY AND OXIDATIVE INJURY

Activation of innate immunity occurs simultaneously with several pathogenic processes and responses to stressors and injury, thereby greatly confounding any clear conclusion about cause-and-effect relationships. For these reasons, we have adopted a simple, but highly specific model of isolated innate immune activation: intracerebroventricular (ICV) injection of low dose lipopolysaccharide (LPS). LPS specifically activates innate immunity through a Toll-like receptor (TLR)e dependent signaling pathway (Imler and Hoffmann, 2001; Akira, 2003). Activation of proteins (CD14 and adaptor protein MyD88), signal transduction cascade, primarily via NF-kB activation but also through c-Fos/ c-Jun-dependent pathways, culminate in the generation of effector molecules, including bacteriocidal molecules. Free radicals generated by NADPH oxidase and myeloperoxidase (MPO), as well as cytokines and chemokines, are known to attract an adaptive immune response (Milatovic et al., 2004). We have employed an ICV model and identified the molecular and pharmacologic determinants of LPSinitiated cerebral neuronal damage in vivo (Montine et al., 2002; Milatovic et al., 2003, 2004). Interestingly, the degree of oxidative damage in this model was equivalent to what we observed in diseased regions of brain from patients with degenerative diseases (Reich et al., 2001). Results from our studies with mice showed that single ICV LPS injections induced delayed, transient elevation in both F2-IsoPs and F4-NeuroPs 24 h after exposure and then returned to baseline by 72 h postexposure (Table 57.2) (Milatovic et al., 2003). Although others have shown that altered gene transcription and increased cytokine secretion occur rapidly and peak within a few hours of LPS exposure, it is likely that the delay in neuronal oxidative damage observed in our experiments is related, at least in part, to the time required to deplete antioxidant defenses. To address whether oxidative damage is related to neurodegeneration, we directly examined the dendritic compartment of neurons, which is largely transparent TABLE 57.2

to the standard histological techniques used so far to investigate ICV LPSeinduced damage. Using Golgi impregnation and Neurolucida-assisted morphometry of hippocampal CA1 pyramidal neurons (Leuner et al., 2003; Milatovic et al., 2010), we first determined the time course of dendritic structural changes following ICV LPS in mice. Our results show a time course similar to neuronal oxidative damage with maximal reduction in both dendrite length and dendritic spine density at 24 h post LPS and, remarkably, a return to baseline levels by 72 h (Table 57.2). Thus, these data strongly imply that neuronal oxidative damage is closely associated with dendritic degeneration following ICV LPS. We, along with others, have shown that primary neurons enriched in cell culture do not respond to LPS (Minghetti and Levi, 1995; Fiebich et al., 2001; Xie et al., 2002); therefore, our results also showed that LPS activated microglialmediated paracrine oxidative damage to neurons. It is becoming increasingly evident that neuroinflammation and associated oxidative damage plays a crucial role in the development and progression of brain diseases. Glia, and in particular microglia, are central to mediating the effects of neuroinflammation. Emerging evidence suggests that the number of activated microglia and the release of inflammatory mediators from these cells increase with age. This amplified or prolonged exposure to inflammatory molecules, including cytokines, chemokines, ROS, and PGs in the aged brain may impair neuronal plasticity and underlie a heightened neuroinflammatory response.

METAL TOXICITY AND OXIDATIVE INJURY Essential metals are crucial for the maintenance of cell homeostasis. However, excessive exposure to some metals, including manganese (Mn) and mercury (Hg), present great health concerns and may lead to pathological conditions, including neurodegeneration.

Cerebral Oxidative Damage and Dendritic Degeneration in Mice 24 h

24 h

72 h

72 h

ICV Saline

ICV LPS

ICV Saline

ICV LPS

3.26  0.19

4.77  0.26*

3.13  0.11

2.98  0.17

F4-NeuroPs (ng/g tissue)

13.91  1.17

58.50  5.98*

12.30  1.18

16.80  0.96

Dendritic length (mm)

1018  113

324  37*

848  60

1030  61

Spine density (spine no./100 mm dendrite)

16.89  1.67

5.86  0.57*

17.09  1.13

16.77  0.87

F2-IsoPs (ng/g tissue)

Effects of ICV saline (5 mL, control) and ICV LPS (5 mg/5 mL) treatment determined at 24 and 72 h following exposure. Each value represents mean  SEM (n ¼ 4e6). *One-way ANOVA showed P < .001 for each end point. Bonferroni’s multiple comparison test showed significant difference (P < .01) compared to vehicle-injected control.

IX. SPECIAL TOPICS

57. BIOMARKERS OF OXIDATIVE/NITROSATIVE STRESS AND NEUROTOXICITY

Neurodegenerative mechanisms and effects of Mn and Hg are associated with oxidative stress. Mn can oxidize dopamine (DA), generating reactive species, and also affect mitochondrial function, leading to accumulation of metabolites and culminating with oxidative damage. Cationic Hg forms have a strong affinity for nucleophiles, such as eSH, and target critical thiol and selenol molecules with antioxidant properties. Therefore, mediation of these processes and control of oxidative stress may provide a therapeutic strategy for the suppression of dysfunctional neuronal transmission and a slowing of the neurodegenerative process.

500

F2-IsoPs Formation (% of control)

1022

* *

200 200

1 mM

*

175

*

150

*

*

500 µM

125 100 µM

100 0

MANGANESE Mn toxicity is primarily associated with neurological effects. Excessive accumulation of Mn in specific brain areas, such as the substantia nigra, the globus pallidus, and the striatum, produce neurotoxicity leading to a degenerative brain disorder. Although the mechanisms by which Mn induces neuronal damage are not well defined, its neurotoxicity appears to be regulated by a number of factors, including mitochondrial dysfunction, oxidative injury, and neuroinflammation. Early studies on the cellular actions of Mn reported that mitochondria are the principal intracellular repository for the metal (Cotzias and Greenough, 1958). More recent data indicate that mitochondria actively sequester Mn, resulting in rapid inhibition of oxidative phosphorylation (Gavin et al., 1992). Manganese is bound to inner mitochondrial membrane or matrix proteins (Gavin et al., 1990) and thus directly interacts with proteins involved in oxidative phosphorylation. Mn directly inhibits complex II (Singh et al., 1974; Liu et al., 2013) and complexes IeIV (Zhang et al., 2003) in brain mitochondria and suppresses ATP-dependent calcium waves in astrocytes, suggesting that Mn promotes potentially disruptive mitochondrial sequestration of calcium (Tjalkens et al., 2006). Elevated matrix calcium increases the formation of ROS by the electron transport chain (Kowaltowski et al., 1995) and results in inhibition of aerobic respiration (Kruman and Mattson, 1999). Our studies with primary astrocytes and neurons have shown that Mn exposure induces an increase in biomarkers of oxidative stress (Milatovic et al., 2007, 2009). We have measured F2-IsoPs (Morrow and Roberts, 1999; Milatovic and Aschner, 2009) and showed that astrocytes exposed to Mn concentrations known to elicit neurotoxic effects (100 mM, 500 mM, or 1 mM) induced significant elevations in F2-IsoPs levels at all investigated exposure times (Fig. 57.5). Thus, increases in ROS, potentially damaging mitochondria directly or through the effects of secondary oxidants such as superoxide, H2O2, or ONOO, mediate Mn-induced oxidative

*

1

2

3 Time (h)

4

5

6

FIGURE 57.5 Effects of MnCl2 on F2-IsoPs formation in cultured astrocytes. Rat primary astrocyte cultures were incubated at 37 C in the absence or presence of MnCl2 (100 mM, 500 mM, or 1 mM), and F2IsoPs levels were quantified at 30 min, 2 h, and 6 h. Data represent the mean  SEM from three independent experiments. *Significant difference between values from control and Mn-treated astrocytes (*P < .05).

damage. Moreover, superoxide produced in the mitochondrial electron transport chain may catalyze the transition shift of Mn2þ to Mn3þ through a set of reactions similar to those mediated by superoxide dismutase and thus lead to the increased oxidant capacity of this metal (Gunter et al., 2006). Consequent oxidative damage produces an array of deleterious effects: it may cause structural and functional derangement of the phospholipid bilayer of membranes; disrupt energy metabolism, metabolite biosynthesis, and calcium and iron homeostasis; and initiate apoptosis (Attardi and Schatz, 1988; Uchida, 2003; Alaimo et al., 2011). Consistently preceding the Mn-induced increase in biomarkers of oxidative damage (F2-IsoPs) (Fig. 57.5), our study showed an early decrease in astrocytic ATP levels (Milatovic et al., 2007). As a consequence, ATP depletion or a perturbation in energy metabolism might diminish the ATP-requiring neuroprotective action of astrocytes, such as glutamate and glutamine uptake and free radical scavenging (Rao et al., 2001). In addition, depletion of high-energy phosphates may affect intracellular Ca2þ in astrocytes through mechanisms involving the disruption of mitochondrial Ca2þ signaling. This assertion is supported by data showing that Mn inhibits Naþ-dependent Ca2þ efflux (Gavin et al., 1990) and respiration in brain mitochondria (Zhang et al., 2004), both critical for maintaining normal ATP levels and ensuring adequate intermitochondrial signaling. A decrease in ATP following Mn exposure is also associated with excitotoxicity, suggesting a direct effect on astrocytes with subsequent impairment of neuronal function. Mn downregulates the glutamate

IX. SPECIAL TOPICS

MANGANESE

transporter GLAST in astrocytes (Erikson and Aschner, 2002) and decreases levels of glutamine synthase in exposed primates (Erikson et al., 2008). Studies with a neonatal rat model indicated that both pinacidil, a potassium channel agonist, and nimodipine, a Ca2þ channel antagonist, reversed Mn neurotoxicity and loss of glutamine synthase activity, further indicating excitotoxicity in the mechanism of Mn-induced neurotoxicity. Excessive Mn may lead to excitotoxic neuronal injury by both decreased astrocytic glutamate uptake and loss of ATP-mediated inhibition of glutamatergic synapses. Oxidative stress as an important mechanism in Mninduced neurotoxicity is also confirmed in our in vivo model. Analyses of cerebral biomarkers of oxidative damage revealed that a one-time challenge of mice with Mn (100 mg/kg, s.c.) was sufficient to produce significant increases in F2-IsoPs (Table 57.3) 24 h following the last injection, respectively. Increased striatal concentrations of ascorbic acid and GSH, antioxidants that when increased signal the presence of an elevated burden from ROS, as well as other markers of oxidative stress have been previously reported (Desole et al., 1994; Dobson et al., 2003; Erikson et al., 2007). Mn-induced decrease in GSH and increased metallothionein were reported in rats (Dobson et al., 2003) and nonhuman primate studies (Erikson et al., 2007). ROS may act in concert with RNS derived from astroglia and microglia to facilitate the Mn-induced degeneration of DAergic neurons. DAergic neurons possess reduced antioxidant capacity, as evidenced by low intracellular GSH, which renders these neurons more vulnerable to oxidative stress and glial activation relative to other cell types (Sloot et al., 1994; Greenamyre et al., 1999; Filipov and Dodd, 2011). Therefore, the overactivation of glia and the release of additional neurotoxic factors may represent a crucial component associated with the degenerative process of DAergic neurons (Filipov and Dodd, 2011). In addition to a decrease in mitochondrial membrane potential and the depletion of high-energy phosphates, Mn-induced ROS generation is also associated with

TABLE 57.3

Cerebral F2-IsoPs and PGE2 Levels in Saline (Control) or MnCl2 (100 mg/kg, s.c.) Exposed Mice

Exposure

F2-IsoPs (ng/g Tissue)

PGE2 (ng/g Tissue)

Control (saline)

3.013  0.03939

9.488  0.3091

Single Mn

4.302  0.3900*

12.030  0.4987*

Multiple Mn

4.211  0.4013*

14.220  1.019*

Brains from mice exposed once or three times (days 1, 4, and 7) to MnCl2 were collected 24 h post last injection. Each value represents mean  SEM (n ¼ 4e6). *One-way ANOVA showed P < .001 for each end point. Bonferroni’s multiple comparison test showed significant difference (P < .05) compared to vehicleinjected control.

1023

inflammatory responses and release of inflammatory mediators, including prostaglandins. Our studies have confirmed that parallel to the increase in biomarkers of oxidative damage, Mn exposure induced an increase in the biomarker of inflammation, prostaglandin E2 (PGE2), in vitro and in vivo (Milatovic et al., 2007, 2009). Results from our in vivo study showed that Mn exposure induced a time-dependent increase in PGE2 (Table 57.3). Previous studies have also shown an inflammatory response of glial cells following Mn exposure (Chen et al., 2006b; Zhang et al., 2009; Zhao et al., 2009). Mn potentiates lipopolysaccharide-induced increases in proinflammatory cytokines in glial cultures (Filipov et al., 2005) and an increase in nitric oxide production (Chang and Liu, 1999). An increase in proinflammatory genes, tumor necrosis factor-a, iNOS, and activated inflammatory proteins such as P-p38, P-ERK, and P-JNK have been measured in primary rat glial cells after Mn exposure (Chen et al., 2006b). However, data from our study indicated that release of proinflammatory mediators following Mn exposure is not only associated with glial response but also with neurons, suggesting that these two events are mechanistically related, with neuroinflammation either alone or in combination with activated glial response contributing to oxidative damage and consequent cell injury. Features of Mn neurotoxicity reflect alterations in the integrity of DAergic striatal neurons and DA neurochemistry, including decreased DA transport function and/or striatal DA levels. The striatum is a major recipient structure of neuronal efferents in the basal ganglia. It receives excitatory input from the cortex and dopaminergic input from substantia nigra and projects to the internal segment of the globus pallidus (Dimova et al., 1993; Saka et al., 2002). Nigrostriatal dopamine neurons appear to be particularly sensitive to Mn-induced toxicity (Sloot and Gramsbergen, 1994; Sloot et al., 1994; Defazio et al., 1996). Intense or prolonged Mn exposure in adulthood causes long-term reductions in striatal DA levels and induces a loss of autoreceptor control over DA release (Autissier et al., 1982; Komura and Sakamoto, 1992). Nigrostriatal DA axons synapse onto striatal medium spiny neurons (MSNs), and these neurons have radially projecting dendrites that are densely studded with spines (Wilson and Groves, 1980). Postmortem studies of PD patients have revealed a marked decrease in MSN spine density and dendritic length (Stephens et al., 2005; Zaja-Milatovic et al., 2005). Similar morphological changes in MSNs were seen in animal models of Parkinsonism (Arbuthnott et al., 2000; Day et al., 2006). Our study investigated the effects of Mn on degeneration of striatal neurons. Representative images of Golgi-impregnated striatal sections with their traced MSNs from control and Mn-exposed animals are presented in Fig. 57.6.

IX. SPECIAL TOPICS

1024

57. BIOMARKERS OF OXIDATIVE/NITROSATIVE STRESS AND NEUROTOXICITY

FIGURE 57.6

Photomicrographs of mouse striatal sections with representative tracings of medium spiny neurons (MSNs) from mice treated with saline (control) (A) or MnCl2 (100 mg/kg, s.c.) (B). Brain from mouse exposed three times (days 1, 4, and 7) to MnCl2 was collected 24 h post last injection. Treatment with Mn induced degeneration of the striatal dendritic system and a decrease in total number of spines and length of dendrites of MSNs. Tracing and counting were done using a Neurolucida system at 100 under oil immersion (MicroBrightField, VT). Colors indicate the degree of dendritic branching (yellow ¼ 1 degree, red ¼ 2 degrees, purple ¼ 3 degrees, green ¼ 4 degrees, turquoise ¼ 5 degrees).

Images of neurons with Neurolucida-assisted morphometry show that Mn-induced oxidative damage and neuroinflammation targeted the dendritic system with profound dendrite regression of striatal MSNs. Although single Mn exposure altered the integrity of the dendritic system and induced a significant decrease in spine number (Fig. 57.7A) and total dendritic lengths (Fig. 57.7B) of MSNs, prolonged Mn exposure led to a further reduction in spine number and dendritic length. Our results indicate that MSNs neurodegeneration could result from loss of spines, removing the pharmacological target for DA-replacement therapy, without

overt MSN death (Stephens et al., 2005; Zaja-Milatovic et al., 2005). Together, several studies suggest that oxidative stress, mitochondrial dysfunction, and neuroinflammation are the underlying mechanisms in Mn-induced vulnerability of DAergic neurons. The mediation of any of these mechanisms and control of alterations in biomarkers of oxidative injury, neuroinflammation, and synaptodendritic degeneration may provide a therapeutic strategy for the suppression of dysfunctional DAergic transmission and slowing of the neurodegenerative process. In addition, multiple mechanisms of Mn action are not

FIGURE 57.7 Total number of spines (A) and total dendritic lengths (B) of MSNs from striatal sections of mice exposed to saline (control), single Mn injection (100 mg/kg, s.c.), or multiple Mn injections (100 mg/kg, s.c.) on days 1, 4, and 7. Mice were sacrificed 24 h after the last injection. *Significant difference between values from control and Mn-treated mice (*P < .01). ^Significant difference between values from single Mn injection versus multiple (8 days) Mn treatment (^P < .001).

IX. SPECIAL TOPICS

1025

MERCURY

500

F2-IsoPs (pg/mg pArotein)

sufficiently known and may vary with environmental factors and susceptibilities, including single nucleotide polymorphisms that may alter Mn homeostasis, transport, and metabolism.

*

400 300

MERCURY

* *

*

200

Mercury (Hg) is one of the most toxic elements in the periodic table. Although Hg is present in nature, it has also been released into the environment for centuries as a result of anthropogenic activities. It exists within the environment in three different chemical forms: elemental mercury vapor, inorganic mercury salts, and organic mercury. The distribution, toxicity, and metabolism of mercury are greatly dependent on its chemical form. Organic mercury compounds, such as methylmercury (MeHg), have been extensively studied because they are able to reach high levels in the central nervous system (CNS), leading to neurotoxic effects (Clarkson and Magos, 2006; Aschner et al., 2007; Dos Santos et al., 2018). Although the precise mechanisms of MeHg neurotoxicity are illdefined, oxidative stress and altered mitochondrial and cell membrane permeabilities appear to be critical factors in its pathogenesis. In vivo and in vitro biochemical studies employing glial and neuronal cultures have shown increased ROS formation with MeHg exposure (Yee and Choi, 1996; Sorg et al., 1998; Mundy and Freudenrich, 2000; Gasso et al., 2001; Shanker and Aschner, 2003; Dos Santos et al., 2018). Mitochondria are believed to be major targets of MeHg-induced toxicity (Limke and Atchison, 2002). Specifically, highly enriched Hg concentrations were found in mitochondrial fractions with the lowest Hg concentration found in the cytosol of livers from Hg-exposed animals (Chen et al., 2006a). Most of the bioenergetic experiments with Hg report the uncoupling of oxidative phosphorylation (Weinberg et al., 1982), inhibition of ATP synthesis (Atchison and Harem, 1994), impairment of the respiratory chain (Santos et al., 1997), and depletion of intracellular ATP and ADP (Palmeira et al., 1997). A study using a selective probe for mitochondrial reactive oxygen intermediates as well as other probes demonstrated a significant MeHginduced increase in intracellular superoxide anion, hydrogen peroxide, and hydroxyl radicals, indicating that the mitochondrial electron transport chain is an early, primary site for ROS formation (Allen et al., 2001; Shanker et al., 2004, 2005). Additionally, MeHg exposure disrupts Ca2þ regulation in mitochondria derived from rat brains by decreasing Ca2þ uptake and inducing Ca2þ release (Levesque and Atchison, 1991). Results from our studies confirmed that MeHg induces increases in ROS and that the lipid peroxidation

6h 1h

100 0.0

2.5

5.0 MeHg (μM)

7.5

10.0

FIGURE 57.8 Effect of MeHg on F2-IsoPs formation in cultured astrocytes. Rat primary astrocyte cultures were incubated at 37 C in the absence or presence of MeHg (1, 5, and 10 mM) and F2-IsoPs levels quantified at 1 and 6 h, respectively. Data represent the mean  S.E. from three independent experiments. *P < .05 versus control by oneway ANOVA followed by Bonferroni multiple comparison tests.

biomarkers of oxidative injury, F2-IsoPs, are increased in MeHg-exposed astrocytes (Fig. 57.8). Another consequence of increased oxidative stress is the induction of the mitochondrial permeability transition (MPT), a Ca2þ-dependent process characterized by the opening of the permeability transition pore (PTP) in the inner mitochondrial membrane. This causes increased permeability to protons, ions, and other solutes 1500 Da (Zoratti and Szabo, 1995), leading to a collapse of the mitochondrial inner membrane potential (DJm). Loss of the DJm results in colloid osmotic swelling of the mitochondrial matrix, movement of metabolites across the inner membrane, defective oxidative phosphorylation, cessation of ATP synthesis, and further generation of ROS. Our studies confirmed a concentration-dependent deleterious effect of MeHg on mitochondrial DJm in cultured astrocytes. It is generally believed that increased [Ca2þ]i triggers ROS formation and increased oxidative stress is a major factor for MPT induction (Castilho et al., 1995; Halestrap et al., 1997) and mitochondrial depolarization. Of particular importance, in vivo studies with rats showed that MeHg combines covalently with sulfhydryl (thiol) groups from plasma cholinesterase, leading to the enzyme inhibition (Hastings et al., 1975). After this important observation, several in vitro and in vivo experimental studies showed that sulfhydrylcontaining enzymes are inhibited by MeHg (Magour, 1986; Kung et al., 1987; Rocha et al., 1993). These observations led to the notion that the direct chemical interaction among MeHg and thiol groups from proteins and nonprotein molecules, such as GSH, plays a crucial role in MeHg-induced neurotoxicity (Aschner and Syversen, 2005). Together, multiple studies demonstrated that MeHg exposure is associated with changes in membrane permeability and glutamine/glutamate cycling,

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increases in ROS formation, and consequent lipid peroxidation. Furthermore, lipid peroxidation increases mitochondrial and cellular permeability alterations involving GSH (Gstraunthaler et al., 1983; Strubelt et al., 1996) and calcium depletion (Strubelt et al., 1996). These outcomes work together to create a continuous cycle where acceleration of the mitochondrial chain induces oxidative stress, lipid peroxidation, and depletion of antioxidant defenses, which, in turn, diminish membrane permeability and accelerate the respiratory chain, thus generating more ROS. Ultimately, the additive or synergistic mechanisms of cellular disruption caused by MeHg lead to cellular dysfunction and cell death.

CONCLUDING REMARKS AND FUTURE DIRECTIONS It is becoming increasingly evident that oxidative stress and associated neuronal damage plays a crucial role in the development and progression of brain diseases. However, measuring oxidative stress in biological systems is complex and requires accurate quantification of either free radicals or damaged biomolecules. One method to quantify oxidative injury is to measure nonenzymatic lipid peroxidation products, F2-IsoPs. The quantification of F2-IsoPs has provided a powerful approach to advance our understanding of the role of oxidative damage in a wide variety of research models and disease states. We have applied this methodology and explored cerebral oxidative damage in several models of neurodegeneration, including excitotoxicity generated by kainic acid, neurotoxicity associated with anticholinesterase agents and metals, and innate immune activation by lipopolysaccharide. Results from our studies supported an association between oxidative stress and neurotoxicity and suggested that oxidative stress, mitochondrial dysfunction, and neuroinflammation are underlying mechanisms in excitotoxicity- and metal-induced degeneration of dendritic systems in different brain areas. Future studies should be directed at deciphering the mechanisms of protection and addressing attenuation of neurotoxicity via radical scavenging mechanisms. Complementary studies should also guide the development of selective and efficacious antioxidant therapies that target neurotoxic mechanisms while maintaining neuroprotective actions.

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C H A P T E R

58 Cytoskeletal Disruption as a Biomarker of Developmental Neurotoxicity Alan J. Hargreaves1, Magdalini Sachana2, John Flaskos3 1

School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom; 2Organization for Economic Cooperation and Development (OECD), Paris, France; 3Laboratory of Biochemistry and Toxicology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

INTRODUCTION The eukaryotic cytoskeleton comprises a network of three interconnected protein filamentous arrays known as microtubules (MTs), microfilaments (MFs), and intermediate filaments (IFs). Many MT and MF arrays are dynamic structures that can undergo changes in organization, activity, and function at key stages in neural cell development. These phenomena are regulated by a variety of posttranslational modifications and interactions with a range of accessory proteins (Carlier, 1998; Joshi, 1998; Biernat et al., 2002; Ishikawa and Kohama, 2007; Akhmanova and Steinmetz, 2008; Janke and Kneussel, 2010; Svitkina, 2018). IFs, however, are relatively stable but may be modulated by cross-linking interactions with associated proteins or by their phosphorylation status (Herrmann and Aebi, 2000; Omary et al., 2006). The cytoskeleton is involved in the control of key cellular processes in nervous system development and maintenance, such as cell division, cell migration, cell differentiation, intracellular transport, and structural support. Its disruption by the interaction of neurotoxins with core proteins or cytoskeletal regulatory systems can therefore be detrimental to a wide range of phenomena including neural development (Hargreaves, 1997; Flaskos, 2014). There is a growing body of evidence for the induction of developmental neurotoxicity via disruption of cell signaling and cytoskeleton-dependent physiological processes by several groups of chemicals including organophosphorus esters (OPs), heavy metals, polybrominated diphenyl ethers (PBDEs), and solvents (Sachana et al., 2017; Pierozan et al., 2017). This chapter focuses on studies showing cytoskeletal disruption

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00058-X

associated with impairment of neural development following exposure to such compounds, supporting the idea that cytoskeletal disruption can be a useful biomarker of developmental neurotoxicity.

MICROTUBULES The main component of the MT network is the heterodimeric protein tubulin, which is composed of a and b subunits that form head-to-tail protofilaments, which in turn come together to make the tubular structure with an external diameter of 25 nm (Amos, 2004). MT assembly requires GTP binding to tubulin and MT dynamics are dependent on GTP hydrolysis, which occurs shortly after subunit addition to the growing MT end (“plus” end) (Carlier et al., 1984). Both a- and b-tubulins have several isoforms encoded by different genes (Luduen˜a, 1998; Amos, 2004; Tischfield and Engle, 2010). Recent findings concerning congenital human neurological syndromes further emphasize the unique roles of specific a- and b-tubulin isoforms during nervous system development (Tischfield and Engle, 2010). For example, isotype III of b-tubulin (bIII-tubulin) appears almost exclusively in neuronal cells, plays an important role in neuritogenesis (Katsetos et al., 2003), and is among the earliest neuronal cytoskeletal proteins to be expressed during CNS development (Lee et al., 1990; Easter et al., 1993). Tubulin can be chemically modified by a variety of posttranslational adjustments, which may affect stability and location of MTs or act as a guidance cue for MT-binding proteins (Janke and Kneussel, 2010; Baas et al., 2016).

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Many proteins can potentially interact with MTs, including the most studied microtubule associated proteins (MAPs). In neurons, these interactions influence MT dynamics, and it is believed that they are necessary for neuronal migration, differentiation, and axon guidance (Wade, 2009; Penazzi et al., 2016). Phosphorylation events have been found to regulate the association of MAPs with MTs, suggesting their potential involvement in cascade events relevant to neuronal development and degeneration (Biernat et al., 2002; Baas and Qiang, 2005). An important member of the MAP family is the protein tau, which has tandem repeats of a tubulin-binding domain and contributes to tubulin assembly. Tau is abundant in neurons and is mainly located in axons, where it is closely associated with MTs. Changes in tau-protein levels and its phosphorylation state have been detected in numerous neurodegenerative diseases (Johnson and Stoothoff, 2004). Phosphorylation is also very important from a developmental point of view and is encountered extensively in fetal rather than adult tau (Watanabe et al., 1993). Indeed, increased phosphorylation of tau and dynamic MTs seem to coexist during brain development (Brion et al., 1994). It has also been shown that MAP-1B is an essential protein for the development and function of nervous system both in vitro (Brugg et al., 1993; Di Tella et al., 1996) and in vivo (Meixner et al., 2000; VillarroelCampos and Gonzalez-Billault, 2014). On the other hand, MAP1A dynamics are very closely associated to spine plasticity and any alterations in MAP1A may indicate changes in synaptic density (Jaworski et al., 2009). As a counter measure to MT-stabilizing MAPs, other groups of MAPs can bind to and destabilize MTs. An example is stathmin, which has been shown to regulate MT stability in the formation of dendritic branches in neuronal cells (Ohkawa et al., 2007). This low molecular weight protein can bind to and sequester tubulin heterodimers and hydrolyze GTP at the growing ends of MTs, leading to reduced MT stability (Howell et al., 1999). The key role of MTs in intracellular transport (e.g., along developing axons) is regulated by another group of MAPs, which act as ATPase motor proteins. Such MAPs include kinesin and dynein, which direct anterograde and retrograde transport along axonal MTs respectively (Vale, 2003), a process which is essential for neurite growth and development. The interaction of such proteins with MTs or their ATPase activities could potentially be affected under conditions where ATP levels are depleted. Several studies have dealt with the effects of established developmental neurotoxicants on MT assembly, organization, protein levels, posttranslational modifications, cell distribution, and gene expression. Altered status or intracellular distribution of MT proteins could reflect a range of adverse effects on the regulation of

neural development. As discussed in the following text, most of the available experimental evidence from cell culture studies suggests that several wellestablished developmental neurotoxicants cause alterations in MTs of neuronal and glial cells under culture conditions, a common finding being a reduction in the levels of MAPs.

Effects of Organophosphorus Esters on Microtubules The effects of several OP pesticides have been studied in the distant past, mainly in relation to posttranslational modifications of MT proteins and their role in organophosphate-induced delayed neuropathy (Abou-Donia, 1993, 1995). More recently, chlorpyrifos (CPF) and diazinon (DZ), the two OPs for which there are supporting data for developmental neurotoxicity, have been investigated regarding their potential effect on MTs both in vitro and in vivo. MAP-2 levels decreased following challenge with chlorpyrifos oxon (CPO) in organotypic slice cultures of immature rat hippocampus (Prendergast et al., 2007), whereas the parent compound CPF caused similar effects in the prefrontal cortex of Wistar rats (Ruiz-Mun˜oz et al., 2011) and in the mouse embryonic stem cells (mESCs) that were differentiated into neuronal cells (Visan et al., 2012). However, a-tubulin levels were not altered by 1e10 mM CPO exposure, suggesting that the general structure of MTs was not modified (Prendergast et al., 2007). Similar findings were reported in the case of N2a cells exposed to CPF at the time of the induction of cell differentiation, as well as 16 h after the induction of differentiation (Sachana et al., 2005). Interestingly, levels of total a-tubulin were also found unaltered in the case of DZ or diazoxon (DZO)-treated differentiating N2a cells (Flaskos et al., 2007; Harris et al., 2009a; Sidiropoulou et al., 2009a), whereas MAP-1B levels were reduced after 10 mM DZ exposure (Flaskos et al., 2007). No change in the levels of b-tubulin isotypes I and III were detected in differentiating N2a cells following exposure to DZ (Harris et al., 2009b). In contrast, in the same cell model, DZO induced a significant reduction in the levels of the bIII-tubulin isotype but had no effect on total b-tubulin levels, suggesting a neuron-specific effect (Sachana et al., 2014) In animal models, oral administration of CPF on postnatal days 1e6 did not affect the expression of the genes coding for the neuronal-specific marker bIII-tubulin (Betancourt et al., 2006). The polymerization of bovine brain tubulin was inhibited by low concentrations of CPO (0.1e10 mM) (Prendergast et al., 2007). The ability of tubulin to polymerize is very important because, apart from contributing toward the maintenance of neuronal

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MICROTUBULES

morphology, MTs also support axonal transport of mitochondria, components of ion channels, receptors, and scaffolding proteins. Perturbation of assembly and transport mechanisms in neurons due to CPF and CPO reaction with tubulin and its organophosphorylation has been suggested from several experimental works both in vivo (Terry et al., 2007; Jiang et al., 2010) and in vitro (Gearhart et al., 2007; Grigoryan et al., 2009b; Grigoryan and Lockridge, 2009). In glial cells, 24 h exposure to CPF or CPO suppresses extension outgrowth in differentiating C6 cells (Sachana et al., 2008). CPO had a stronger morphological effect than CPF that has been associated with a significant decrease in the levels of tubulin and MAP1B (Sachana et al., 2008). Similarly, only the in vivo metabolite of DZ, DZO, triggered inhibition of the development of C6 cell extensions, an effect linked also to the reduction in the levels of tubulin (Sidiropoulou et al., 2009b). Immunofluorescence staining revealed normal MT networks in control and CPFtreated cells and, although there was no evidence for a major collapse of the MT network, there were increased levels of localized patchy staining compared to the control, particularly in CPO-treated cells (Sachana et al., 2008).

Effects of Heavy Metals on Microtubules MT proteins have also been investigated in relation to methylmercuryeinduced developmental abnormalities of the nervous system. Exposure of N2a neuroblastoma cells to methylmercury revealed significant disruption in MT organization after staining the cells with antibody that recognizes b-tubulin (Kromidas et al., 1990). The same research group further emphasized the predominant effect of methylmercury on MTs compared to IFs and MFs by using scanning electron microscopy (Trombetta and Kromidas, 1992). In the same cell line, cells demonstrated decreased reactivity against C-terminally tyrosinated a-tubulin following only 4 h exposure to a sublethal concentration of methylmercury chloride compared to controls, which was associated with inhibition of neurite outgrowth (Lawton et al., 2007). The importance of the cytoskeleton in methylmercury neurotoxicity was further emphasized in a study by Castoldi et al. (2000) using primary cultures. In this study, rat cerebellar granule cells exhibited MT depolymerization within 1.5 h of exposure to 1 mM methylmercury and long before disturbance of neurite processes (Castoldi et al., 2000). Methylmercury was found to suppress tubulin polymerization in vitro, to disrupt the MT network, and to reduce tubulin synthesis in mouse glioma cells (Miura et al., 1984; Miura and Imura, 1987). This corresponded

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to a decrease in tubulin mRNA levels but no effect on the transcription rates of b-tubulin genes were found, suggesting that exposure had disrupted the autoregulatory control of tubulin synthesis in a manner similar to that described for the antimitotic agent colchicine (Miura et al., 1998). It is well documented that lead exposure can cause learning and memory impairment, particularly in developing organisms. However, the molecular mechanisms are not fully understood, although several studies investigated the potential role of MTs in disruption of memory formation. In human primary cultures, exposure to biologically relevant concentrations of lead (5, 10, 20, and 40 mg/dL) was associated with hyperphosphorylation of tau protein, as determined by Western blotting and immunocytochemistry, due to upregulation of protein phosphatases (Rahman et al., 2011). These findings were also confirmed in both Wistar rat and mouse pups, emphasizing the importance of tau hyperphosphorylation in cognitive impairment (Li et al., 2010; Rahman et al., 2012). Recent data further support this as the pre- and neonatal exposure to lead causes a significant increase in the phosphorylation of tau and upregulates tau protein level in the rat brain cortex and cerebellum (Gąssowska et al., 2016). In a study by Scortegagna et al. (1998), the detectable levels of MAP-2b and MAP-2c were found to decrease 24 h after a 3 h exposure to 3 or 6 mM lead in serumfree medium maintained E14 mesencephalic rat primary cultures. However, in the same study, these protein levels remained similar to controls in serum-cultured cells, suggesting that a serum factor prevents cytoskeletal changes otherwise noted in this primary culture containing differentiating neurons and proliferating astrocytes (Scortegagna et al., 1998). On the other hand, lead (II) acetate reduced the number of MAP-2 stained cells and the mRNA levels of MAP-2 in a concentration-dependent manner, by applying default differentiation of mESCs (Beak et al., 2011; Visan et al., 2012). Inorganic lead had no effect on the in vitro assembly of MTs from porcine brain, whereas trimethyl lead blocked and completely inhibited MT assembly at 300 mM, as monitored by turbidity measurements and electron microscopy (Roderer and Doenges, 1983). In contrast, triethyl lead chloride inhibited MT assembly and depolymerized preformed MTs in porcine brain preparations (Zimmermann et al., 1988), whereas the same research group reported no change in MT network in mouse N2a neuroblastoma cells after exposure to the same organic lead compound (Zimmermann et al., 1987). Regarding arsenic, it was shown that tau gene expression can be increased in ST-8814 schwannoma and SK-N-SH neuroblastoma cell lines by exposure to inorganic trivalent and monomethyl pentavalent

IX. SPECIAL TOPICS

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CYTOSKELETAL BIOMARKERS OF DEVELOPMENTAL NEUROTOXICITY

arsenic metabolites, respectively (Vahidnia et al., 2007b). In addition, the phosphorylated state of MAPtau has been found altered in both in vitro and in vivo studies (Vahidnia et al., 2007a). More specifically, Giasson et al. (2002) reported hyperphosphorylation of tauprotein in Chinese hamster ovary cells after treatment with inorganic trivalent arsenic. Similarly, subchronic exposure of rats to this arsenic metabolite increased the phosphorylation of tau in sciatic nerves (Vahidnia et al., 2008a). However, a more recent study shows that sodium arsenite causes neurite inhibition in N2a cells that was associated with alterations in cytoskeletal proteins. More specifically, sodium arsenite decreased the mRNA levels of tau and tubulin in a dosedependent manner but had no significant effect on the mRNA levels of MAP-2 (Aung et al., 2013). The inhibitory effect of arsenic trioxide on the migration of primary neurons established from the brains of neonatal rats has also been recently investigated; the study revealed that it was associated with a decrease in the protein levels of doublecortin, which is a MTassociated protein expressed during the neuronal migration (Zhou et al., 2015).

Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Microtubules Ethanol is the only organic solvent studied in relation to its neurotoxic potential against MTs in in vitro models. Ethanol had no effect on the rate and extent of bovine tubulin polymerization in vitro, whereas the ethanol metabolite acetaldehyde inhibited MT formation (Jennett et al., 1980). Similarly, McKinnon et al. (1987) demonstrated that acetaldehyde had an inhibitory effect on the polymerization of MT protein derived from calf brains, further emphasizing the acetaldehydemediated alteration of cytoskeletal MTs. Continuous exposure of developing neural crest cells to ethanol has also been found to cause MT disruption (Hassler and Moran, 1986). In PC12 cells, chronic exposure to ethanol led to alterations in the balance between free tubulin in the cytoplasm and tubulin polymerized into MTs, enhancing the content of the latter, possibly through phosphorylation (Reiter-Funk and Dohrman, 2005). In contrast, chronic alcohol exposure was found to decrease the levels of polymerized tubulin in cultured hippocampal neurons and simultaneously to reduce the amount of MTs and the levels of MAP-2 in dendrites (Romero et al., 2010). The same research group also described impairment of MT dynamics and reassembly in primary cultures of rat astrocytes treated with ethanol (Tomas et al., 2003). However, in a short-term exposure of rat C6 glioma cells to acute levels of ethanol (50, 100, and

200 mM), there was no detectable change in the MT network (Loureiro et al., 2011). In whole cerebral hemisphere model, developing Layer 6 neurons exposed to ethanol exhibited diminished MAP-2 levels in dendritic processes (Powrozek and Olson, 2012). Similarly, MAP-2 immunostaining intensity was reduced in hippocampal slice cultures from neonatal Wistar rats subjected to ethanol treatment (200 mM) for up to 4 weeks (Noraberg and Zimmer, 1998). MAP-2 immunolabeling has also been used to determine the effect of ethanol (70 mM) on dendrites of rat embryonic hippocampal pyramidal neurons in culture, revealing decreases in both length and number of dendrites compared with controls (Lindsley et al., 2002; Lindsley and Clarke, 2004). Stimulation of MAP-2 phosphorylation has been detected in MT preparations from rat brain exposed to low and biologically relevant doses of ethanol (6, 12, and 24 mM), whereas higher doses (48, 96, 384, and 768 mM) decreased phosphorylation (Ahluwalia et al., 2000). In the same study, MAP-1 was found to show increased phosphorylation with only 12 and 24 mM of ethanol and tubulin only from the lower dose tested (6 mM) (Ahluwalia et al., 2000). More recently, the application of neural stem cell technology indicated disturbance of neuronal differentiation from noncytotoxic concentrations of alcohol (25e100 mM), as recorded by reduced immunostaining and levels of MAP-2 protein (Tateno et al., 2005). Immunolabeling of axons derived from rat hippocampal pyramidal neurons with a bIII-tubulin antibody was used to assess the effect of ethanol on the length of axons (VanDemark et al., 2009). Indeed, ethanol alone had no effect on axon length, whereas carbachol-treated cells in the presence of ethanol (50 and 75 mM) did cause shortening of axons compared to controls (VanDemark et al., 2009). In the P7 rodent model, which is extensively used for mechanistic elucidation of ethanol-induced neurodevelopmental toxicity, it was found that ethanol elevated the phosphorylation state of tau, as demonstrated by two different phospho-specific antibodies (Saito et al., 2010). Similarly, in a human neuroblastoma cell line after tau induction, ethanol caused a dose-dependent increase in tau levels and cell mortality (Gendron et al., 2008). A more extensive overview on the effects of ethanol on the neuronal cytoskeleton, covering both in vivo and in vitro studies, can be found in Evrard and Brusco (2011).

MICROFILAMENTS MFs are formed by the polymerization of monomeric G-actin into filaments with a diameter of 5 nm (Dominguez and Holmes, 2011). MF assembly is

IX. SPECIAL TOPICS

MICROFILAMENTS

ATP-dependent, and the dynamic properties of MFs are dependent on the hydrolysis of actin-bound ATP following incorporation at the filament plus end (Carlier, 1998). MF dynamics are also influenced by a wide variety of actin binding proteins (ABPs) that regulate its organization and function by acting as either nucleating factors (e.g., ARP 2/3, formin), crosslinking proteins (e.g., fascin), destabilizing factors (e.g., ADF/cofilin, gelsolin, fragmin), membrane cross-linkers (e.g., spectrin, GAP-43), or MFassociated motor proteins (e.g., myosin) (Ishikawa and Kohama, 2007; Dominguez, 2009; Lee and Dominguez, 2010; Dominguez and Holmes, 2011; Jansen et al., 2011; Svitkina, 2018). The interaction between actin and its ABPs is in turn regulated by cell signaling pathways (Endo et al., 2003; Ishikawa and Kohama, 2007). MFs play key roles in mitosis, neural cell differentiation, and the regulation of cell migration (Gungabissoon and Bamburg, 2003; Kunda and Baum, 2009). The inhibition of neurite outgrowth from explants of embryonic chick spinal cord cultured in the presence of the MT-stabilizing agent taxol and the MF-disrupting agent cytochalasin D clearly demonstrates the importance of both MT and MF integrity in the developmentally important process of neurite outgrowth (Ro¨sner and Vacun, 1997). Given the important roles played by MFs in cytokinesis, receptor trafficking, and neurite outgrowth, their disruption by toxin exposure could have major effects on neural development.

Effects of Organophosphorus Esters on Microfilaments In early work, Carlson and Ehrich (2001) tested the ability of several OPs including paraoxon, parathion, diisopropyl fluorophosphate (DFP), phenyl saligenin phosphate (PSP), triorthotolyl phosphate (TOTP), and triphenyl phosphite at 0.1e1 mM to disrupt the filamentous actin (F-actin) network in mitotic SH-SY5Y cells using a fluorescently labeled phalloidin probe. Significant decreases in the levels of F-actin were observed within 30 min exposure of cells treated with PSP and TOTP, whereas other OPs required longer exposure times and DFP had no observable effect. The data clearly support the idea that some OPs can disrupt the MF network, although the concentrations used were relatively high and cytotoxic within a few hours, as determined by protein assay (Carlson and Ehrich, 2001). However, proteomic studies on differentiating N2a neuroblastoma cells exposed to sublethal neurite inhibitory concentrations of the OP pesticide DZ showed that inhibition of neurite outgrowth by 10 mM DZ was associated with increased levels but decreased LIM kinaseemediated

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phosphorylation of the actin-destabilizing protein cofilin (Harris et al., 2009a). Although total actin levels were unaffected by DZ, the altered levels and phosphorylation status of cofilin, together with reduced staining intensity of neurites with antiactin antibody, suggest that DZ exposure leads to a reduction in the levels of F-actin in neurites due to disruption of MF dynamics caused by altered expression and phosphorylation of cofilin. Another important modulator of MF organization in the axonal growth cone of developing neurons is growth associated protein 43 (GAP43), which has been shown to exhibit reduced mRNA and/or protein levels following exposure to sublethal neurite outgrowth inhibitory concentrations of OPs (Sachana et al., 2003; Flaskos et al., 2011; Ta et al., 2014). A transient reduction in GAP43 protein levels was also observed to be associated with the retraction of neurites in predifferentiated N2a cells exposed to sublethal concentrations of CPF and CPO (Sindi et al., 2016). It has been suggested that these phenomena may, at least in part, be the result of upstream events such as OP-induced autophagy or disruption of Ca2þ homeostasis, which could impact on the activity of Ca2þ-dependent ABPs and other MF-regulatory proteins (Chen et al., 2013; Fernandes et al., 2017). Thus, OPs can disrupt MF organization and functions by interacting with ABPs and/or signaling pathways that modulate MF dynamics. However, in vitro studies by Grigoryan et al. (2009a) and Schopfer et al. (2010) demonstrated a covalent interaction of OPs with lysine and tyrosine residues on actin, raising the possibility that a direct interaction with actin might also be involved in the disruption of MFs by OPs. Further in vivo and in vitro studies to determine the role of OP-actin adduct formation would be worthwhile.

Effects of Heavy Metals on Microfilaments Many cytoskeleton-related developmental neurotoxicity studies with heavy metals have focused on the MT network. However, micromolar concentrations of mercurial compounds can block SH groups on purified actin, thereby inhibiting its ability to interact with myosin and induce myosin ATPase activity in vitro (Perry and Cotterill, 1964; Martinez-Neira et al., 2005). The ability of mercury (as well as cadmium, copper, and zinc) ions to interfere with actineABP interactions has also been demonstrated by native gel electrophoresis (Kekic and dos Remedios, 1999), suggesting that direct binding of heavy metal ions to actin, ABPs, or other MF-regulatory proteins could induce toxic effects directed at the MF network. Studies on the inhibition of glioma cell migration by arsenic suggested that this heavy metal compound may be able to disrupt F-actin by interference with

IX. SPECIAL TOPICS

1038

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CYTOSKELETAL BIOMARKERS OF DEVELOPMENTAL NEUROTOXICITY

MF-regulatory cell signaling pathways such as protein kinase C (Lin et al., 2008). Sodium arsenite was also observed to inhibit neurite outgrowth in differentiating N2a neuroblastoma cells; although this was not found to involve alterations in mRNA levels for b-actin (Aung et al., 2013), the possibility that changes to MF dynamics may involve alterations to the levels and posttranslational modification of ABPs or other regulatory pathways cannot be ruled out and warrants further investigation. Changes were observed in the levels of mRNA for NFL and NFM (both elevated) and for tubulin and tau (both reduced) in arsenic-treated cells; the possibility that these changes, if they are reflected at the protein level, may cause disruption of F-actin organization could also be further investigated. In a study of the effects of methylmercury chloride on postnatal rat brain development, it was found that the degradation of the ABP a-spectrin by m-calpain was significantly greater in cerebral cortex extracts from treated animals than in controls at postnatal day 16 (Zhang et al., 2003). Calpain activation could also target a range of other cytoskeletal proteins that act as substrates for calpain. In this respect it is interesting to note that exposure to other metal compounds has also been linked to the activation of calpain in brain (Zhang et al., 2012), neural cells (Vahidnia et al., 2008b; Rocha et al., 2011), or in other tissues (Lee et al., 2007). Furthermore, studies in primary cultures of fetal mouse brainederived cerebellar granule cells and human placental tissue showed that submicromolar levels of methylmercury chloride induced a significant decrease in the phosphorylation of the ABP cofilin and the translocation of actin and cofilin to mitochondria (Vendrell et al., 2010; Caballero et al., 2017). The fact that these changes were associated with elevated levels of protein carbonylation (detected by immunoassay) and could be blocked by cotreatment with the antioxidant probucol, suggest that they were triggered by elevated levels of protein oxidation (Caballero et al., 2017). Moreover, evidence for the inhibition of both neurite outgrowth and neuronal cell migration via disruption of MFs through altered regulatory signaling pathways comes from a combination of cell culture and in vivo developing rodent brain studies following exposure to methylmercury chloride (Fujimura and Usuki, 2012; Usuki and Fujimura, 2012; Guo et al., 2013; Herna´ndez et al., 2018). Thus, direct binding to SH groups, elevated protein oxidation, and disruption of MF-regulatory signaling pathways have a major impact on the regulation of MF dynamics, suggesting that exposure to heavy metal compounds can disrupt the regulation of MF dynamics in a variety of ways.

Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Microfilaments Chronic ethanol exposure has been shown to be associated with disruption of the MF network in cultured PC12 cells and in primary cultures of hippocampal neurons. In PC12 cells, chronic exposure was found to reduce dopamine release via protein kinase Ce dependent pathways, an effect that was attenuated by cotreatment with the MF-disrupting agent cytochalasin, indicating the need for MFs in this exocytotic process (Funk and Dohrman, 2007). In ethanol-exposed hippocampal neurons, a reduction was observed in the levels of F-actin compared to untreated control cell cultures, as determined by FITC-labeled phalloidin staining, which corresponded to reduced protein levels of total Rac1, RhoA, and cdc42 (small GTPases known to be involved in the regulation of MF assembly and dynamics), as determined by G-LISA assays (Romero et al., 2010). However, although the levels of activated (GTPbound) forms of Rac1 and cdc42 were reduced, ethanol had no significant effect on the levels of active RhoA, suggesting that only the inactive form of this GTPase was downregulated by ethanol treatment. Western blotting analysis indicated that the levels of total cofilin (which destabilizes MFs) were unchanged, although the levels of inactive (phospho-) cofilin were not assessed (Romero et al., 2010). Chronic exposure to ethanol was also found to inhibit endocytotic uptake of serum albumin and transferrin by cultured fetal rat hippocampal neurons (Marı´n et al., 2010) This effect was associated not only with altered levels of proteins involved in vesicle formation and docking but also with proteins involved in the regulation of MF assembly and dynamics, including Arf6 (upregulated), cdc42, and RhoA (both downregulated). Furthermore, the ability of ethanol to inhibit neural cell differentiation in a human embryonic stem cell model of early brain development was also associated with disruption of the MF network (Tale´ns-Visconti et al., 2011). In a study of chronic ethanol exposure on cultured rat astrocytes, the impairment of glucose uptake was associated with disassembly of actin stress fibers; the fact that lysophosphatidic acid attenuated these effects by stabilizing the MF network suggested that a major disruption of MF dynamics was caused by ethanol exposure (Tomas et al., 2003). Although there was no detectable change in the MT network following short-term exposure of rat C6 glioma cells to acute levels of ethanol, the resultant formation of reactive oxygen species was associated with major disruption of the MF network (Loureiro et al., 2011). Taken together, these data illustrate not only the fact that MFs are disrupted by this

IX. SPECIAL TOPICS

INTERMEDIATE FILAMENTS

solvent but also that MF-dependent processes of importance in neural development are impaired when this occurs.

INTERMEDIATE FILAMENTS The most abundant IF proteins found in differentiating and mature neurons are the three neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H), whereas in glial cells the most important IF protein is GFAP. Nestin is the main IF specifically expressed in immature neural cells. Indeed, most of the genes coding for IF proteins are expressed in a tissue- or cell typeespecific manner, except for the nuclear lamins. Thus, the NF proteins are present only in neurons and GFAP only in glia (and more specifically in astrocytes). As a result, NF proteins and GFAP have been widely used in neurotoxicology as markers for specific effects on neurons and glia, respectively. In the context of developmental toxicology, changes in NF proteins and GFAP have been commonly employed as a measure of the capacity of a toxicant to interfere with neuronal and glial differentiation. Although NFs are typically considered to be more stable than MFs and MTs, their dynamic capability is demonstrated by their reorganization that occurs during several neurodevelopmental stages including proliferation (mitosis), apoptosis, and axonogenesis (Omary et al., 2006). NFs can facilitate axonal elongation by stabilizing the axonal cytoskeleton and inhibiting the retraction of long axons (Lariviere and Julien, 2003). Apart from their use in developmental neurotoxicity studies as a neuronal differentiation marker, NF proteins may constitute a mechanistically relevant marker for assessing specific biochemical cytoskeletal effects in some cases. This is particularly true for the studies on OPs and arsenic discussed in the following paragraphs, where NFs have been proposed to be a direct target for these neurotoxicants. NF parameters assessed in neurodevelopmental toxicity studies include NF protein levels, distribution, assembly, transport, phosphorylation, and expression of NF genes. A review of the available data on established developmental neurotoxicants (see the following) indicates that in most cases there is a decrease in the levels of at least one of the three NF proteins, whereas NF gene expression data are inconsistent, with both increases and decreases in NF mRNA levels caused by the toxicants. The distinct features of the three NF proteins in terms of structure, properties, and function and their differential expression during neuronal development imply that assessment of toxicant-induced changes in one NF protein cannot substitute for measurements in the other two. Finally, because NF protein phosphorylation is known to be the major factor in the

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regulation of the dynamics and function of NFs (Omary et al., 2006; Sihag et al., 2007), assessment of changes in NF phosphorylation-related parameters (determination of relevant kinases and upstream cell signaling molecules) may provide valuable markers for developmental neurotoxicity. In developmental neurotoxicity studies both protein and mRNA levels of GFAP have been assessed. These studies have usually reported decreases in these parameters, indicating specific repression of glial cell differentiation, whereas any increases obtained have been attributed to reactive gliosis because of high dosing and primary damage to neurons.

Effects of Organophosphorus Esters on Intermediate Filaments NF parameters have been assessed to a larger extent in toxicological studies involving OP pesticides than other developmental neurotoxicants. This may be partly because of the prior existence of data implicating NF (and other cytoskeletal) abnormalities as being etiologically important in delayed OP neurotoxicity (Abou-Donia, 1993; Jiang et al., 2010). In this context, in some OP neurodevelopmental studies NF parameters have not been employed as a common marker for neuronal cell-specific differentiation but as a mechanistically relevant marker for specific biochemical effects on the neuronal cytoskeleton. Parameters assessed in OP neurodevelopmental studies include the levels and intracellular distribution and posttranslational modification (phosphorylation) of NF proteins and the expression of NF genes. Exposure of both mitotic and differentiating rat PC12 pheochromocytoma cells to a sub-lethal concentration of CPF (30 mM), led to the upregulation of nfl and nef3 genes (which encode NF-L and NF-M, respectively), whereas expression of the nefh gene (which encodes NF-H) was unaffected (Slotkin and Seidler, 2009). On the other hand, exposure of PC12 cells to 30 mM DZ under the same conditions caused upregulation of the nef3 but had no effect on the expression of nfl and nefh genes (Slotkin and Seidler, 2009). The expression of genes encoding NF-L and NF-H, used as a marker for neuronal differentiation, was studied in primary neuronal cultures of cerebellar granule cells prepared from 7-dayold rat pups treated with parathion (Bal-Price et al., 2010). Exposure to this OP, used at concentrations of 10e50 mM, for up to 12 days caused a concentrationdependent decrease in mRNA levels for both NF-L and NF-H. A series of studies have assessed changes in several NF parameters following exposure of differentiating mouse N2a neuroblastoma cells to CPF and DZ, the two OPs for which there is the most evidence for

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CYTOSKELETAL BIOMARKERS OF DEVELOPMENTAL NEUROTOXICITY

developmental neurotoxicity. In these mechanistic studies of OP-induced developmental neurotoxicity, determination of NF (and other cytoskeletal) parameters were extended to include the influence of CPO and DZO, the two in vivo oxon metabolites of CPF and DZ, because there is now considerable evidence to suggest that these compounds can interfere with neuronal differentiation and development (Flaskos, 2012). These studies have demonstrated decreases in the levels of NF-H protein by both CPF and DZ. Thus, exposure of N2a cells to 3 mM CPF for 8 h (Sachana et al., 2001) and to 10 mM DZ for 24 h (Flaskos et al., 2007) leads to reduced NF-H protein levels. Indirect immunofluorescence studies also showed that, apart from an alteration in the expression of NF-H, there was a change in the intracellular distribution of this protein, with NF-H located mainly in cell body aggregates of DZ-treated N2a cells (Flaskos et al., 2007). NF parameters were also affected by exposure to CPO and DZO. For example, at a concentration of 10 mM, CPO exposure for 24 h reduced the total levels of NFH and disrupted NF-H intracellular distribution in differentiating N2a cells (Flaskos et al., 2011). On the other hand, CPO had no effect on the levels of phosphorylated NF-H under these experimental conditions, in which the OP was added at the point of induction of cell differentiation. However, in N2a cells induced to differentiate for 20 h prior to OP exposure, CPO induced a transient increase in ERK 1/2 activation and NF-H phosphorylation after 2 h, which preceded neurite retraction (Sindi et al., 2016), suggesting a mechanistic link between NF-H hyperphosphorylation and neurite destabilization. In contrast, the oxon metabolite DZO, applied at concentrations of 5 and 10 mM for 24 h, had no effect on total NF-H levels, but increased phosphorylated NF-H levels in differentiating N2a cells compared to the control (Sidiropoulou et al., 2009a). Under these conditions, DZO had no effect on the total levels of NF-L and NF-M (Sachana et al., 2014). Further work is required to determine the molecular basis of these effects in more detail. Determination of GFAP in neurodevelopmental studies of OPs has involved measurement of both its protein and mRNA levels. Following developmental exposure to OPs, GFAP protein and mRNA levels exhibited both decreases and increases, the latter being attributed to the occurrence of reactive gliosis because of high dosing. Thus, in aggregating brain cells of fetal rat telencephalon, GFAP levels were found to be increased following parathion treatment, indicative of gliosis (Zurich et al., 2000). Postnatal exposure of rats to CPF for 4 days initially decreased GFAP levels, indicating specific repression of normal glial (astrocytic) development. At a later stage, however, increases in the levels of GFAP occurred, typical of reactive gliosis

following neuronal cell damage (Garcia et al., 2002). Postnatal administration of CPF to rats for 6 days at doses high enough to cause significant cholinesterase inhibition also led to increased GFAP mRNA levels, reflecting increased astrocyte reactivity (Betancourt et al., 2006). On the other hand, administration of DZ to neonatal rats for 4 days resulted in a decrease in the expression of the gene coding for GFAP (Slotkin and Seidler, 2007). Similarly, the in vivo metabolite of DZ, DZO (at 1e10 mM) caused, after 24 h in N2a cells, a reduction in GFAP protein levels, indicating repression of specific glial cell/astrocytic differentiation (Sidiropoulou et al., 2009b).

Effects of Heavy Metals on Intermediate Filaments Both NF-L and NF-M levels were found to be altered in a number of mammalian cell lines after exposure to mercuric oxide. Thus, exposure of differentiating human SK-N-SH neuroblastoma cells for 6 days to mercuric oxide decreased particularly NF-L levels (Abdulla et al., 1995). Because this effect correlated well with effects on neurite outgrowth, it was suggested that determination of NF-L might afford a rapid measure of effects on neuronal differentiation. More recently, methylmercury exposure was also found to decrease the mRNA levels for NF-L and NF-H in primary cultures of rat cerebellar granule cells, whereas GFAP mRNA expression was unaffected (Hogberg et al., 2010). NF organization was disrupted following exposure of N2a cells to triethyl lead (Zimmermann et al., 1987). NF assembly was also disrupted. These effects led to suggestions that interaction of triethyl lead with NFs may be responsible for triethyl lead neurotoxicity in vivo. In addition, in rats exposed for 13 weeks to lead acetate, transport of NF proteins was retarded, indicating impairment of slow axonal transport (Yokoyama and Araki, 1992). Apart from NF organization, assembly, and transport, NF protein phosphorylation was also affected by lead; exposure of mice to lead acetate throughout gestation and postnatally led to increased phosphorylation of both NF-M and NF-H in auditory brainstem nuclei (Jones et al., 2008). Lead also affects GFAP, as suggested by decreased GFAP expression in four human and two rat glioma cell lines, indicating interference with glial cell differentiation (Stark et al., 1992). The mRNA levels for GFAP were also reduced after lead treatment of primary cultures of rat cerebellar granule cells for 12 days (Bal-Price et al., 2010). In neurotoxicity studies involving arsenic, NF parameters constitute a mechanistically relevant marker because NFs (and other cytoskeletal elements) have been suggested to represent a possible target in arsenic neuropathy. Although these studies have been not

IX. SPECIAL TOPICS

CONCLUDING REMARKS AND FUTURE DIRECTIONS

always carried out in a developmental context, the known significance of NFs in neurodevelopment implies that any neurotoxic effects on NFs obtained in adult animals or differentiated neurons may be of potential relevance in neurodevelopment. Both acute (Vahidnia et al., 2006) and subchronic (Vahidnia et al., 2008a) administration of arsenite to adult rats induced a reduction in NF-L levels in the sciatic nerve. In contrast, NF-M and NF-H expressions remained unchanged. In addition, NF-L was found to be hyperphosphorylated (Vahidnia et al., 2008a). These NF-L changes have been proposed to contribute to the disruption of the NF network, ultimately leading (in combination with other cytoskeletal changes) to the axonal degeneration seen in arsenic neuropathy (Vahidnia et al., 2007a, 2008a). However, in a study by the same group involving cell lines and assessment of NF gene expression, the results obtained were not compatible with the above data. In this case, exposure of SK-N-SH neuroblastoma and ST-8814 schwannoma cells to arsenite for up to 48 h had no effect on the expression of genes coding for NF-L and NF-M (Vahidnia et al., 2007b). However, the metabolites monomethyl- and dimethyl-arsenic induced alterations in the expression of NF genes in both cell lines and particularly in the expression of the gene coding for NF-H. Mechanistically important NF parameters assessed under the influence of arsenite also included axonal transport and phosphorylated NF distribution. For example, exposure of differentiated mouse NB2/dl neuroblastoma cells and dorsal root ganglion neurons cultured from embryonic day 12 chicks to arsenite decreased NF transport into axons and caused accumulation of phosphorylated NFs in the perikaryon leading to changes in NF dynamics that may contribute to arsenic neuropathy (De Furia and Shea, 2007).

Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Intermediate Filaments Ethanol exposure inhibited the expression of NF proteins in N2a cells, indicating disruption of neuronal differentiation (Chen et al., 2009). In contrast, at a concentration of 100 mM, ethanol induced an increase in GFAP levels in differentiating neural stem cells, which was thought to imply increased glial differentiation as a compensatory mechanism to repair the impaired neuronal differentiation (Tateno et al., 2005). However, exposure to environmentally relevant low levels of toluene (down to 0.2 ppb), decreased GFAP levels during differentiation of mouse embryo cells into an astrocytic lineage in serum-free medium (Yamaguchi et al., 2002, 2003).

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In a study that adopted a proteomic approach, exposure of neonatal mice to 2,20 ,4,40 ,5-pentabromodiphenyl ether (PBDE 99) induced significant alterations in several cytoskeletal and other proteins in the cerebral cortex. However, one of the greatest changes noted was an increase in the levels of NF-L (Alm et al., 2008).

CONCLUDING REMARKS AND FUTURE DIRECTIONS It is well established that the cytoskeleton plays a key role in a range of cellular processes involved in neural development. From the mechanistic studies of developmental neurotoxicity to date, there is now a significant body of evidence pointing to the disruption of one or more of the cytoskeletal networks following exposure to a range of developmental neurotoxicants, with changes at the protein level currently being more consistent than those at the level of gene expression. Taken together, the findings from studies discussed in this chapter strongly suggest that cytoskeletal disruption is a common feature of adverse outcome pathways associated with chronic exposure to many developmental neurotoxicants. Thus, despite the diversity of molecular initiating events associated with exposure to different developmental neurotoxins, subsequent molecular changes invariably converge on pathways that regulate the cytoskeleton, causing cytoskeletal disruption. In some cases, the molecular initiating event may be direct binding to cytoskeletal proteins themselves. A schematic view of cytoskeletal disruption as a convergence point in developmental neurotoxicity is summarized in Fig. 58.1. Further work to characterize the ability of wellestablished developmental neurotoxins to disrupt cytoskeletal elements would be worthwhile, as this would help to identify the key events in each adverse outcome pathway in more detail. This could, for example, involve the study of upstream events such as kinase/phosphatase activities in cases where the phosphorylation status of cytoskeletal proteins is disrupted, or RT-PCR and/or proteolytic enzymes (calpain, proteasomes, etc.) cases where protein levels are significantly affected by exposure to toxin. The monitoring of these molecular events in high throughput screening platforms would help to establish a more comprehensive battery of rapid tests. In summary, the monitoring of cytoskeletal disruption is an integral part of mechanistic studies of developmental neurotoxicity and is becoming an increasingly important component in a battery of in vitro tests to rapidly screen large numbers of compounds for their ability to induce developmental neurotoxicity.

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CYTOSKELETAL BIOMARKERS OF DEVELOPMENTAL NEUROTOXICITY

Cytoskeletal disruption as a convergence point in developmental neurotoxicity pathways Developmental neurotoxicants Mitochondrial dysfunction Elevated ROS Multiple molecular initiating events

CYTOSKELETAL DISRUPTION

Disrupted Ca2+ homeostasis Disruption of cell signaling

Impairment of cytoskeleton-mediated processes:

Direct binding to cytoskeletal proteins

Intracellular transport Membrane trafficking Neurite outgrowth Synaptogenesis Mitosis Cell migration

FIGURE 58.1 Simplified schematic representation of the involvement of cytoskeletal disruption in developmental neurotoxicity. Developmental neurotoxicants may act via many different molecular initiating events, such as direct binding to membrane or nuclear receptors and disruption of gene expression patterns. They may also induce the generation of increased ROS and/or cause disruption of mitochondrial function, both of which could interfere with a range of cellular activities, including intracellular transport, protein folding, and degradation. Similarly, the disruption of Ca2þ homeostasis could interfere with a host of Ca2þ dependent activities, including cell signaling pathways, proteolytic degradation (e.g., by calpain), and cytoskeletal dynamics. Finally, direct binding of toxins to cytoskeletal proteins could induce conformational changes that affect subunit assembly and/or the interaction of subunits with accessory proteins that regulate cytoskeletal dynamics.

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ventral hippocampus after acute lead exposure. Exp. Toxicol. Pathol. 64, 619e624. Zhang, J., Miyamoto, K., Hashioka, S., 2003. Activation of calpain in developing cortical neurons following methylmercury treatment. Dev. Brain Res. 142, 105e110. Zhou, H., Liu, Y., Tan, X.J., et al., 2015. Inhibitory effect of arsenic trioxide on neuronal migration in vitro and its potential molecular mechanism. Environ. Toxicol. Pharmacol. 40, 671e677.

Zimmermann, H.-P., Plagens, U., Traub, P., 1987. Influence of triethyl lead on neurofilaments in vivo and in vitro. Neurotoxicology 8, 569e578. Zimmermann, H.-P., Faulstich, H., Hansch, G.M., et al., 1988. The interaction of triethyl lead with tubulin and microtubules. Mutat. Res. 201, 293e302. Zurich, M.G., Honegger, P., Schilter, B., et al., 2000. Use of aggregating brain cell cultures to study developmental effects of organophosphorus insecticides. Neurotoxicology 21, 599e605.

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59 MicroRNA Expression as an Indicator of Tissue Toxicity and a Biomarker in Disease and Drug-Induced Toxicological Evaluation Gopala Krishna1, Saurabh Chatterjee2, Priya A. Krishna1, Ratanesh Kumar Seth2 1

Nonclinical Consultants, Ellicott City, MD, United States; 2Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC, United States

INTRODUCTION Noncoding RNA sequences, termed microRNAs (or miRNAs or miRs), have been shown to posttranscriptionally regulate messenger RNA (mRNA). They contain a seed sequence (positions 2e7) that binds to the complementary sequence of mRNA causing an alteration in gene expression. miRNAs are short (w22 nucleotides) and relatively stable. Studies have shown their presence in plasma with a half-life of hours to days (Creemers et al., 2012). Because of their inherent stability and ease of sampling, the number of studies evaluating miRNAs as potential biomarkers both for disease- and drug-induced states has markedly increased. For a biomarker to be successfully utilized, it must satisfy a number of criteria such as ease of sampling, rapid detection, injury or disease sensitivity and specificity; discrimination between variations in the injury or disease; and stability within the biological matrix (Etheridge et al., 2011). miRNAs fulfill a number of these requirements. Much effort has been spent in making correlations between various disease states and miRNA levels in both biological fluids and tissues (Mikaelian et al., 2013). These efforts have also been extended to include not just biomarker data in support of diagnosis but also more recently in the drug development process, where relationships between miRNA levels and toxicologic or pathologic signs (Wang et al., 2013) may be used to generate earlier safety data in advance of more expensive animal or clinical trials. Perhaps most importantly, Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00059-1

a biomarker must be associated with the biological mechanisms of a disease or treatment, and it must be possible to make a statistical correlation between the biomarker and the clinical effect. Although the bulk of data reported in the literature refers to the relationship between miRNAs and therapeutic effects, less has been published regarding the relationship between changes in miRNA expression following administration of xenobiotics and the utility these extracellular molecules may have a correlating drug effect with, for example, a toxicologic endpoint.

REGULATORY MECHANISMS OF MIRNA BIOGENESIS miRNAs are an important regulatory molecule in many biological processes and pathways. Being said that, the biogenesis and expression profile of the miRNAs are changed considerably in various diseases (O’Reilly, 2016). The miRNA profiling has been extensively studied in several diseases especially in cancer and the results of these studies indicate that miRNA expression profiling is an important tool for diagnostic and therapeutic strategy. Therefore, it is important to illustrate the molecular mechanism that regulates the miRNA expression, and it would allow an explanation of variation in protein coding genes (Gulyaeva and Kushlinskiy, 2016). The two steps in such regulation involve (1) transcriptional regulation and (2) posttranscriptional regulation (Fig. 59.1). Endogenous and/or xenobiotic

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FIGURE 59.1

Schematic representation of miRNA biosynthesis and regulation: transcription, cleavage, transport to cytoplasm, and targeting of the target gene mRNA 30 UTR (Winter et al., 2009).

stimulation either activates transcription factor (TF) binding to the promotor region to facilitate miRNA transcription or methylation of promotor region of intergenic or intragenic miRNA genes to inhibit miRNA transcription (Gulyaeva and Kushlinskiy, 2016). The posttranscription regulation represents changes in miRNA processing and stability. The transcript of intronic region of host gene or independent miRNAs gene is known as mirtrons or primary miRNAs (primiRNAs). The Pri-miRNA further processed by Drosha and DGCR8 into precursor miRNA (pre-miRNA) inside the nucleus. DGCR8 contains RNA binding domain and it binds with pri-miRNA to stabilize it and facilitate the cleavage by Drosha (Yeom et al., 2006). The pre-miRNA

was then exported to cytoplasm, where Dicer cleaved pre-miRNA to form mature miRNA (Merritt et al., 2010). Therefore, changes in miRNA profile at processing levels depend on availability and activity of these enzymes. The mature miRNA forms RNA-induced silencing complex (RISC), which binds with complementary sequence of mRNA and cleaves the mRNA using RNase or destabilize or inhibit translation of mRNA (Mendell and Olson, 2012; Vermeulen et al., 2005). The Ingenuity Pathway Analysis tool by Qiagen Inc. can precisely predict the target mRNA (Bam et al., 2017). In summary, the endogenous molecules such as hormones and cytokines, and exogenous molecules such as xenobiotics, can alter miRNA expression. These

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comprehensive mechanisms of miRNA regulation solely depend on cell type, physiological conditions, and external or internal factors (Gulyaeva and Kushlinskiy, 2016). Thus, it is clear that the study of miRNAs expression profile enables us for the diagnosis, prognosis, and treatment of a wide variety of diseases.

STANDARD TOXICITY MEASUREMENTS (TISSUE AND CIRCULATING BIOMARKERS) For many years, the standard continuum approach employed in the discovery and development of new chemical or molecular entities (NCEs or NMEs) has been to first screen large numbers of molecules using in vitro (biochemical and cell-based) assays followed by testing a smaller number of active compounds in preclinical efficacy, safety, and toxicity models. In general, it is only at the in vivo testing stage that biomarkers are given the attention they deserve. Within the drug development setting, it has long been standard practice to focus on easily accessible matrices, for example, blood, saliva, and urine, as potential reservoirs of information for drug safety and efficacy and as correlates for disease progression. It is perhaps interesting to note that although analytical technologies have advanced significantly in biomarker measurement (genomics, metabolomics, proteomics, etc.), the corresponding information technology tools required to process the large amounts of information generated have lagged (Starmans et al., 2012).

TOXICOLOGY Toxicological evaluations typically include body weights, clinical observations, food consumption, ophthalmologic examination, clinical pathology, body temperatures, electrocardiographic evaluation (large animals only), neurobehavioral evaluation (usually only done in rodents), toxicokinetic evaluation, organ weights, and gross and microscopic pathology of tissues. Several of the items listed above are typical standard biomarkers utilized both preclinically, as part of a drug development program, and clinically, for both injury and disease diagnosis and monitoring. Further details are highlighted below.

CLINICAL PATHOLOGY Overview The standard clinical pathology analyses routinely performed include hematology and clinical chemistry

evaluations and, less frequently, urine and coagulation analyses. The results of these noninvasive tests may suggest a pathology that impacts the function of various organs. Limitations exist in the sensitivity and specificity of these analyses and, as such, they are used primarily to screen for health problems. An advantage, however, is that information on general animal health may be gathered via a nonterminal bleed throughout the duration of a study.

HEMATOLOGY Hematological tests are used to examine the components of blood, specifically erythrocytes, leukocytes, and platelets, as well as specific parameters related to each cell type. The most common test is the complete blood count/hemogram, which is used as a broad screening panel that provides information such as, but not limited to, the erythrocyte: red blood cell count, hematocrit, hemoglobin, mean cell volume, and reticulocyte percentage, and count; leukocyte: count and differential; platelet: count and mean platelet volume. This information is used to screen for processes such as anemia, infectious or noninfectious inflammation, and thrombocytopenia or thrombocytosis. Microscopic examination of the peripheral blood may also be performed. This provides information regarding erythrocyte, leukocyte, and platelet number and morphology. Certain insults may result directly or indirectly in alterations in cellular morphology (e.g., Heinz body or eccentrocyte formation in erythrocytes resulting from oxidative damage) that may provide additional information regarding the pathologic process.

CLINICAL CHEMISTRY A variety of different serum clinical chemistries may be analyzed to provide an overview of the general health status of the individual including the electrolyte balance and the status of several organs. Interpretation of the quantitative levels of the individual analytes may be affected and complicated by factors such as the site of production (i.e., a single cell type/organ that is the origin vs. multiple potential cell types/organs) and physiological regulation critical for homeostasis, as well as potential sites of loss or processing by various organ systems. Generally, there is no single analyte that is specific in indicating the dysfunction of one organ system; as such, multiple different analytes are typically measured. For example, blood urea nitrogen (BUN) and creatinine classically increase with kidney failure (renal azotemia). In contrast, if there was a concern for

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hepatic toxicity, specific chemistry tests of interest would include analytes such as albumin, total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase, and bilirubin. Therefore, if the target organ of interest is known, it is possible to rank the chemistry analytes based on the degree of importance.

URINALYSIS Urine is produced by the kidney and is a means by which the body eliminates waste products and regulates fluid homeostasis. A complete urinalysis is composed of three types of examination (visual, chemical, and microscopic) during which the levels of various biochemical products and cellular components are determined. Therefore, a urinalysis may function as a screening or diagnostic procedure in the assessment of renal as well as various other metabolic functions. Currently, the chemical portion of urinalysis is semiquantitative and utilizes dry-chemistry methodology via a commercially manufactured reagent stick. Quantitative measurements of selected analytes may be performed using the Cobas c501 chemistry analyzer, a wet-chemistry based methodology.

COAGULATION TESTS Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen levels are assays commonly performed in various species. These tests are ex vivo screening measures used to assess coagulation. Multiple factors are involved in the cascade of reactions that result in clot formation in the PT and aPTT test(s). A significant decrease (w50% is remarkable. Arg is a safe and inexpensive intervention with narcotic-sparing effects that may be a beneficial adjunct to standard therapy for sickle cellerelated pain in children. A large multicenter trial is warranted to confirm these observations (Morris et al., 2013). Further studies are needed to confirm these observations. A recent systematic review showed that Arg (oral, topical or nasal), with or without hydroxyurea (HU), increased NO levels and improved patient clinical condition in sickle cell disease. The parameters shown to be improved were reduction in pain and opioid use [remarkably up to 50% reduction in hospitalizations and emergency visits (Morris et al., 2013)], improvement in heart rate indices, and healing of leg ulcers (topical use of butyrate Arg cured 30% of the patients at 12 weeks and 78% after 3 months). Because it is known that oral HU used for this disorder acts by multiple direct effects such as increasing the synthesis of fetal hemoglobin, reducing infraerythrocytes, and HbS polymerization in deoxygenation conditions, the number of neutrophils is decreased along with erythroid hydration and the expression of adhesion erythrocytes, and the synthesis of NO and its bioavailability is increased. However, being a chemotherapeutic agent HU is both cytotoxic and genotoxic, hence further strategies need to be explored to improve quality of life in these patients (Eleuterio et al., 2016). Citrulline and Cardiovascular Effects NO, synthesized by the endothelial cells using Arg as a substrate, contributes to regulation of vascular tone in humans that has been demonstrated by showing that inhibition of its synthesis results in significant vasoconstriction and impaired endothelial regulation of vascular tone in patients with systemic arterial hypertension (Cardillo and Panza, 1998). Thus, increasing Arg availability either by Arg therapy or arginase inhibition may provide therapeutic benefits in future patients with hypertension. NO activates sGC in smooth muscles, leading to an increase in intracellular cGMP, causing vasodilation. This process is essential for endothelial function, and disturbed NO production in the human endothelium is attributed to endothelial dysfunction.

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Citrulline in Hypertension It has been observed that hypertensive patients had a lower ArgeCit ratio than normotensive patients associated with NO deficiency, oxidative stress, increased ROS, and a disturbed constrictoredilator balance in the kidneys. Melatonin was found to exert an antihypertensive effect in young spontaneously hypertensive rats (SHRs) because of restoration of the NO pathway by reduction of plasma ADMA, preservation of renal Arg availability, and attenuation of oxidative stress (Kaore et al., 2013). Studies with both Cit and nitrates consumption significantly decreased diastolic blood pressure, both decreased vascular compliance but had no effect on either pulse pressure or rate-pressure product, whereas Cit also decreased systolic pressure. Cit significantly decreased ReR interval 9% at rest and increased heart rate in addition to significantly decreasing pulse transit duration by 6%. QRS duration decreased by 5% with reduction in ReR interval (this seems to be a direct result of the modest tachycardia caused by Cit). HR augmentation in response to exercise was approximately reduced to half in Cit-treated subjects, without the desensitization observed for glyceryltrinitrate (Alsop and Hauton, 2016). Hence, Cit shows benefit in human subjects for the improvement of oxygen delivery in heart failure. Another study for effective therapies for infants with forms of pulmonary hypertension that develop or persist beyond the first week of life needs to be worked out. Cit improves NO signaling and ameliorates pulmonary hypertension in newborn animal models. Hence, strategies that increase the supply and transport of Cit merit pursuit as novel approaches to managing infants with chronic, progressive pulmonary hypertension (Cit has proved better than Arg supplementation). More clinical trials are needed for safety and efficacy of Cit therapy in human infants with and at risk of developing chronic, progressive forms of pulmonary hypertension deserve consideration as a novel and potentially cost-effective approach to treat this lifethreatening condition in term and preterm infants (Fike et al., 2014). Therapeutic strategies that increase the transport of Lcitrulline may represent a novel approach to the management of pulmonary hypertension in infants. Another study evaluated Cit in preventing deterioration of pulmonary hypertension and explored combination therapy of tadalafil þ Cit versus tadalafil þ Arg in 4-week-old rats. Echocardiographical examination suggested that the ratios of RV to LV weight in a tadalafil group and a tadalafil þ Cit group were significantly lower than other groups. The estimated pulmonary artery pressure in a Cit and tadalafil þ Cit group

seem to be lower than those in other groups because of Cit-induced vasodilation that reduces right ventricular function with improved survival rate attributed to tadalafil þ Cit group due to low RV to LV ratio and decreased pulmonary artery pressure. The survival rate of tadalafil þ Cit was higher than other groups with lower ratio of RV to LV weight (Ishikura et al., 2015). Preliminary results from a case report clearly indicates that L-citrulline supplemetatation might be a potential therapy for chronic PH in infants with BPD. In this case report, for the first time a premature newborn (study in newborn animals was supporing) was given oral Cit along with sildenafil, hydrocortisone, inhaled nitric oxide. Oral Cit, from beginning of the sixth month of hospitalization, single dose of 150 mg/kg/day was introduced and continued for 70 days; patient was weaned from mechanical ventilation and never intubated again until discharge (Lauterbach et al., 2018). A study explored and concluded that both NOSdependent and -independent approaches in the prehypertensive stage toward augmentation of NO can prevent the development of hypertension in young SHRs. The study was done in male SHR and normotensive control rats where Cit was replaced with sodium nitrate. Rats were sacrificed at 12 weeks of age. It is noteworthy that Cit therapy also reduced levels of Arg and ADMA (endogenous inhibitor of NOS) and increased the ARGeADMA ratio in SHR kidneys, whereas nitrate treatment reduced plasma levels of ARG and ADMA concurrently in SHRs (Chien et al., 2014). This further proves its beneficial effect in hypertension at the molecular level operating in this condition. Hence, Cit is a safer way of delivering Arg for endothelial and immune cells as well as preventing excessive uncontrolled NO production (Cynober et al., 2010) that exerts deleterious effects of its own and is an important coadjuvant in the treatment of stable systolic heart failure patients. Citrulline in Diabetes Cit is shown to benefit the underlying endothelial dysfunction (Hecker et al., 1990) of diabetes, in addition to producing a reduction in glucose levels. Watermelon juice exerts beneficial effects by increasing Arg availability, improving glycemic control and vascular dysfunction in type 2 diabetes mellitus through intake of Cit from watermelon (Wu et al., 2007). Cit (50 mg/kg/day, p.o.) and arginase inhibitors are found to be of benefit in endothelial dysfunction and improve impaired vasodilation of coronary arteries in diabetes (Romero et al., 2008). Thus, studies suggest Cit may be of benefit in diabetes by improving glycemic control and in diabetes and metabolic syndrome by ameliorating underlying endothelial dysfunction. Large-scale clinical trials and mechanistic studies are required to evaluate the therapeutic strategies.

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Citrulline as Immunomodulator Cit production is significantly reduced in sepsis because of diminished de novo conversion of Arg and NO, reducing Cit and Arg availability in septic patients and transgenic mice, in part due to ADMA release. In sepsis, patients are found to have significantly decreased levels of Cit (by 56%), glutamate (by 48%), and Arg (by 47%) in comparison to healthy control subjects. A study in rats revealed that Cit supplementation reduced cell membrane lipid peroxidation and enhanced SOD activities in septic rats. It also decreased the release of TNF-a, IL-6, and IL-1b in early stages of sepsis (Cai et al., 2016). Thus, Cit may be a safe means of immunomodulation that preserves the antiinflammatory mediator response. The concept of immunonutrition with formulation containing medium dose Arg and high dose Arg (20 g/d) is emerging, which significantly lessens the overall complications, and also improves local wound complications as well as reduces the length of hospital stay in postoperative cancer patients (De Luis et al., 2009; Rodera et al., 2012). This has been supported by another study (containing Arg, zinc, and antioxidants within a high-calorie, high-protein formula) that showed improvement in healing of pressure ulcers (PU) in malnourished patients resulting in greater reduction in PU area at 8 weeks (Cereda et al., 2015). Citrulline Therapy May Decrease Severity of Sepsis Sepsis is a major health problem due to its high morbidity and mortality rate and may be considered to be an Arg deficiency state (Luiking et al., 2004). Arg availability is increased in sepsis mainly by protein breakdown, but Arg catabolism is also increased due to its enhanced utilization for NO synthesis and higher arginase activity. Therefore, Arg availability is reduced in sepsis because of lower plasma Arg concentrations and a slower Arg production rate (Luiking et al., 2009). Because of this, Cit production is very low, but because data for Cit in intensive care units are not available, large clinical trials are warranted before any reliable recommendations can be made for Cit-enriched diets in this situation. At least reinforcement can be suggested for Cit supplementation. Its utility as a marker in sepsis or critically ill patients is doubtful because of associated vascular abnormalities, acute renal failure (ARF) and/ or multiple organ failure, or comorbid conditions (Cynober, 2013). Further studies are warranted to either prove or disprove the use of Cit therapy as an Arg precursor in this condition. Citrulline in Arginase-Associated T-Cell Dysfunction It is interesting to note that T lymphocytes depend on Arg for their proliferation, zeta-chain peptide formation,

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T-cell receptor complex expression, and development of memory. This makes it obvious that T-cell abnormalities such as decreased proliferation and loss of zeta chain are observed in cancer and after trauma, and they may provide new insights into T-cell dysfunction (Kaore et al., 2013). Cit may serve as a substitute for arginaseassociated T-cell dysfunction, the basis for which is the molecular capability of T-cells to increase Cit membrane transport and upregulate arginosuccinate synthase (ASS) expression and thus convert Cit to Arg in in vitro studies, escaping the ill effects of Arg depletion (Bansal et al., 2004). Cit supplementation can preserve T-cell proliferation and prevent the loss of CD3 zeta chain under conditions of low Arg. New drug targets for improving T-cell associated dysfunction may be explored in the future, and further research may open novel mechanistic insights in this regard. Citrulline: Role in Dementia of Alzheimer’s Disease and in Multi-Infarct Dementia Cit levels in the cerebrospinal fluid were significantly higher in MID in comparison to a healthy control group (Mochizuki et al., 1996). Further, it was found that there is elevated NOS activity in microvessels of the brain in AD, raising vascular NO production, exerting neurotoxicity, and being responsible for susceptibility to neuronal injury and cell death in AD (Kaore et al., 2013). Some studies contradict this notion and suggest decreased NO production to play a role in neurodegenerative disorders. Arg fortification with a lysine (Lys)-restricted diet is one of the emerging strategies for a rare autosomal recessive disorder, pyridoxine-dependent epilepsy (PDE), which is resistant to conventional antiepileptic drugs but responsive to pharmacological dosages of pyridoxine (van Karnebeek and Jaggumantri, 2015). Further research may prove or disprove the use of Cit modulators in brain lesions or in PDE. Citrulline: Can It Improve Aerobic Function/ Energy Production? L-Citrulline malate can develop beneficial effects on the elimination of ammonia in the course of recovery from exhaustive muscular exercise (Sureda and Pons, 2012). Cit is also shown to be a more suitable substrate than Arg to restore NO production in microcirculation during endotoxemia (Wijnands et al., 2012). Cit is shown to enhance protein expression of the myofibrillar constituents (Faure et al., 2013), and protein synthesis occurs through a mechanism of action mediated by the mTOR signaling pathway (Bahri et al., 2013). Citrulline malate (CM) ingestion significantly reduced fatigue sensation, improved aerobic energy production in muscles, and improved exercise tolerance (Sureda and Pons, 2012), thus greatly enhancing aerobic

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performance and prolongation of onset of muscular fatigue (Kaore et al., 2013). CM also limits muscle fatigue or skeletal muscle dysfunction induced by bacterial endotoxins (Kaore et al., 2013). Recent studies show ergogenic effects on Cit supplementation with improvement of muscular contraction efficiency and of athletic performance in mice (Giannesini et al., 2011; Sureda and Pons, 2012). Thus, Cit may be used as an agent to increase exercise capacity for various reasons. However, a recent study found conflicting results where CM supplementation (single 6 g dose preworkout) does not improve the muscle recovery process following a highintensity resistance exercise session in untrained young adult men (Da Silva et al., 2017). Citrulline for Urea Cycle Disorders Cit may be used to treat the inborn errors of metabolism of the urea cycle responsible for removal of ammonia that, fortunately, are quite rare. The first step in the urea cycle is the reaction of ammonia and bicarbonate using ATP, catalyzed by the enzyme carbamoyl phosphate synthetase (CPS) to produce carbamoyl phosphate. The second step is the reaction of carbamoyl phosphate and L-ornithine to produce L-Cit, catalyzed by the enzyme ornithine transcarbamylase. It is interesting to note that deficiencies of either of these two enzymes lead to low serum levels of L-Cit. Treatment is with oral L-Cit. In long-term management, dietary treatment must be individualized (Kaore et al., 2013). Recently, a breakthrough in these disorders has emerged in the form of FDA approval of Cit for certain UCDs. Citrulline for Acceleration of Wound Healing NO is an integral part of the inflammatory phase, functioning as a regulatory mechanism to mediate epithelialization, angiogenesis, and collagen deposition crucial to the proliferative phase. NO-induced vasodilatation acted as a host-protective agent by killing pathogens in an in vitro study (Norris et al., 1995) and increasing blood flow to wounds (Stechmiller et al., 2005). This notion is supported by a clinical study in graft patients suggesting that Arg metabolism is involved in tissue repair, including angiogenesis, epithelialization, and collagen formation as increased levels of Cit and other metabolites were found in wound fluid versus plasma (Debats et al., 2009). Citrulline in Intestinal Pathology Studies suggest that Cit has a strong potential in total parenteral nutrition after massive intestinal resection (SBS), causing an increase in the Arg pool and acting to restore nitrogen balance. Long-term supplementation of Arg intraduodenally in rats failed to show beneficial results in intestinal ischemia and reperfusion injury (Lee et al., 2012). Cit pretreatment improves integrity

of the gut barrier and helps to preserve ileum mucosa in experimental studies, thus reducing bacterial translocation. Similar effects are seen with Arg as well. This understanding of molecular mechanisms may help in developing new strategies that could be extended to malnourished patients with compromised intestinal functions. Citrulline: Effects of Amino Acids on Hair Strength Human studies report considerable amounts of Arg deposition on or in hair fibers from coloring agents, whereas a decreased amount of Arg and Cit was found in damaged hairs in users of “relaxers.” Decreased Cit has been associated with inflammation. Study reports indicate that when coloring agents were partially replaced with Arg, it decreased the oxidative change in tensile strength of the hair by preventing the undesirable attack by hydrogen peroxide on hair proteins and hair surface lipids. Therefore, prospective studies need to be undertaken to understand whether or how “relaxers” induce inflammation and whether Arg or Cit can substantially reduce hair damage and fragility (Kaore et al., 2013). Citrulline’s Effect on Protein Synthesis Cit is found to increase protein synthesis during refeeding in rodents with SBS, aging, and malnutrition, and it improves nitrogen balance in healthy humans (Thibault et al., 2011). Cit is a conditionally essential AA in stress, or when intestinal function is compromised. This notion is proved in SBS in rats, where Cit is able to restore nitrogen balance, increase mucosal protein content in the ileum, increase Arg levels, and increase muscle protein content, as well as muscle protein synthesis (þ90%) in elderly malnourished rats. Moreover, it also has potential as a supplement for total parenteral nutrition in SBS patients because it additionally prevents muscle atrophy, not seen with Arg supplementation (Osowska et al., 2008; Kaore et al., 2013). Thus, Cit plays a crucial role in maintaining protein homeostasis and improving muscle mass related to malnutrition. Further understanding of molecular mechanisms may help in developing new strategies for malnourished patients with compromised intestinal functions (Box 60.2). Citrulline as a Biomarker in Diseases Cit is not involved in protein synthesis or nutrition products, but it may serve as a biomarker in various diseases such as RA, intestinal dysfunction, or sepsis. It is now an established biomarker of enterocyte functional metabolic mass in adults and children, which can be used in assessing the remnant length of small bowel in intestinal diseases such as short bowel, extensive enteropathies, intestinal toxicity of chemotherapy, and radiotherapy. Normal plasma Cit levels range

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THERAPEUTIC APPLICATIONS OF CITRULLINE

Citrulline supplementation exploited for benefits in a variety of conditions Cardiovascular conditions

Neurodegenerative conditions • Alzheimer’s disease • Multi-infarct dementia

Parasitic infections

• Hypertension • Right ventricular dysfunction • Hyperlipidemia

• Malaria • Trypanosoma cruzi • Toxoplasma gondii

Hematological disorders

Others

• Sickle cell anemia

• • • • • •

Metabolic disorders • Diabetes • Urea cycle disorders

between 30 and 50 mmol/L independent of nutritional status. Levels less than 10 mmol/L may indicate an objective threshold for parenteral nutrition in case of intestinal failure that allows monitoring of intestinal function, except in cases of significant renal failure (Crenn et al., 2011). This reveals that decreased plasma Cit concentration reflects a decrease of functional mass of enterocytes provided renal function is normal (Kaore et al., 2013) (Box 60.3). Citrulline as a Biomarker in Intestinal Disease Serum Cit is emerging as an innovative biomarker candidate for assessment of intestinal function. It has been further assessed as a marker of intestinal failure and for monitoring of bowel function. Serial plasma Cit assay helps to monitor residual small bowel adaptation in children. Studies show serum Cit to be significantly lower in SBS patients, and thus it could be accurately taken as a simple, noninvasive biomarker for assessing the severity of intestinal failure. It may prove to be a candidate marker for the gut-trophic effects of bowel rehabilitation therapies. Studies show postabsorptive plasma Cit concentration to be a marker of functional absorptive bowel length that also allows distinction of transient to permanent intestinal failure in SBS patients, and also to be a marker of reduced enterocyte mass in patients with villous atrophy diseases (Kaore et al., 2013). Decreased Cit levels also correlate to the intensity of acute mesenteric ischemia and duration of intestinal damage, for which Cit has a potential future role (Cakmaz et al., 2013).

Wound healing As an antioxidant Erectile dysfunction Sepsis (as an immunomodulator) Arginase associated T-cell dysfunction Cancer chemotherapy (in certain tumors)

Plasma Cit is a reliable biomarker of enterocyte functional mass in HIV patients. It can discriminate between protease inhibitor (PI) toxic diarrhea and infectious enteropathy and quantify the functional consequences, which makes it an objective indicator of the need for parenteral nutrition. In HIV enteropathy, Cit concentration