Principles of food toxicology [Second edition] 9781466504110, 1466504110

Table of contents :
Front Cover......Page 1
Contents......Page 9
Preface to the Second Edition......Page 17
Preface to the First Edition......Page 19
Acknowledgments......Page 21
Author......Page 23
Chapter 1 - Introduction......Page 27
Chapter 2 - Routes of Xenobiotics in an Organism......Page 51
Chapter 3 - Toxic Response......Page 89
Chapter 4 - Analytical Toxicology......Page 133
Chapter 5 - Evaluation of Toxicity of Substances......Page 141
Chapter 6 - Toxicological Safety and Risk Analysis......Page 165
Chapter 7 - Endogenous Plant Toxicants......Page 181
Chapter 8 - Geochemical Pollutants That Plants Absorb from Soil......Page 223
Chapter 9 - Environmental Pollutants......Page 233
Chapter 10 - Mycotoxins......Page 255
Chapter 11 - Animal Endogenous Poisons......Page 273
Chapter 12 - Food Toxicants from Aquatic Animals......Page 279
Chapter 13 - Pesticide Residues......Page 293
Chapter 14 - Veterinary Drugs and Feed Additives......Page 307
Chapter 15 - Toxicants Unintentionally Formed during Processing, Storage, and Digestion of Food......Page 315
Chapter 16 - Food Additives......Page 357
Chapter 17 - Vitamins......Page 373
Chapter 18 - Food Adulteration......Page 385
Glossary......Page 395
Back Cover......Page 407

Citation preview

SECOND EDITION

PRINCIPLES OF FOOD

TOXICOLOGY Tõ n u P ü s s a

SECOND EDITION

PRINCIPLES OF FOOD

TOXICOLOGY

SECOND EDITION

PRINCIPLES OF FOOD

TOXICOLOGY Tõ nu Pü ssa

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2014 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20130709 International Standard Book Number-13: 978-1-4665-0411-0 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

To my wife, Tiia

Contents

Preface to the Second Edition xv Preface to the First Edition xvii Acknowledgments xix Author xxi I. Basics of Toxicology Related to Food  1 1. Introduction 3 1.1 What Is Toxicology?  3 1.2 Short History of Toxicology  6 1.3 Toxicity, Dose, and Response  8 1.4 Integrated Effect of Toxic Substances  16 1.5 Classification of Toxicants  18 1.6 Some Toxicology-Related Principles of Cellular Biology and Biochemistry  18 1.6.1 Structure of Cellular Membranes  18 1.6.2 Transport of Substances across Biomembranes  19 1.6.3 Receptors 23 1.6.4 Ion Channels  25 References 26 2. Routes of Xenobiotics in an Organism 27 2.1 Entry and Absorption of Foreign Compounds  27 2.1.1 Digestive Tract  28 2.1.2 Lungs 30 2.1.3 Skin 30 2.2 Distribution of Xenobiotics in the Organism  31 2.2.1 Blood Supply and Membrane Barriers  31 2.2.2 Role of Lymph in Absorption and Distribution of Xenobiotics  34 vii

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2.2.3 Binding of Xenobiotics to Charged Particles  34 2.2.4 Bioaccumulation of Xenobiotics  35 2.3 Metabolism of Xenobiotics  36 2.3.1 General Principles  36 2.3.2 Phase I Reactions  39 2.3.2.1 Enzyme Superfamily CYP  40 2.3.2.2 Other Enzymes Catalyzing Oxidation  41 2.3.2.3 Examples of Phase I Reactions  42 2.3.3 Phase II Reactions  45 2.3.4 Induction and Inhibition of Metabolic Enzymes  50 2.3.4.1 Induction of Enzymes  50 2.3.4.2 Inhibition of Enzymes  52 2.3.5 Participation of Enteric Microflora in the Metabolism of Xenobiotics  53 2.3.6 Influence of Diet on Metabolism  54 2.4 Elimination of Xenobiotics and/or Their Metabolites from the Organism  56 2.4.1 Kidneys 56 2.4.2 Liver 58 2.4.3 Intestines 59 2.4.4 Lungs 59 2.5 Biomagnification 59 2.6 Antidotes 61 2.6.1 General Methods  61 2.6.2 Specific Mechanisms of Antidote Action  62 References 63 3. Toxic Response 65 3.1 Variability in Toxic Response  65 3.2 Physiological Classification of Toxic Responses  66 3.2.1 General Principles  66 3.2.2 Direct Injury to a Cell or a Tissue  69 3.2.3 Biochemical Damage  70 3.2.4 Neurotoxicity 72 3.2.5 Immunotoxicity 73 3.2.6 Teratogenicity 74 3.2.7 Genotoxicity and Mutagenicity  77 3.2.8 Carcinogenicity 79 3.2.9 Reproductive and Developmental Toxicity  87 3.2.9.1 Reproductive Toxicity  88 3.2.9.2 Developmental Toxicity  88 3.2.10 Multiorgan Toxicity  89 3.3 Molecular Mechanisms of Toxicity  90

Contents

3.3.1 3.3.2 3.3.3 3.3.4 3.3.5

Disturbance of Cell Homeostasis  91 Receptor-Mediated Mechanisms  91 Other Toxic Effects Mediated by Cellular Membranes 94 Alteration of Cell Energetics  95 Covalent Binding to Essential Cellular Macromolecules 95 3.3.6 Endocrine Disruption  96 3.3.7 Oxidative Stress  101 3.3.8 Inhibition of DNA Repair  103 3.3.9 Multiple Interorgan Effects  103 3.4 Biomarkers of Toxic Effect  104 References 106 4. Analytical Toxicology 109 4.1 Introduction 109 4.2 Sample Preparation  109 4.3 Mouse Bioassay: Functional Analysis  110 4.4 Biological Methods: Biosensors  112 4.5 Immunochemical Methods: Immunosensors  112 4.6 Spectrophotometric (Colorimetric) Methods  113 4.7 Electrophoretic Methods  113 4.8 Chromatographic Methods  113 References 114 5. Evaluation of Toxicity of Substances 117 5.1 Epidemiological Studies  117 5.2 Animal Tests  119 5.2.1 General Principles  119 5.2.2 Organism-Depending Factors, Influencing the Compound Toxicity  124 5.2.2.1 Dependence on Species  124 5.2.2.2 Genetic Variabilities  125 5.2.2.3 Generic Variabilities  126 5.2.2.4 Dependence on the Age  127 5.2.2.5 Dietary Conditions  127 5.2.2.6 Health Conditions  129 5.2.2.7 Simultaneous Contact with Several Xenobiotics 129 5.3 Cell Culture Tests  130 5.4 Computer Calculations  133 5.5 Acute Toxicity Tests  134 5.6 Subacute/Subchronic Toxicity Tests  136

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5.7 Chronic Toxicity Tests: Acceptable Daily Intake  136 5.8 Specific Toxicity Tests  138 References 139 6. Toxicological Safety and Risk Analysis 141 6.1 Toxicological Safety  141 6.2 Risk Assessment  142 6.2.1 Hazard Identification: Principle of the Three R’s  145 6.2.2 Demonstration of a Dose–Response Relationship  145 6.2.3 Assessment of Exposure  147 6.2.4 Risk Characterization  148 6.3 Risk–Benefit Analysis  151 References 153 II. Main Groups of Food Toxicants 155 7. Endogenous Plant Toxicants 157 7.1 Lectins or Hemagglutinins  157 7.2 Enzyme Inhibitors  159 7.3 Alkaloids 160 7.3.1 Pyrrolizidine Alkaloids  160 7.3.2 Solanine-Group Glycoalkaloids  162 7.3.3 Xanthine Alkaloids  164 7.3.4 Ephedrine Alkaloids  167 7.4 Cyanogenic Glycosides: Toxicity Mechanism of HCN  168 7.5 Phytoestrogens 171 7.6 Glucosinolates 172 7.7 Coumarin 174 7.8 Thujones 175 7.9 Toxic Amino Acids  177 7.10 Toxic Lipids  179 7.10.1 Erucic Acid  179 7.10.2 Sterculic and Malvalic Acids  180 7.10.3 Polyunsaturated Fatty Acids  180 7.11 Oxalates 181 7.12 Fluoroacetates 182 7.13 Bracken Toxins  183 7.14 Saponins 184 7.15 Grayanotoxin 186 7.16 Soybean as a Potential Source of Versatile Possible Toxicants  187 7.17 Mushroom Toxins  188 7.17.1 General Principles  188 7.17.2 Amatoxins 190

Contents

7.17.3 Muscarine 191 7.17.4 Isoxazoles 192 7.17.5 Other Mushroom Toxins  193 References 195 8. Geochemical Pollutants That Plants Absorb from Soil 199 8.1 Arsenic 199 8.2 Selenium 202 8.3 Fluorine 204 References 206 9. Environmental Pollutants 209 9.1 Toxic Elements  209 9.1.1 Mercury 209 9.1.2 Lead 212 9.1.3 Cadmium 214 9.1.4 Chromium 216 9.1.5 Copper 217 9.1.6 Nickel 218 9.2 Radionuclides 219 9.3 Polychlorinated Biphenyls  221 9.4 Polychlorinated Dibenzodioxins and Dibenzofurans  223 References 228 10. Mycotoxins 231 10.1 Overview 231 10.2 Aflatoxins 233 10.3 Ochratoxins 236 10.4 Sterigmatocystin 238 10.5 Zearalenone 239 10.6 Fumonisins 240 10.7 Trichothecenes 240 10.8 Patulin 242 10.9 Citrinin and Citreoviridin  242 10.10 Ergot Toxins  243 10.11 Other Mycotoxins  245 10.12 Combined Toxicity of Mycotoxins  246 References 246 11. Animal Endogenous Poisons 249 11.1 Prions 249 11.2 Lactose 251 11.3 Phytanic Acid  252

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11.4 Avidin 253 11.5 Vitamins of Animal Origin  253 References 254 12. Food Toxicants from Aquatic Animals 255 12.1 Introduction 255 12.2 Shellfish Toxicants  255 12.2.1 Paralytic Shellfish Poisoning  255 12.2.2 Diarrhetic Shellfish Poisoning  258 12.2.3 Neurotoxic Shellfish Poisoning  260 12.2.4 Amnesic Shellfish Poisoning  261 12.2.5 Microcystins and Nodularins  262 12.3 Fish Toxins  263 12.3.1 Tetrodotoxin 263 12.3.2 Ciguatoxin 265 References 267 13. Pesticide Residues 269 13.1 Overview 269 13.2 Insecticides 277 13.3 Herbicides 278 13.4 Fungicides 279 References 280 14. Veterinary Drugs and Feed Additives 283 14.1 Antibiotics 284 14.2 Hormones 285 14.3 Other Veterinary Drugs  286 14.3.1 Coccidiostatics 286 14.3.2 Anthelmintics 287 14.3.3 β-Agonists 288 14.3.4 Glucocorticoids 288 14.3.5 Thyreostatics 288 References 289 15. Toxicants Unintentionally Formed during Processing, Storage, and Digestion of Food 291 15.1 General 291 15.2 Polycyclic Aromatic Hydrocarbons  295 15.3 Alcohols 298 15.4 Bacterial Toxins  302 15.4.1 Exotoxins 303 15.4.1.1 Staphylococcus 303 15.4.1.2 Clostridia 304 15.4.1.3 Bacillus cereus 308

Contents

15.4.1.4 Campylobacters  308 15.4.1.5 Listeria 309 15.4.2 Endotoxins 310 15.5 Biogenic Vasoactive Amines  312 15.5.1 Scombroid Poisoning  313 15.6 Nitrates, Nitrites, and Nitrosamines  316 15.7 Acrylamide 319 15.8 Trans Fatty Acids  321 15.9 Chlorinated Propanols  322 15.10 Food Contact Materials  323 15.10.1 Phthalates  323 15.10.2 Bisphenols  326 15.10.3 Aluminum from Cookware  327 References 328 16. Food Additives 333 16.1 Traditional Food Additives  333 16.1.1 General Principles: ADI  333 16.1.2 Colorants 336 16.1.3 Artificial Sweeteners  337 16.1.4 Preservatives 340 16.1.5 Antioxidants 340 16.1.6 Glutamates 340 16.2 Functional Additives  341 16.2.1 General Principles  341 16.2.2 Functional Additive–Drug Interactions  345 References 346 17. Vitamins 349 17.1 General 349 17.2 Vitamin A: Phenomenon of Smokers  350 17.3 Vitamin D  353 17.4 Vitamin E  354 17.5 Vitamin K  355 17.6 Vitamin B2 356 17.7 Vitamin B6 356 17.8 Vitamin C  357 17.9 Vitamin B3 358 17.10 Diagnosing and Therapy of Vitamin Intoxications  359 References 359 18. Food Adulteration 361 18.1 What Is Food Adulteration?  361 18.2 Melamine and Cyanuric Acid  361

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18.2.1 Toxicity of MEL  363 18.2.2 Toxicity of Cyanuric Acid  363 18.2.3 Combined Toxicity of Triazine Compounds  364 18.3 Anisatine 365 18.4 Toxic Oil Syndrome  366 18.5 Epidemic Dropsy  367 References 368 Glossary 371

Preface to the Second Edition

Several years have passed since the publication of the first edition of this book. The developing food technology has adopted new processing techniques that may generate totally new food constituents calling for a special toxicological evaluation. For example, trans fatty acids resulting from the chemical hydrogenation of unsaturated fats, or 3-monochloropropanediol arising from the chemical hydrolysis of proteins have recently been identified as toxic substances in processed food. On the other hand, toxicology, hand in hand with other natural sciences and largely owing to the developments in chemical analysis, is ever more ready for these new challenges, including re-examination of the former data and even theories. For example, it turned out that the hitherto infamous nitrite ions are perhaps not so bad at all, but in fact possess beneficial physiological properties as well. Also, the customary NOAEL (no-observed adverse effect level) values have started to shake. The risk−benefit analysis has become a constituent part of toxicological evaluation for any substance or mixture. There is a widespread view that the safety of traditional foods has already been fully proven by their long-term nonproblematic consumption. Nevertheless, thanks to progress in chemical analysis techniques, it has been discovered that foodstuffs consumed for centuries may still contain remarkable amounts of toxicants and/or antinutritional substances. One of the recently reported ­examples of process-derived chemical hazards in food is the formation of acrylamide in baked products. Although a similar process had been used for centuries for baking bread, potatoes, and other starch-based foods, this drawback went unnoticed until 2002. An increased knowledge of long-term and chronic toxic effects enables us to be aware of the health hazards that were earlier either unknown or underestimated. Even very small quantities of toxic substances in prepared food can, as a result of continuous long-term consumption and accumulation in the organism, become hazardous for humans. Moreover, life standards, expectations, and conceptions of life and health have also substantially changed over the past few decades. Diets have changed, and there has been an upsurge in vegetarianism and the consumption of exotic, minimally prepared, and fast foods. An emerging area of further concern that has xv

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Preface to the Second Edition

recently become a more serious issue for food packaging material manufacturers is the migration of hazardous chemicals from packaging materials into foods. Against this background, new conceptions of food safety are emerging. Acute food intoxications are nowadays relatively rare; considerably more frequent and more significant issues are represented by the long-term and often hard-to-diagnose effects of plant and animal toxicants that have already developed extensively by the time of discovery. In comparison with the first edition, the material presented here has been revised and updated. A completely new chapter “Food Adulterants” and new sections, such as “Reproductive and Developmental Toxicity” and “Risk−Benefit Analysis,” have been included. The list of literary sources, mainly referring to recent scientific, in particular, reviewing articles, has been sufficiently expanded to facilitate deeper immersion of the reader into the exciting details of food chemical safety.

Preface to the First Edition

Food is an extremely complex and complicated system that consists of a practically endless number of high- and low-molecular substances, mostly of natural origin. A majority of these compounds are indispensable for the normal functioning of a human, either as a source of energy or body building material or as a normal source of taste—the main function of which is to turn eating into a pleasure and to improve digestion. Some of the food components also perform the task of turning foods healthier and safer and prolonging the storage time or “shelf life” (“best before”). On the other hand, food always contains substances that are capable of evoking smaller or bigger health disorders, that is, that are toxic. Poisonous compounds may originate from the raw materials of food, but they may get into food during its processing, transportation, or storage. Compounds, often synthetic, that are intentionally added to food may also be toxic. Although these substances, called food additives, undergo, nowadays, an exhaustive toxicological examination, one can never be absolutely sure that a long-known food constituent can be regarded as safe in a new environment, where it can turn toxic by itself or synergetically enhance the toxicity of another which was thought to be a nontoxic food component. Food is never ready to be consumed; various physical and biochemical processes occur continuously in it that may provide new and not always harmless substances. The so-called health-promoting functional additives may also elicit toxicological problems. This book is an attempt to put into one pot principles of general- and food toxicology and to spice them up with the most important and vivid examples of food-related poisons and poisonings from all over the world. Owing to the rapid development of food toxicology, it is not usually possible to present the ultimate truth about toxic effects and their mechanisms. And this is good because it makes the reader think with us. Special attention is paid to the biochemical mechanisms of the toxic effects as much as they are known. Knowledge of the mechanisms helps toxicologists to scientifically perform the risk assessment. The first part of the book is dedicated to the introduction of principles of toxicology at the molecular, cellular, and organism level, as much as possible in xvii

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relation with food. At times, examples taken from the second part are used to illustrate the principles. The second part is a systematic characterization of the most important food-borne toxicants, closely connected with the first part of the book. This book is a thoroughly revised and updated translation of a book written in Estonian that is being used in the author’s course on Food Toxicology at the Estonian University of Life Sciences. It will be of interest to students of food science and technology and to professional food scientists, manufacturers, and regulatory agency personnel.

Acknowledgments

The author is grateful to his colleagues who helped with the manuscript. The author also wishes to specially thank his doctoral student Piret Raudsepp for the preparation of figures and his daughter Triina for the linguistic proofreading of the translation.

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Author

Tõnu Püssa, PhD, is a professor of toxicology at the Department of Food Hygiene of the Estonian University of Life Sciences in Tartu, Estonia. After graduating from Tartu University as an organic chemist in 1969, he received his PhD in chemistry from the same university in 1973. During his work at the Department of Organic Chemistry and Laboratory of Chemical Kinetics and Catalysis of Tartu University as a research fellow and associate professor, his research interests were connected with chemical and enzymatic catalysis, algal carbohydrates, and proteins of the endocellular matrix. During one year, he was a guest researcher at the Finnish Red Cross Blood Transfusion Service in Helsinki. In 1996–2001, Tõnu Püssa worked at the Estonian University of Life Sciences (former Agricultural University) as the head of the Laboratory of Environmental and Ecological Chemistry. He has taught student courses in organic chemistry, analytical biochemistry, hydrochemistry, food- and environmental toxicology, and food safety (chemical hazards). His present scientific interests are connected to functional foods, particularly the mechanism of interaction between herbal antioxidant polyphenols and the peroxidation system of polyunsaturated fatty acids producing mutagenic epoxyacids.

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I BASICS OF TOXICOLOGY RELATED TO FOOD

1 Introduction

1.1  WHAT IS TOXICOLOGY? The term “toxicology” is a combination of two Greek words: τοξικον = poison or toxicant and λογος = word, reason, or science. The word τοξικον means a toxic substance into which arrowheads were dipped, whereas τοξικος means a bow. A toxicant or poison is a chemical substance which, after entering an organism, is capable of eliciting smaller or larger adverse changes in the functioning of cells, tissues, or even the whole organism, resulting in the most severe and untreated cases in the death of the organism. The term “toxicant” denotes, above all, a synthetic substance causing adverse health effects. A similar term “toxin” usually refers to any proteinaceous poison produced by living organisms, particularly microorganisms such as bacteria in the body of a host. In a broader approach, toxin is any poisonous substance, irrespective of the molecular size and structure, produced by an organism. When used nonscientifically, the term toxin is often applied to any toxic substance. The term “venom” (from Latin venemus) or zootoxin denotes a poisonous matter (toxin) normally secreted by snakes, scorpions, bees, and so on. Homo sapiens, similar to other living organisms in the contemporary world, are exposed to diverse substances or compounds, which are foreign particles for the organism, called xenobiotics, which may either be simple metals or inorganic compounds, as well as organic molecules of different complexities and sizes. These substances can be • Natural—produced by another organism • Anthropogenic—produced during or due to diverse human activities Toxicology is a science that studies • The formation, composition, and properties of the harmful substances, the mechanisms of adverse action of these substances on biological systems (organisms, organs, cells), and the ways of assessment and 3

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Principles of Food Toxicology

r­ eduction of the adverse effect, methods of prophylactics, and in case of necessity, the medication of the toxic results. • The toxicokinetics and toxicodynamics of the substances elaborate the sensitive and exact methods for the determination of threshold doses, as well as concentrations, in the case of both short-term (acute) and longterm (chronic) effects of xenobiotics. • The cellular toxicity, mutagenicity, teratogenicity, carcinogenicity, and other adverse effects of xenobiotics. Substances, which are intrinsic to an organism (e.g., histamine and other vasoactive amines (see Section 15.5) or selenium (see Section 8.2)) can also be harmful to this organ, when their concentration in the organism rises impermissibly due to either (uncontrolled) intensification of an internal process or due to the devouring of an extra quantity of the substance into the organism. Toxicology is closely related to pharmacology. Toxicology makes a greater use of the methods of pharmacology for estimating the substance’s toxicity, for investigating its mechanism of action, and for obtaining the data characterizing the disorders caused by the interaction of xenobiotics with an organism. The main difference is that while pharmacology studies the desirable changes in an organism leading to its recovery, toxicology is busy with the study of undesirable changes causing damage to a cell, an organ, or an organism. While toxicology primarily studies the manifestations of an extreme action of substances often, with symptoms preceding the death of a test animal, pharmacology mostly investigates the smaller alterations in the functioning of the organism. In the latter case, the research focuses on substances designed for therapy. At the same time, a modern toxicologist must be interested in determining the effect of small doses of a toxicant just as a pharmacologist is supposed to determine the toxicity of the drugs. If, during the past centuries, toxicology mainly studied highly poisonous substances, most of which had a lethal effect, then, in recent times, the main subjects are regarding the substances causing, in case of a contact with an organism, relatively weak, yet adverse, physiological changes which lower the quality of life, but usually do not pose any serious threats to one’s life. To continue more, the term “morbidity” is used instead of “mortality” in the toxicological literature. It has become possible essentially because of the fast development of methods for chemical analysis, which provide opportunities to measure much smaller concentrations of different substances in various matrices. And conversely, the growing demands of pharmacology as well as toxicology have contributed to the rapid development of methods in analytical toxicology (see Chapter 4). Toxicology has always been closely connected with other disciplines such as physiology, pathology, epidemiology, biochemistry, molecular biology, genetics, immunology, microbiology, ecology, statistics, and so on. Toxicology as an extensive and highly practice-oriented science has a number of subdisciplines or subdivisions such as analytical, biochemical, clinical,

Introduction

environmental, industrial, and forensic toxicology. In this list, food toxicology occupies a significant position. Food toxicology investigates • The ways and mechanisms of the entry of toxic substances into food or the generation of toxicants during food processing and storage, and the ways of avoiding or reducing food contamination. • The methods for the assessment of toxicity and risk of food components. • The adverse effects on an organism produced by harmful components of foods as well as beverages which can lead to functional disturbances of varying degree in the organism, even death. Since the environment is an endless source of various food toxicants, food toxicology is inseparable from environmental toxicology. Food is a material consumed either in a natural or in a processed form to satisfy the substantial and energetic needs of the consumers. However, food consumption can cause a variety of health problems, in which either the whole organism or at least a specific part of it can be shifted from its normal physiological state—homeostasis. This shift may, in a simple case, cause only some discomfort, but, in more serious cases, be life threatening. An initial light discomfort can strengthen over time and develop into a situation, where the question “to be or not to be” is to be answered. In these cases, we are dealing, in a broader sense, with the nonconformity of the food with the organism eating it. The nonconformities can be divided into toxic and oversensitivity phenomena. The latter may either be immune-system dependent or independent. Our prehistoric ancestors obviously tried to eat various plants, which they picked, and animals, which they managed to catch, as much as possible, to diversify and optimize their diet. So, by the trial and error method, which had sometimes fatal results, they learnt which food more efficiently satiated hunger without causing any illness or even death. The criteria of choice also certainly included the pleasantness of the food, its smell, and taste. In this way, our ancestors developed their nutritional habits, enabling them to survive and grow. Owing to their sedentary way of life, their habits and customs certainly depended on the local possibilities. Later on, the food components, especially additives such as spices, turned into commodities of long-distance trade. Time and increased trade brought about a homogenization of sorts, as most foodstuffs that were once unique to specific locales are now made globally available. Nowadays, the incidence of food-related illnesses is increasing again after a long period of decline. Food intoxications have turned into global issues; some of them, although considered to be defeated, are growing into problems again. Agricultural production methods and consumption habits have been changed; modern technologies of crop harvesting, processing, and packaging may often cause an emergence of new food-borne pathogens. Intensified travelling and

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Principles of Food Toxicology

trade facilitate the spread of pathogens and the outbreak of diseases. All communities in a nation are served by components of the same food system, even in geographically large countries such as Canada and the United States. Often, consumer preference for fresh food means a large-scale transport of such commodities from tropical and subtropical areas that are prone to both infectious agents and fungal toxins. Biochemical terrorism can turn into a serious food toxicological issue (Möller, 2000). Food toxicants can be divided into three groups: 1. Unconditional (real) toxicants which, in normally accessible foods and quantities, are unconditionally either acutely or/and chronically toxic for every human or even mammal. In the case of these poisons (botulinum toxin, tetrodotoxin, anatoxins, dioxins, mycotoxins, PAHs, etc.), no risk and benefit problems usually arise (see Section 6.3), although many of them can in small doses serve as remedies. A substance that, being used in sufficiently high quantities, leads an organism out of equilibrium may help in returning it back to equilibrium if administered in lower doses. 2. Conditional (possible, probable) toxicants are those for which the risk and benefit studies are essential and the debate about the usability and acceptable doses of these toxicants may last for years. Sometimes, toxicological science alternately announces them toxic and nontoxic in the normally applicable doses and the consumption of the food matrix. For example, multiple normal food components, such as caffeine (see Section 7.3.3) or coumarin (see Section 7.7), and food additives, such as colorants, synthetic sweeteners, flavor enhancers (see Chapter 16), nitrite ions, and so forth. 3. Selective toxicants such as lactose in the case of respective intolerance (Section 11.2), phytanic acid in the case of Refsum disease (Section 11.3), or vicin and covicin in the case of favism (Section 7.9). Usually, these toxicities are based on genetically governed enzyme deficiencies and those substances are poisonous only for a definite group of humans. 1.2  SHORT HISTORY OF TOXICOLOGY Early historical writings tell us that many centuries ago, humans were already acquainted with the toxic effect of a number of natural materials, including foodstuffs. The oldest recipes, more than 800 (!), of poisons such as belladonna (deadly nightshade) and opium alkaloids, lead, copper, and antimony can be found in Ebers Papyrus written in 1552 bc. The ancient Egyptians were able to distill hydrocyanic acid (HCN) from peach stones. The medical science of ancient India was aware of poisons such as arsenic, opium, and the extract of wolfsbane or

Introduction

monkshood (Aconitum napellus L.). The latter was used as an arrow poison in ancient China. The American Indians still use plant seed extracts containing poisonous glycosides as a weapon. In the ancient Greece, where intentional poisoning was quite an everyday issue, both poisons and their antidotes were known. King Mithridates VI (132–63 bc) used criminals to search for poisons and antidotes (mithridatics). He protected himself against poisoning by a mixture of 50 different antidotes. According to a legend, he developed serious problems owing to self-poisoning that it was necessary to commit suicide. It was in Greece that the shoots of contemporary toxicology sprouted. The writings of Hippocrates (460–377 bc), an outstanding physician of those times, demonstrate a true professional knowledge of poisons and toxicology among the Greeks. The most famous victim of intoxication is Socrates (470–399 bc), who was killed by the poison of hemlock (Conium maculatum L.), containing conium and other alkaloids such as toxic components (Hayes and Gilbert, 2009). In the Middle Ages, political poisoning turned into a real cult in Italy. For example, the Town Council of Venice or the Council of Ten (Il Consiglio dei Dieci) concluded contracts for the poisoning of their political opponents. The proceedings of this council are available from the detailed notes which include the victim’s name, the contractual partner, type and quantity of the poison used, and the outcomes. There was a historically well-known family of Borgia (Cesare, Lucretia, etc.), who poisoned spouses, lovers, political opponents, clergymen, and others. The study of toxicology also owes to “scientists” such as Catherine de Medici (1519–1589), the spouse of French King Henry II, who prepared poisons and tested them on the poor and the sick of France, noting all the clinical signs and symptoms. All of this helped the Swiss doctor Paracelsus (1493–1541) in formulating his famous postulates, like: “Was ist das nit gifft ist? alle ding sind gifft/und nichts ohn gifft/Allein die dosis macht ein ding kein gifft ist–” (Martinetz, 1982) or What is not poisonous? Everything is a poison/and there is nothing without poison/Only the dose permits something not to be poisonous. Paracelsus developed the meaning of the term “dose”; modern methods of risk assessment of chemicals, including terms such as threshold dose, safe, and nontoxic levels, are based on his postulate. Considering it very broadly, there is no single substance that does not possess any toxic action above its specific threshold dose. Even water is a poison if one drinks it too much. The oxygen we breathe and which is absolutely necessary for our life is poisonous for anaerobic organisms and even for us via the oxidative stress. Aerobic organisms have, during their evolution, created special mechanisms to control the toxicity of the free radicals produced by the oxygen. Paracelsus also believed that illnesses are localized in specific organs and that the poisons act in target organs as well. Unrecognized during his lifetime, the

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role of Paracelsus in the development of medicine as well as toxicology is enormous (Pappas et al., 1999). Toxicology as an individual discipline was first recognized by the Spanish scientist Matthieu Joseph Bonaventura Orfila (1787–1853), the physician-in-ordinary of Louis XVIII, king of France, who elaborated the first methods for the chemical detection of toxic substances. He was the founder of analytical toxicology. Orfila used dogs as the test animals. His main achievement in toxicology was the discovery that the ingested toxins do not stay and accumulate in the stomach, but after absorption move by the blood into other organs. Since then, toxicology started to develop as a science, also dealing with investigation of the mechanisms of toxicity of substances. So, the French scientist Claude Bernard (1813–1878), one of the founders of physiology and experimental pathology, believed that by studying the toxic effects of substances on biological systems, we obtain much useful data about the structure and functioning of these systems. From a later period, a very good example of the success of this approach is the study of the action of cyanide ion on cells, providing researchers with data about the functioning of the electron transport chain in mitochondria (see also Section 7.4). As it is known from biochemistry, specifically, the study of inhibitor reactions (quite often a poison is an inhibitor of a definite enzyme) provides us with much more information about the structure of an enzyme’s active center and the catalytic mechanism than the respective substrate reaction. An important milestone in the history of toxicology was the discovery and characterization of ricin, the toxic protein of castor oil plant (Ricinus communis) at Tartu University, Estonia, in 1888, by Peter Herman Stillmark and Eduard Rudolf Kobert (Kobert, 1906; Stillmark, 1888). Some years later, under the supervision of professor Kobert, the second toxic plant lectin abrin from Abrus precatorius was isolated by Heinrich Hellin. For more information about lectins, see Section 7.1. 1.3  TOXICITY, DOSE, AND RESPONSE Toxicity is 1. The capacity of a chemical substance to cause adverse or deleterious effects on a living organism or on a part of it. 2. The degree to which a substance is toxic. The toxicity of a substance depends on many different factors, such as • Chemical structure of the compound, distribution of charges in the molecule, and hence, the polarity and reactivity. • Route of administration (i.e., the substance may be applied to the skin, ingested, inhaled, or injected).

Introduction

• • • •

Period (time) of exposure (a brief encounter or a long-term one). Number of exposures (a single dose or multiple doses over time). Physical form of the toxicant (solid, liquid, or gas). Genetic constitution of an individual, an individual’s overall health, and so forth.

To reveal the toxicity of a substance, the organism must be exposed to this substance. The exposure to (contact with) the toxicant can be acute, subacute, subchronic, or chronic. Among other parameters, toxicity depends on the dose of the toxic substance. Dose is the total amount of a biologically active (toxic) compound administered to the organism, expressed usually in micrograms (µg) or milligrams (mg) per kg of the body weight (bw); in the case of a toxicant, dose is one of the most important determinants of its toxicity. The dose of the compound can enter the organism (to be administered) perorally, intrapulmonarily, intravenously, intramuscularly, percutaneously, intraperitoneally, and so forth. Response (effect)—Alteration of the biochemical or physiological parameters of the organism exposed to the biologically active (toxic) substance, which has reached the specific (super)molecular action points in the organism. The intensity of the response depends on the concentration of the toxic substance or its active metabolite in this action (target) point. Response can be either all-or-nothing as the death of the organism or graded, for example, alterations of activity of an enzyme or hormone. A dose can be either external or internal. In routine toxicological studies, the net amount of a compound administered per kilogram of the test animal weight is regarded as the dose. It can also be called the external dose. Ingested or inhaled substances are still conventionally considered to be outside the body until they have crossed such formidable barriers as the mucous membranes of the gastrointestinal tract or respiratory organs or the skin. Most substances, excluding local irritants or oxidants, will cause the toxic effect only after they

1. Get absorbed into the general bloodstream. The absorbed part of the external dose is called the absorbed or internal dose. 2. Reach the action point(s) or targets in the organism by the bloodstream.

There are a number of reasons why a foreign substance administered to an animal by any of the aforementioned routes does not reach this action point or reaches only in parts. Bioavailability is the fraction of the administered external dose that reaches the systemic circulation in unchanged form. Understandably, there is a better correlation between the internal dose and toxic response of the substance. Nevertheless, as it can be more precisely determined, in the toxicological studies of a chemical compound, the external dose is usually regarded as an organism-independent toxicological parameter.

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As already stated (see Section 1.2), classically, every substance has low and safe doses and high and definitely toxic doses. The threshold dose is the minimal amount of a substance, the administration of which will cause adverse alterations beyond the borders of the physiological adaptation in the animal organism, that is, the start of a concealed temporarily compensated disease. However, in the recent years, this monotonic/linear dose–response paradigm has been challenged by the so-called low-dose hypothesis. According to this hypothesis, hormonally active agents and endocrine-disrupting chemicals (EDCs—see Section 3.3.6) may exert low-dose effects, that is, in the range of typical human exposure, and may display a nonmonotonic U-shaped or inverted U-shaped dose–response profile. A nonlinear relationship between dose and response would not allow, for a given effect, the extrapolation from high to low doses. This would, in turn, challenge the key assumption of a threshold which is implicit in the current risk assessment process for most substances. Several chemicals that can be present in food have been claimed to possess endocrine-active properties and to produce “low-dose effects.” These substances include a number of pesticides, dioxins, polychlorobiphenyls (PCBs), and bisphenol A, as well as natural hormones (Vandenberg et al., 2012). There is a high scientific and public interest in how this information is taken into account for chemical risk assessment and food safety. This hypothesis has been challenged (Rhomberg and Goodman, 2012) and has still not been widely recognized. Nevertheless, the European Food Safety Authority (EFSA) has recently launched a full re-evaluation process of bisphenol A (BPA) as an EDC (see also Section 15.10.2) focusing on exposure and just on low-dose effects (http://www.efsa.europa.eu/en/press/news/120424.htm). It should be remembered that in the case of any substance, no single dose exists that will cause toxic symptoms in all the test animals. Actually, an interval of doses exists in which different individuals of a test group respond similarly, that is, develop similar symptoms of intoxication. What can be the toxic effect we could measure? The measurable effect can be either discontinuous like the death of an organism or continuous such as an alteration of blood pressure or respiration rate or concentration or activity of a physiologically relevant molecule such as a hormone or enzyme. Very often a question arises—if adverse changes occur simultaneously in several organs, which of them is the most important? Answer: The most vitally important effect. Exposure to the toxicant (contact with) can be 1. Acute: Contact time below 24 h, mostly by a single exposure, but there can also be repeated exposures during 24 h. 2. Subacute: Usually repeated contacts during 1 month. 3. Subchronic: Continuous contact during 1–3 months. 4. Chronic: Continuous contact during more than 3 months, most often a daily contact via food, air, and so forth. In the case of animal experiments, it means during the life span of the organism.

Introduction

In some textbooks on toxicology, a slightly different list of the types of exposure can be found; sometimes subacute and subchronic exposures are considered together as one type, sometimes only two—acute and chronic exposures—are presented. In the case of the absorption of sufficiently high doses of a toxicant, an adequate adverse response of the organism emerges. The following responses may occur: 1. Acute: Develops quickly, usually with severe symptoms. For example, an exposure to sufficiently high doses of potassium cyanide is followed by death within few minutes. 2. Subacute: The effects are generally the same as in the case of an acute response, but with weaker symptoms which establish over a longer period (during some weeks). 3. Chronic: Symptoms develop slowly in the case of a systematic long-term absorption of relatively small amounts of the toxicant. At the time of diagnosis, confusion with other pathologies may occur. For example, a tumor initiated by the ingestion of aflatoxins may develop into cancer over decades (see Section 10.2). Acute contact or exposure may cause either an acute or chronic response (intoxication). Acute intoxication is an illness, which forms after a single or repeated exposure of an organism to the toxicant during a short period (some days). Well-known examples are severe anoxemia, caused by carbon monoxide, and tetrodotoxin intoxication, emerging after the intake of toxic puffer fish (see Section 12.3.1), or botulism (see Section 15.4.1.2). Many acute poisonings can result in permanent health disorders even if there is a general recovery. For instance, after acute carbon disulfide intoxication, reflex, excitability, visual, and psychical disorders may persist over a long period. Subacute intoxication may occur in the case of frequent short-term contacts with the toxicant during a period of 28–90 days. It may happen quite often in the case of agricultural workers dealing with pesticides (see Section 13.1). Chronic exposure or contact may cause either a chronic or acute response (intoxication). Chronic intoxication is a disease, formed by a long-term (months or years) effect of small, separately taken harmless daily doses of a toxic substance. Since no signs of poisoning would be visible after a single or even multiple entrance of the substance, it is often very difficult to estimate the genuine reason of the chronic intoxication. A fast-developing chronic intoxication is also referred to as subacute. Chronic intoxication develops in the case of either an accumulation of a toxic substance in the organism (material cumulation) or due to an accumulation of initially unimportant functional alterations in the organism (functional cumulation). In most cases, the second situation prevails. This is the mechanism of the

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effect of fat-soluble substances such as chlorinated hydrocarbons, benzene, or trinitrotoluene. Long-term exposure to low doses of organophosphorus inhibitors depresses cholinesterases to such a low level that symptoms of intoxication start to appear. In the case of toxic heavy metals (Pb, Hg, Cd, etc.), which have a long half-life in the organism, mainly material cumulation occurs, although functional cumulation also plays a definite role in the development of an intoxication. Chronic intoxication occurs chiefly as an occupational disease in the case of workers at the factories producing these toxic metals; in the case of environmental toxicants as DDT, PCB-s, cadmium, or mercury, the possible array of the sufferers is much broader. Some substances such as HCN, due to their fast modification or excretion, never cause chronic intoxication. On the contrary, compounds such as silicon dioxide, which dissolve only slightly in the body liquids, are capable of causing only chronic intoxication. Response (intoxication) can be local or systemic, immediate or delayed, and reversible or irreversible. Local response emerges in the same organ that was in direct contact with the poison, and is systemic in the other distant organs. Many compounds cause damage in the region of the skin or mucous membrane which is directly exposed to the toxicant. The general symptoms of intoxication either reflect the development of pathological processes (like leucocytosis or inflammation in the case of corrosion) or are a result of disruption in the functioning of the injured organ (such as anoxia in the case of pulmonary edema). Many poisons have only a systemic effect. These compounds do not cause any tissue damage before they reach either blood or tissue liquid. An immediate response follows very shortly after contact with the toxicant, whereas a delayed response occurs considerably later. Thus, for example, the symptoms of radiation sickness, like loss of hair or a malignant tumor, may develop only over a long period after the contact with acute irradiation. Toxicants can predominantly produce either

1. A selective effect on one or another organ or system or 2. A simultaneous injury of several organs or systems (polythropic effect).

In the case of many poisons, a molecular or supermolecular site, receptor, exists in the cell, and this should either be reversibly or irreversibly contacted by the toxicant to cause a toxic response (see Section 1.6.3). The intensity of this response depends on the toxicant concentration in the vicinity of the receptor, which, in turn, depends on the dose of the toxicant. A toxicant can have the same point of attack in the case of acute and chronic intoxications; these points can also be different. For example, if in the case of acute benzene intoxication, the central nervous system (CNS) becomes damaged, then a chronic intoxication with benzene injures the hematopoietic system.

Introduction

Repeated attacks of a toxicant may cause the development of tolerance, a phenomenon of a reduced response to the action of the toxicant in case there has been a preceding contact with the same (or similar) compound. This tolerance can be caused either by

1. The induction of enzymes metabolizing the toxic compound (see Section 2.3.4) or 2. Change of the number or binding capacity of specific receptor groups.

On one hand, the second mechanism is connected with (partly) irreversible changes in the spatial structure of the binding site of the receptor during the first contact. As a result, the receptor molecule becomes “damaged” and is not able to have a close contact with the toxic molecule as it did in the first time. Both changes are induced by the first contact with the toxicant molecule. On the other hand, a repeated contact with a toxicant can sometimes promote physiological accumulation and, hence, the amplification of the toxic effect. The simplest acute toxic effect to estimate is the stopping of the organic life of an organism—its death. Death is the severest parameter, which actually should be used as little as possible. Much frequent use of animal tests, in general, conflicts with the Animal Welfare Act enforced by the United States Department of Agriculture (USDA) and by the European Council Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes as well as with ethical norms (Council Directive, 1986). In addition, the use of a lethal dose (LD) provides usually less information about the mechanisms of a toxic effect (see also Section 5.2). Mostly, it is not obligatory to know the LD, but it is necessary to study what happens to the test animals at those doses of a toxicant, which can be contacted by humans or animals in their real life. For the assessment of a chemical’s toxicity, very often it is more beneficial to use a nonfatal biochemical or pharmacological change in the organism such as a decrease of enzyme activity or the appearance of a molecule in blood, characteristic of a definite pathological disorder. An LD is the amount of a substance (usually in logarithmic scale) causing the death of an animal in the absence of antidotes or treatment. Absolute (LD100), minimal (LDmin), and median (LD50) lethal doses (Table 1.1) can be distinguished. In the experimental toxicology, LD50, causing the death of 50% of animals of a test group at an acute contact with a toxicant, has been the most popular dose, LD100 belongs to the most durable, and LDmin to the weakest animal. Instead of doses, concentrations can be used—the respective parameters will be LC50, LC100, and LCmin (mmol/L, μg/m3). The classical LD50 test, which requires torture and sacrifice of a large number of test animals and which had caused long scientific and social debates, was finally canceled by the end of 2002. The alternative animal acute-oral toxicity tests, such as the fixed-dose procedure (FDP), acute toxic class (ATC) method, and up-and-down procedure (UDP), have been developed (see Section 5.4).

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Table 1.1  Approximate LD50 Values for a Selection of Chemical Compounds Chemical Compound

LD50 (mg/kg, rat, orally)

Ethanol Sodium chloride Paracetamol Malathione Lindane Morphine sulfate Caffeine, aspirin Sodium nitrite DDT Arsenic Dieldrine Strychnine sulfate Aflatoxin B1 Nicotin Tetrodotoxin Tetrachlordibensodioxin (TCDD) Botulinum toxin

10,000 4000 1900 1200 1000 900 200 180 100 48 40 2 1.2 1 0.1 0.001 0.00001

For the estimation and comparison of acute toxicities of substances, dose– response curves are still very often used, where the percentage of the response is plotted against the logarithm of the dose (see Figure 1.1). What do the location and shape of the curve tell us? • The slope of this curve speaks about the predictability. For example, if two toxicants have the same LD50, but the slope of curve B is steeper than 100

% Mortality

14

B

50

A 0

Dose

Figure 1.1  Relationship between percentage of mortality and log dose. (From http://www.fao.org/ docrep/008/y5723e/y5723e0l.htm.)

Introduction

in the case of toxicant A (Figure 1.1), a change in the dose necessary to produce the same response is smaller when compared to toxicant A. In the case of equality of all other parameters, the degree of predictability is higher in the case of toxicant B. • The location of the curve with respect to the x-axis shows the potency of a toxicant to induce toxic responses, that is, its toxicity. The curve, representing the substance with a higher toxicity, is located more left. • Sometimes, instead of the usual S-shaped curve as shown in the Figure 1.1, a curve resembling the shape of a saxophone appears. In this case, low doses cause effects favorable for the organism, turning into adverse ones only at higher doses of the chemical. Such a phenomenon is called hormesis. Botulinum toxin, which can emerge in food as a result of contamination with bacterium Clostridium botulinum (Section 15.4.1.2), is acutely the most toxic substance known hitherto. Instead of death, any other exactly fixable physiological (biochemical) parameter can be used for toxicity assessment. The corresponding statistical parameter will then be ED50—dose that evokes 50% of the possible alteration of the parameter or IC50 in case of inhibition of some physiological (biochemical) process. IC50 corresponds to the inhibitor dose causing 50% of the maximum inhibition. In the case of oral intoxications as it occurs with food-borne poisons, the toxicant doses are expressed either in milligram or microgram per kilogram body weight or per whole conventional person with a body weight of 70 kg (in the case of pesticides—50 kg). For the estimation of severity of an intoxication and efficacy of the treatment, it is important to measure the toxicant concentration in the blood plasma as well as in various other tissues. The threshold doses (concentrations) inducing acute severe intoxications are always remarkably higher than the doses causing chronic intoxications. The most often used modes of expression of threshold doses/concentrations are • NOEL (no-observed effect level)—The highest dose/concentration, not causing any effect in any animal in a test group. • NOAEL (no-observed adverse effect level)—The highest dose/concentration, not causing any adverse (toxic) effect in any animal in a test group; in food toxicology, NOAEL is the basis for counting of acceptable daily intakes (ADI). ADI is a factor, used for expressing safe consumption of food that contains contaminants such as pesticide or veterinary drug residues or food additives (see Chapters 6, 14 through 16). • LO(A)EL—The lowest observed (adverse) effect level—the lowest dose causing an observable effect, at least in part of the animals. • BMD—Benchmark dose for low toxicant doses. This is the dose in the point of departure from background level response, based on all the available data in a dose–response series, the statistical estimate of

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Principles of Food Toxicology

100 80

% Affected

16

60 40 NOAEL 20 LOAEL

0 BMD BMDL

Dose

Figure 1.2  Comparison of the threshold and benchmark doses.

the NOAEL (Figure 1.2). BMD generally estimates an increased risk of 10% over background for the response of interest. While different levels of excess risk may be used, this level of risk is presumed to be at or near the limit of sensitivity for most cancer and some noncancer, bioassays. The benchmark dose lower confidence limit (BMDL) attempts to increase the confidence of the estimate by taking into account the variability of the data. The BMDL typically uses the lower bound of a 95% confidence limit on the BMD. An improved understanding of the mechanisms of toxicity, the use of biologically based models, and other tools may help in extending the dose–response curve to lower levels of exposure, for example, to help in addressing low-level veterinary drug residues. Other approaches such as sensitive biomarkers (see Section 3.4), biochemical changes in response to chemical exposure, are also being developed. Changes to biomarkers may occur at exposures well below those required to cause the changes observed in traditional toxicological studies. As a result, the use of biomarkers may be particularly useful in characterizing response to very low residues of veterinary drugs. More about the benchmark dose can be read, for example, in the review paper by Sand et al. (2008). 1.4  INTEGRATED EFFECT OF TOXIC SUBSTANCES An organism can be attacked very often simultaneously by several potentially toxic compounds. Such a situation may occur, if, for example, a patient must take

Introduction

several drugs or in the case of contact of a human being or an animal with environmental or industrial toxicants. The resultant toxic effect of such a mixture can be essentially different from the effect if every toxicant acted separately and very often unpredictably. The following situations can occur in this case: 1. Additivity of responses. Often the toxic effects can be simply summarized as 1 + 1 = 2. This situation occurs when the mechanisms of action of the compounds are similar. 2. Synergism of responses. In this case, the combined effect of two different substances is bigger than the simple sum of the separate effects (1 + 1 > 2). The toxic effect of a chemical substance can be even potentiated by another compound, which otherwise is harmless in the concentration used. Examples: • Piperonyl butoxide (PB) is added to some insecticides (pyrethrins, pyrethroids, rotenone, and carbamates) to enhance their insecticidal effect. PB inhibits metabolic detoxification of the pesticide by the insect, reducing the amount of the active ingredient required. • Carbon tetrachloride and ethanol together have a much higher hepatotoxicity than either of the substances taken separately. • Sulfirame, used for the treatment of alcoholism, potentiates the toxicity of ethanol. By inhibiting aldehyde dehydrogenase, catalyzed oxidation of acetaldehyde, sulfirame promotes its accumulation in the organism (see Section 15.3). The process is accompanied by an extremely poor feeling. 3. Antagonism of responses. The toxic effect of chemical A can be weakened (inhibited) by the addition of another toxicant B (antagonist). 1 + 1  C1. Fick’s relationship applies to a system at a constant temperature and for diffusion over the unit distance. In the case of hydrophobic substances, factor K depends mainly on three factors: • On the relative lipid solubility of this compound, often characterized by its water–octanol distribution constant Kow = Co/Cw, where Co and Cw are the compound’s equilibrium concentrations, respectively, in n-octanol and water. The higher the Kow is, the easier and faster the molecules of this substance diffuse through a lipid membrane. Usually, log P (decimal logarithm of Kow) is used. Log P can be calculated directly from the molecular structure of the compound using various mathematical models that utilize molecular fragmentation schemes and other structure-based indexes (Zhu et al., 2005).

Introduction

• On the dimensions of the diffusing molecule. Small molecules penetrate the membrane faster. • On the degree of ionic dissociation of the diffusing molecule. Many substances exist in two forms—ionized and nonionized. Diffusion of the ionized form through the membrane is relatively impeded, since ions adsorb better to the polar membrane surfaces and move slower in the nonpolar lipid phase.   In a normally functioning dynamic biological system, there is always a difference (gradient) of substance concentrations between the two sides of the membrane. The formation of this gradient is caused by the differences in H+ (H3O+) ion concentrations (pH values) of the liquid in these spaces, governing the dissociation of the other ionizable molecules.   The degree of ionization of acidically dissociable molecules is described by the Henderson–Hasselbach equation:



[A − ] log ------ = pH − pK a , [HA]

where pKa is the negative decimal logarithm of the acidic dissociation constant of an acid HA. [HA] and [A−] are the equilibrium concentrations of the nonionized and ionized forms of the substance, respectively. If pH = pKa, then half of the substance molecules are in a dissociated ionic form, increase or decrease of pH causes an increase or decrease of the number of the molecules in the ionized form, respectively.   In the case of basically ionizing molecules:



[AH + ] log ------- = pK a − pH, [A]

where [A] and [AH+] are the equilibrium concentrations of the nonionized and ionized forms of a base A, respectively. The increase of pH now causes a reduction of the number of ionized molecules and vice versa.   Since in both cases the membranes are preferentially penetrated by more lipophilic nonionic particles, the weak acids are concentrated in a space with a high pH and the weak bases in the space of low pH. Particles, having strongly and pH-independently ionizing groups such as sulfate or tetra-ammonium ions, are not able to pass the membranes by passive diffusion at any physiological pH. 3. Facilitated (favored) diffusion. In this case, concentration gradient and transporting molecule are needed, and the process can be saturated at high concentrations of the diffusing substance. This process is faster

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than passive transport and, as in the case of other diffusions, chemical energy is not required. This is how usually endogenous (organism-borne) substances and structurally similar exogenous substances (xenobiotics) are transported through the biomembranes. So, for example, the transport of glucose and amino acids from enteric cells to the bloodstream is organized. Active transport of molecules through the membrane, carried out against the concentration gradient, is a selective process requiring metabolic energy. Two main mechanisms are known: 1. Simple active transport. A molecule passes through a membrane complexed with a specific transport molecule residing on the surface of the membrane. Similar molecules must compete for their common transporters. Since there can occur a shortage of the carrier molecules in the case of higher concentrations of molecules to be transported, the process is saturable and preferably of zero order, and is inhibitable by the metabolic poisons inhibiting the production of ATP, such as cyanide, azide, or dinitrophenol.   The transport molecules can be uniports, symports, or antiports. The uniports transfer one molecule in one direction, symports and antiports two molecules in the same or opposite directions. This type of membrane transport has been built up by the organism for specific endogenous and nutritious compounds, but in the same way, similar exogenous molecules and ions, such as lead from the stomach, can be transported. Active transport is essential in the case of xenobiotics moving into the liver, kidney (elimination), and the CNS, and for the maintenance of the electrolyte and nutrient balance. 2. The subtypes of endocytosis are phagocytosis and pinocytosis. Phagocytosis is anticipated for the transport of large particles (microorganisms, cell debris, etc.), pinocytosis for the transport of dissolved macromolecules such as insulin in the CNS or botulinum toxin in the gut. Endocytosis is a complex type of transportation, starting with invagination of the membrane part, named as coated pit, to enclose a particle or droplet, followed by detachment in the form of a coated vesicule from the remaining membrane and entering the cell. Many molecules and particles, endocytozed by a cell, are transported into lysosomes. In the cell, the membrane bag delivers its content and returns to the outer membrane forming a new coated pit there. In most animal cells, endocytosis represents a very selective mechanism of concentration of compounds, used by the cell to collect specific macromolecules from its environment. Actually, the forming vesicule also includes extracellular liquid and nonspecific, highly concentrated molecules. Obviously, such transport cannot be very extensive, but

Introduction

is still considerable. For example, a macrophage is able to internalize a membrane with an area equal to its own surface area in 30 min. This is the way the particles of asbestos and uranium dioxide enter the pulmonary cell and are transported through membrane peptides and antigen–antibody (AG:AB) complexes. Most eukaryotic cells pinocytose continuously; phagocytosis is characteristic only of specialized cells, such as mammal macrophages and neutrophiles—both of which have been developed from the same ancestor. Phagocytosis, different from pinocytosis, requires induction, for example, by antibodies. 1.6.3 Receptors On the outer surface of a cell, there are numerous biochemically sensitive and active sites called receptors, consisting of proteins (protein complex, glycoprotein, and lipoprotein) that usually extend throughout the membrane to the cytosol. Toxicologically important receptors may be located also in the cytoplasm. Binding of a specific complementary (matching in dimensions and in charge distribution) molecule called a ligand to the binding site of this receptor complex will change the conformation of this site. This transformation initiates consecutive alterations (signal transductions) along the whole integral receptor complex up to its intracellular end. This process triggers a chain of biochemical reactions leading to alterations in the physiological (biochemical) state of the whole cell. If this alteration is necessary and favorable for this cell or the respective organ or even for the whole organism, we have a normal regulation of cell and organism life. It is also beneficial if the ligand is a drug molecule, the right dose of which enables to correct the physiological state of a sick cell. But, the same receptor complex can be attacked by substances, capable of giving a signal for taking the cell significantly out of its normal physiological state. In this case, we have to do with a toxicant–receptor interaction, which, depending on the toxicant’s dose and other circumstances, can lead to the death of the cell, organ, or even the organism. The ligand–receptor interaction and the respective response can be described by the following simplified scheme: LR L + R  LR K → RESPONSE



where KLR is the equilibrium constant of the ligand–receptor complex LR. The target molecules of the ligand–receptor interaction, followed by conveying of the conformational alterations in the receptor, can be • • • •

Cellular enzymes (reduction or magnification of their activity) Other cellular proteins (tubulin and carrier molecules) Other cellular macromolecules (DNA and RNA) Other receptors located at the same cellular membrane

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A ligand–receptor complex, LR, can be formed by a combination of the following bonds (the respective approximate bond energies in kcal/mol is given in brackets): 1. Covalent bond (100)—A common electron cloud between two atoms, seldom in LR, irreversible. 2. Ionic bond (5)—Between oppositely charged atoms, usual in LR, reversible. 3. Hydrogen bond (2–5)—Binding through a common H-atom, usual in LR, reversible. 4. Hydrophobic interaction (1)—A weak binding between nonpolar groups, entropic, usual in LR, reversible. 5. van der Waals bond (0.5)—Weak electrostatic attractive power, usual in LR, reversible. The higher the bond energy is, the stronger and more stable, both chemically and thermally, the bond will be. The covalent bond is by far the strongest of them and, therefore, it forms the basis for the stable structure of a molecule. Formation of a covalent bond between a ligand and receptor most often means that this receptor molecule gets irreversibly blocked and it is very difficult, if not impossible, to regenerate the initial molecule, capable of building up native LR complexes. In the case of normal physiological processes, there is no place for covalent bonds in LR complexes. Weaker noncovalent bonds of types 2–5 can be complementarily combined to form a physiological receptor–ligand complex. The mutual binding sites of a receptor and its genuine ligand molecules are related to each other similar to a key and its respective lock. The differences from a usual door lock and key are as follows:

1. The cellular lock is not too precise, enabling the formation of complexes with slightly different molecular keys. 2. The molecular key is able to modify slightly the structure of the cellular lock.

Just as these properties form the basis for the ability of a ligand molecule to direct the intracellular processes from the outside, a not too high specificity of a receptor enables the existence of antagonists of the ligand–receptor interaction. The degree of fitness of the ligand binding site with the binding site of the receptor is characterized by the term affinity, a measure of which is the constant KRL (see Equation 1.4). The bigger the KRL is, the stronger the link between the receptor and the ligand will be. Actually, the link must be of physiologically substantiated right strength. In addition to specific ligand(s), a receptor also has specific agonists and antagonists. Agonists are exogenous substances that also selectively bind to the receptor and activate it by triggering a response in the cell. They mimic the action

Introduction

of endogenous biochemical molecules (such as hormones or neurotransmitters) that bind to the same receptor. Antagonists are, in turn, substances that, binding too tightly to the receptor, do not activate it; since they hinder ligand binding, they inactivate the receptor. The role of receptors in the induction of enzymes of biotransformation will be discussed in Section 2.3.4.1 and in the development of toxic responses in Section 3.3.2. 1.6.4  Ion Channels Ion channels are pore-forming transmembrane protein assemblies that help in establishing and controlling the voltage gradient across the plasma membrane of cells by allowing the flow of ions down their electrochemical gradient. Ion channels are present in all membranes surrounding the biological cells. They have very important role in the development of (neuro)toxic responses. There are four main types of ion channels. 1. The voltage-gated channels such as the sodium and potassium channels of the nerve axons and nerve terminals, the target of toxic action, for example, of tetrodotoxin (Section 12.2.1), ciguatoxins (Section (12.2.2), saxitoxins (Section 12.1), and grayanotoxin (Section 7.15). 2. The extracellular ligand-activated channels, such as GABA (γ-aminobutyric acid) and glycine receptor channels, most of which are regulated by ligands that are “neurotransmitters.” These channels are often named according to the ligand they bind to. Poisons of the extracellular-activated channels are, for example, thujones (GABA—Section 18.3) or cyclodiene-type insecticides. 3. Intracellular ligand-gated ion channels. These include cystic fibrosis transmembrane conductance regulator (CFTR) and some other ABC family members as well as ion channels involved in sense perception. These are often activated indirectly by G-protein-coupled receptors (GCPRs). Other common intracellular ligands which activate these kinds of channels include calcium ions, ATP, cyclic AMP and GMP as well as phosphatidyl inositol (PI). 4. The mechanosensory and volume-regulated channels have their own grouping, but they are still in the process of being classified. It has been shown by sequence comparison that ion channels within the above groups also will show the greatest sequence similarity and are, therefore, most likely to have descended from a common ancestor. A fifth “catch-all” group, which includes any ion channels, is not included above. This group includes the GAP junctions, peptide ion channels, such as gramicidin, and various venomous insect toxins like the conus toxins from cone shells.

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REFERENCES Council Directive. 1986. European Council Directive of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/EEC) http://ec.europa.eu/food/fs/aw/aw_legislation/ scientific/86–609-eec_en.pdf Harrington, J.M. and Gill, F.S. 1992. Occupational Health Pocket Guide, 3rd ed. Blackwell Scientific Publishers, Oxford. Hayes, A.N. and Gilbert, S.G. 2009. Historical milestones and discoveries that shaped the toxicological sciences, In Molecular, Clinical and Environmental Toxicology, Vol. 1., Molecular Technology, Luch, A, (Ed.)., Birkhäuser Verlag, Switzerland. Kobert, R. 1906. Lehrbuch der Intoxikationen, Ferdinand Enke; Stuttgart, Germany (in German). Martinetz, D. 1982. Arsenik, Curare, Coffein, Gifte in unserer Welt, Urania-Verlag, Leipzig (in German). Möller, L., Ed. 2000. Environmental Medicine, Karolinska University Press, Stockholm. Pappas, A.A., Massoll, N.A., and Cannon, D.J. 1999. Toxicology: Past, present and future, Ann. Clin. Lab. Sci., 29, 253–262. Rhomberg, L.R. and Goodman, J.E. 2012. Low-dose effect, non-monotonic dose–response of endocrine disrupting chemicals. Has the case been made? Regul. Toxicol. Pharmacol., 64, 130–133. Sand, S. et al. 2008. The current state of knowledge on the use of the benchmark dose concept in risk assessment, J. Appl. Toxicol., 28, 405–421. Stillmark, H. 1888. Ueber Ricin, ein giftiges Fragment aus den Samen von Ricinus communis L. und einigen anderen Euphorbiaceen, Kaiserliche Universität zu Dorpat; Tartu, Estonia (in German). Vandenberg, L.N. et  al. 2012. Hormones and endocrine-disrupting chemicals: Low-dose effects and nonmonotonic dose responses, Endocr. Rev., 33, 378–455. Zhu, H. et al. 2005. A new group contribution approach to the calculation of LogP, Curr. Comput. Aided Drug Des., 1, 3–9.

2 Routes of Xenobiotics in an Organism Ingested and inhaled substances are considered outside the body until they cross the cellular barriers of the gastrointestinal tract (GIT), the respiratory system, or the skin. When a xenobiotic is in the GIT, in the lungs, or on the skin, it finds itself in the physiological route of absorption–distribution–metabolism–(new distribution)–excretion (ADME). Now, its toxicity depends on many factors, including the speed and the exact way of moving along this labyrinth. Since a free toxicant or its active metabolite is at the action point (tissue) in a dynamic equilibrium with free toxicant in the plasma, in most cases, the toxic effect is proportional to the toxicant’s concentration in the blood. The intensity of the toxic effect depends on the pharmacokinetics of the xenobiotic, that is, how the compound enters the organism and the bloodstream (absorption), how and in which (original or metabolitic) form it moves along the body (distribution and metabolism), and how the xenobiotic and/or its metabolites leave the organism (excretion). The pharmacokinetics of a toxicant is substantially influenced by the age, gender, lifestyle, nutritional condition, and dietary habits of the individual, tissue (liver, kidneys) functions and conditions, and so forth. Three of the blocks in the aforementioned scheme—absorption, distribution, and excretion—are connected with the transposition of the substance in the organism, which comprises crossing of biomembranes (see Section 1.6). 2.1  ENTRY AND ABSORPTION OF FOREIGN COMPOUNDS Xenobiotics can enter the body of a mammal through three main gateways (portals)—skin, lungs, and GIT—besides intravenously, intramuscularly through injections, and so on. Since most foreign substances, including toxicants, enter perorally, it is the GIT that is the most important gateway, where the primary toxic effects of xenobiotics manifest. Respiratory pathways, including the lungs, are the entering portal and site of absorption of volatile toxicants such as carbon monoxide, nitrogen oxides, and so forth. Transdermal entry is essential for organic solvents, detergents, and other fat-soluble lipophilic substances, which can, by dissolution of the skin fats, cause local irritation and dermatitis. 27

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Toxic compounds may exert their primary adverse effects at the entry to the body, but to cause a systemic physiological effect, they must first pass through membranes into the bloodstream by absorption. The role of the interstitial fluids and lymph in the transport of substances is generally less important (see Section 2.2.2). For crossing of membranes, a substance always selects the easiest way (see Section 1.6.2). Mammals have no special system for the absorption of xenobiotics; these cross the membranes through the same passages as the endogenous compounds. Most of the toxicants are absorbed by passive diffusion. Absorption is usually a complicated multistep process, wherein the substances often get metabolized that may lead to their reduced bioavailability. On the contrary, many compounds such as plant polyphenols that enter the GIT as glycosides may be deglycosylated by enzymes of either enteric flora or cells of gastric wall into more hydrophobic aglycones that will have a higher bioavailability than respective glycosides (D’Archivio et al., 2007). 2.1.1  Digestive Tract Various food components and drugs enter the organism via the oral cavity and the digestive tract (esophagus, stomach, small and large intestines, and rectum), which is specifically adapted for the digestion and absorption of food. The digestive tract also forms the first line of defense in the body against the main load of xenobiotics; the gastrointestinal mucosa has several mechanisms for the modification of foreign compounds. The ingested food is first digested through physical and chemical processes, followed by the absorption of the refined macro- and micronutrients, mainly in the duodenum and in the upper jejunum. Absorption is mostly performed by enterocytes using passive diffusion, transcytosis, and active transporters. During this process, nutrients are usually intensively metabolized into a form suitable for transport to the target organs or even by phase I and phase II metabolic enzymes (see Section 2.3) (Langerholc et  al., 2011). Actually, the bioavailability of any compound depends on a large number of different factors, including chemical structure, concentration in food, interaction with numerous other food compounds such as proteins or lipids, intestinal factors such as activity of enzymes and microflora, intestinal transit time, and systemic factors, such as gender and age, genetics, pathologies, and so forth. The digestive tract is also the site of absorption of food toxicants. Lipophilic substances, including toxicants such as phenols and cyanide ions that easily cross the biomembranes, are usually absorbed already in the oral cavity. For instance, there is a positive correlation between the partition coefficient Kow (see Section 1.6.2) and sublingual absorbtivity of alkaloids. To achieve the “desired” physiological effect, the sublingual dose of lipophilic cocaine must be twice the hypodermic dose, and for a considerably more hydrophilic morphine, the respective ratio is 10. Oral absorption eliminates the destructive effect of gastric and intestinal juices as well as hepatic metabolism of a toxicant that sometimes may increase the toxicity of xenobiotics.

Routes of Xenobiotics in an Organism

Here, we again encounter (see Section 1.3) the important toxicological term bioavailability that denotes part of the oral dose (possible interval 0–100%) that reaches the bloodstream. The fact that bioavailability is generally smaller than 1 (or 100%) is caused by partial enzymatic destruction in the GIT, incomplete absorption of the compound, and its metabolism or biotransformation in the enteric cells. The gastrointestinal epithelium is only one cell thick and has a large surface area in different parts. Since, by moving along the tract, the pH changes substantially, it is possible to find a suitable absorption site even for ionizable substances. At the very beginning of their journey down the alimentary canal, the ingested substances contact the oral cavity, where the pH is near 7 in humans and it is more basic in rats and some other species. Ethanol, hydrocarbons, and other neutral nonionizable substances begin to be absorbed/digested. Weak acids diffuse across the membrane best of all in the stomach, where the pH is approximately 2 in humans, and in many other mammals, weak bases pass the membranes in the intestine, where the pH ≈ 6. However, if the molecule of a weak acid for some reason was not absorbed in the stomach, it can be absorbed in the intestine that has a good supply of blood and, due to the folding of the intestine wall and villi, a large surface area. A lipid-soluble nonionizable substance absorbs equally well in any part of the digestive tract, and its absorbtivity increases with an increase in the Kow, that is, the lipophilicity of the substance. This rule does not apply for every lipophilic (Kow > 3000) molecule that tends to form in the stomach and intestine supramolecular colloidal solution, the dispersed particles of which, micelles, are too large to cross the membrane. Molecules with a mass of over 3000 Da are not able to cross the digestive tract wall by simple diffusion. For nutrients and endogenous substances (saccharides, amino acids, amines, inorganic salts, etc.), there are specialized transport systems in the cell membranes of the digestive tract. For example, P-glycoprotein facilitates the absorption of many compounds. The dependence on the throughput of specialized membranous systems may reduce the degree of absorption, and, vice versa, factors such as saturation and competition can increase the absorption. Metals are absorbed mainly in the first part of the small intestine—chromium, manganese, and zinc in the ileum; iron, copper, mercury, thallium, and antimony in the jejunum. Alkali metals are absorbed quickly and completely, and alkaline earth metals to the extent of 20–30% as hydroxides or poorly soluble phosphate complexes. Metals may, during the absorption process, change their valence form; for example, bivalent iron can be oxidized into trivalent iron, and insoluble inorganic salts of lead may be changed into lead-organic substances, which will absorb much better than inorganic lead. Absorption is favored by ulcers and irritations of the GIT as well as by starvation, but hindered by ample food and vomiting. In contrast, food can accelerate the absorption of a substance that dissolves in fat, especially in milk fat. Milk proteins, in turn, adsorbing various low-molecular-weight substances,

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may remarkably reduce their absorbtivity, although contrary results have been obtained through in vitro study of green tea catechins (Xie et al., 2012). The rate of absorption is also influenced by the intensity of the blood supply in the stomach mucosa, the formation of mucus, and peristalsis. In the case of drugs, the vehicle, added to facilitate the suspension or dissolution of the active substance, may have a significant effect on the toxicity of the medicine, influencing its absorption and distribution in the body. The exact absorption site plays a significant role in the fate of a xenobiotic. Therefore, a substance can be hydrolyzed in the acidic content of the stomach that may play a role, for example, in the inactivation of a snake toxin or other proteinaceous toxin. A foreign compound can be hydrolyzed by the enzymes produced by the intestinal wall cells, the pancreas, or the intestinal bacteria. For the majority of compounds, oral administration will be followed by absorption into the portal vein supplying blood from the region of the digestive tract to the liver. 2.1.2 Lungs Lung tissue has a very large effective area (50–100 m2 in the case of humans) and an excellent blood supply. The pulmonary alveolus is separated from the blood vessels by a layer with a thickness of one to two cells. Therefore, the absorption in the lungs is very fast and effective. The air we breathe contains various toxic substances of different potencies and particles such as carbon monoxide (CO), nitrogen oxides (NOx), sulfur dioxide (SO2), vapors of organic solvents, aerosols, and solid particles (asbestos and pollen). Two essential factors influencing the absorption of substances from the pulmonary alveoli are the speed of breathing and the speed of blood flow. In the case of substances that are poorly soluble in blood, the speed of blood circulation is crucial; in the case of well-soluble substances, the speed of breathing is more important. The capability of the substance to form complexes with blood plasma (especially transport) proteins is also a very important factor. Small fat-soluble molecules are absorbed very fast. For these molecules, the pulmonary route is the most important way of entering the organism. Hence, the water-insoluble particles of uranium dioxide can, on being absorbed via lungs, initiate liver damage. Lead is also absorbed from the air through the lungs. Pulmonary absorption of compounds and particles can occur by pinocytosis or phagocytosis, respectively (see Section 1.6.2). Phagocytosis can harmlessly leave toxic asbestos particles in the wall of the pulmonary tissue for a long period. Carcinogenesis may start much later without any extra alarm. 2.1.3 Skin Skin is a complex membrane structure consisting of thick layers of various cells and intercellular cavities. Although the skin, potentially, has the highest contact with gases, solvents, and dissolved substances, and its area is quite considerable (approximately 2 m2 in the case of a medium-sized human), skin serves, due to its

Routes of Xenobiotics in an Organism

Table 2.1  Dependence of the Interval between Contact with a Toxic Compound and Death of the Organism on the Absorption Site Absorption Site

Interval (min)

Lungs Eye Digestive tract Skin

15–30 15–30 30–120 60–240

multiple cell layers (epidermis, derma, and subcutaneous adipose tissue), with a poor blood supply as an efficient barrier to the penetration of substances. Human skin permits two distinct routes of absorption:

1. Through the epidermis (in most cases) 2. Through the sweat glands and follicula (e.g., lead)

The skin can be permeated in considerable amounts only by lipophilic solvents such as dimethyl sulfoxide (DMSO). The skin itself can be damaged by corrosive substances such as strong acids or alkalis. Ionic substances are either not able to penetrate the skin or they do so extremely slowly. Nevertheless, cases of percutaneous intoxication with pesticides (e.g., with insecticide parathion) have been reported. Percutaneous absorption is favored by warm skin, sweating, rash, scratches, and injuries of the skin. A review of absorption through the skin and in vitro approach has been published by Barbero and Frasch (2009). In the case of lipid-soluble substances, the route of entry is of high importance for the starting moment of the toxic effect, since the time after the exposure, during which it is possible to weaken the toxic effect which depends on the absorption site. Table 2.1 provides some very approximate information about this issue. 2.2  DISTRIBUTION OF XENOBIOTICS IN THE ORGANISM 2.2.1  Blood Supply and Membrane Barriers When a substance is absorbed, it passes through the cell linings of the absorbing organ into the interstitial fluid surrounding the cells (about 15% of the body weight). The other body fluids are the intracellular fluid inside the cells (about 40% of the body weight) and the blood plasma (about 8% of the body weight). These body fluids actually represent one large pool. The first two of the fluids are quite motionless, and their constituents are not mechanically transported as in the blood. They can leave the interstitial fluid by entering either local tissue cells or blood capillaries and further to the general circulatory system or into the lymphatic system. Once a substance is in the bloodstream, it may

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be further distributed between various tissues, excreted from the organism or stored, and biotransformed into different metabolites; the substance or its metabolites may interact or bind with cellular components of tissues where they were distributed. The further journey of the substance particles (molecules, ions), either in a bound or in an unbound form, is governed by the properties of various cellular membranes along the circulatory system and binding in the body liquids. Since virtually all tissues have a blood supply, all organs and tissues of the body are potentially exposed to any absorbed substance. Differences in the blood supply of different tissues are also of high importance in the distribution of foreign compounds. The largest volume of the blood crosses the liver, kidneys, brain, and skin, while much less blood circulates in the fat (adipose) tissue and bones. In the absence of selectivity, the highest amounts of a foreign substance should reach the first four tissues. Relatively small organs such as the thyroid gland and pancreas get much blood owing to their low weight. The distribution of xenobiotics is a dynamic process. It depends on the ratio of the rates of absorption, metabolism, and elimination (excretion), on the physical and chemical properties of the substance, and the particular environment in various body compartments. The distribution of a chemical to body cells again requires penetrating a series of cell membranes, first of the cells of capillaries and then the cells of target organs. Knowing its concentration in the plasma, it is possible to calculate important pharmaco- or toxicokinetic parameters of a substance such as its half-life, area under the curve (AUC), volume of distribution, and the body burden. Half-life (τ1/2) is the time during which one-half of the initial dose of a substance becomes degraded, metabolized, or eliminated from the organism (Figure 2.1). Half-life is determined by metabolism and excretion rates. The AUC is the area in a plot of the concentration of a xenobiotic in the plasma (y-axis) against time (x-axis). AUC has the unit (mass/volume) × time and expresses the total amount of a xenobiotic absorbed by the body, irrespective of the rate of absorption (Figure 2.2). C0

C0/2 C0/4 τ1/2

2τ1/2

3τ1/2

4τ1/2

Figure 2.1  Explanation of the term “half-life.” Co is the concentration of a substance at moment 0.

Routes of Xenobiotics in an Organism

Cmax

Plasma concentration

Absorption phase Elimination phase (τ1/2)

AUC Tmax

Cmin Time

Figure 2.2  Explanation of the term “area under the curve” (AUC).

The volume of distribution VD (liters) is the volume of body liquid in which a foreign compound is distributed.

VD =

Dose (mg) Plasma concentration (mg/L)

If a toxicant is distributed only in the blood plasma, a low VD will result, but if it is distributed in all the body fluids, or concentrated (accumulated) in some solid tissue (Section 2.2.3), such as adipose tissue, VD will be high. Body burden is the total amount of foreign chemicals that are present in a body at a given point in time. Sometimes, it is also useful to consider the body burden of a specific single chemical, such as lead, mercury, or dioxin.

Body burden (mg) = Plasma concentration (mg/L) × VD (L)

Organisms have metabolizing membrane barriers such as placental (fetomaternal) or blood–brain (hematoencephalous) barriers that hinder the penetration of various foreign substances into the fetus and brain, respectively. Unfortunately, these barriers are not perfect. Hence, the blood–brain barrier is easily crossed by toxicants such as organic methyl mercury, lead, aluminum, and ethanol. Ethanol even enhances the permeability of this barrier, thus increasing the toxicity of other poisons to the brain cells. Since the brain is not fully developed in newborns, they are especially sensitive to the toxic effects of chemical substances. The placental barrier is crossed by simple diffusion by small (M