A TEXTBOOK OF - Action Modulerswikimariner.actionmodulers.com/wiki/images/8/80/Modern... · 2017-11-17 · PART III TOXICANT PROCESSING IN VIVO 77 5. Absorption and Distribution of

A TEXTBOOK OF MODERN TOXICOLOGY

A TEXTBOOK OF MODERN TOXICOLOGY

FOURTH EDITION

Edited by

Ernest HodgsonNorth Carolina State UniversityRaleigh, North Carolina

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2010 John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.Published simultaneously in Canada.

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifi cally disclaim any implied warranties of merchantability or fi tness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profi t or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com.

Library of Congress Cataloging-in-Publication Data:

A textbook of modern toxicology / edited by Ernest Hodgson. —4th ed. p. cm. ISBN 978-0-470-46206-5 (cloth) 1. Toxicology. I. Hodgson, Ernest, 1932– RA1211.H62 2010 615.9—dc22 2009045883

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

www.copyright.comhttp://www.wiley.com/go/permissionhttp://www.wiley.com/go/permissionwww.wiley.com

CONTENTS

PREFACE TO THE FOURTH EDITION xxi

CONTRIBUTORS xxiii

PART I INTRODUCTION 1

1. Introduction to Toxicology 3Ernest Hodgson

1.1 Defi nition and Scope 31.2 Relationship to Other Sciences 91.3 A Brief History of Toxicology 101.4 Dose–Response Relationships 111.5 Sources of Toxic Compounds 121.6 Movement of Toxicants in the Environment 12Bibliography and Suggested Reading 13Sample Questions 14

2. Introduction to Biochemical and Molecular Methods in Toxicology 15Ernest Hodgson, Gerald A. Leblanc, Sharon A. Meyer, and Robert C. Smart

2.1 Introduction 152.2 Cell Culture Techniques 15

2.2.1 Suspension Cell Culture 162.2.2 Monolayer Cell Culture 162.2.3 Indicators of Toxicity in Cultured Cells 162.2.4 Use of Stem Cells 172.2.5 Cell Culture Models as “Alternative” Toxicity Tests 19

2.3 Molecular Techniques 192.3.1 Molecular Cloning 202.3.2 cDNA and Genomic Libraries 202.3.3 Northern and Southern Blot Analysis 212.3.4 PCR 222.3.5 Evaluation of Gene Expression, Regulation, and Function 22

2.4 Immunochemical Techniques 232.5 Proteomics 262.6 Metabolomics 262.7 Bioinformatics 262.8 Summary and Conclusions 27

v

vi CONTENTS

Bibliography and Suggested Reading 27Sample Questions 27

PART II CLASSES OF TOXICANTS 29

3. Exposure Classes, Toxicants in Air, Water, Soil, Domestic, and Occupational Settings 31W. Gregory Cope

3.1 Air Pollutants 313.1.1 History 313.1.2 Types of Air Pollutants 323.1.3 Sources of Air Pollutants 333.1.4 Examples of Air Pollutants 343.1.5 Environmental Effects 37

3.2 Water and Soil Pollutants 383.2.1 Sources of Water and Soil Pollutants 383.2.2 Examples of Pollutants 39

3.3 Occupational Toxicants 423.3.1 Regulation of Exposure Levels 433.3.2 Routes of Exposure 443.3.3 Examples of Industrial Toxicants 44

Bibliography and Suggested Reading 46Air Pollutants 46Water and Soil Pollutants 47Occupational Toxicants 47

Sample Questions 47

4. Classes of Toxicants: Use Classes 49W. Gregory Cope and Ernest Hodgson

4.1 Introduction 494.2 Metals 49

4.2.1 History 494.2.2 Common Toxic Mechanisms and Sites of Action 504.2.3 Lead 514.2.4 Mercury 524.2.5 Cadmium 534.2.6 Chromium 534.2.7 Arsenic 544.2.8 Treatment of Metal Poisoning 54

4.3 Agricultural Chemicals (Pesticides) 554.3.1 Introduction 554.3.2 Defi nitions and Terms 564.3.3 Organochlorine Insecticides 59

CONTENTS vii

4.3.4 Organophosphorus (OP) Insecticides 604.3.5 Carbamate Insecticides 614.3.6 Botanical Insecticides 614.3.7 Pyrethroid Insecticides 624.3.8 New Insecticide Classes 624.3.9 Herbicides 624.3.10 Fungicides 644.3.11 Rodenticides 644.3.12 Fumigants 654.3.13 Conclusions 65

4.4 Food Additives and Contaminants 654.5 Toxins 66

4.5.1 History 664.5.2 Microbial Toxins 674.5.3 Mycotoxins 674.5.4 Algal Toxins 684.5.5 Plant Toxins 694.5.6 Animal Toxins 70

4.6 Solvents 714.7 Therapeutic Drugs 714.8 Drugs of Abuse 724.9 Combustion Products 724.10 Cosmetics 74Bibliography and Suggested Reading 74

General 74Metals 74Pesticides 75Toxins 75Solvents 75Therapeutic Drugs 75

Sample Questions 75

PART III TOXICANT PROCESSING IN VIVO 77

5. Absorption and Distribution of Toxicants 79Ronald E. Baynes and Ernest Hodgson

5.1 Introduction 795.2 Cell Membranes 805.3 Mechanisms of Transport 82

5.3.1 Passive Diffusion 835.3.2 Carrier-Mediated Membrane Transport 86

5.4 Physicochemical Properties Relevant to Diffusion 875.4.1 Ionization 885.4.2 Partition Coeffi cients 89

viii CONTENTS

5.5 Routes of Absorption 905.5.1 Extent of Absorption 915.5.2 Gastrointestinal Absorption 925.5.3 Dermal Absorption 945.5.4 Respiratory Penetration 97

5.6 Toxicant Distribution 995.6.1 Physicochemical Properties and Protein Binding 995.6.2 Vd 106

5.7 Toxicokinetics 108Bibliography and Suggested Reading 112Sample Questions 113

6. Metabolism of Toxicants 115Ernest Hodgson and Randy L. Rose

6.1 Introduction 1156.2 Phase I Reactions 116

6.2.1 The Endoplasmic Reticulum, Microsomes, and Monooxygenations 116

6.2.2 The CYP-Dependent Monooxygenase System 1186.2.3 The FMO 1326.2.4 Nonmicrosomal Oxidations 1356.2.5 Co-oxidation by Cyclooxygenase (COX) 1376.2.6 Reduction Reactions 1386.2.7 Hydrolysis 1406.2.8 Epoxide Hydration 1426.2.9 DDT Dehydrochlorinase 143

6.3 Phase II Reactions 1436.3.1 Glucuronide Conjugation 1436.3.2 Glucoside Conjugation 1456.3.3 Sulfate Conjugation 1456.3.4 Methyltransferases 1476.3.5 GSTs and Mercapturic Acid Formation 1496.3.6 Cysteine Conjugate β-Lyase 1516.3.7 Acylation 1526.3.8 Phosphate Conjugation 154


7. Reactive Metabolites 157Ernest Hodgson and Randy L. Rose

7.1 Introduction 1577.2 Activation Enzymes 1587.3 Nature and Stability of Reactive Metabolites 1607.4 Fate of Reactive Metabolites 161

7.4.1 Binding to Cellular Macromolecules 1617.4.2 Lipid Peroxidation 161

CONTENTS ix

7.4.3 Trapping and Removal: Role of Glutathione 1627.4.4 Trapping and Removal: Role of Epoxide Hydration 162

7.5 Factors Affecting Toxicity of Reactive Metabolites 1627.5.1 Levels of Activating Enzymes 1637.5.2 Levels of Conjugating Enzymes 1637.5.3 Levels of Cofactors or Conjugating Chemicals 163

7.6 Reactive Oxygen Species 1637.7 Examples of Activating Reactions 164

7.7.1 Piperonyl Butoxide 1647.7.2 Chlorpyrifos 1647.7.3 Vinyl Chloride 1657.7.4 Methanol 1657.7.5 Afl atoxin B1 1657.7.6 Carbon Tetrachloride (Tetrachloromethane) 1667.7.7 Acetylaminofl uorene 1667.7.8 Benzo(a)pyrene 1677.7.9 Acetaminophen 1687.7.10 Cycasin 169

7.8 Summary and Conclusions 170Bibliography and Suggested Reading 171Sample Questions 171

8. Chemical and Physiological Effects on Xenobiotic Metabolism 173Andrew D. Wallace and Ernest Hodgson

8.1 Introduction 1738.2 Nutritional Effects 173

8.2.1 Protein 1738.2.2 Carbohydrates 1748.2.3 Lipids 1748.2.4 Micronutrients 1758.2.5 Starvation and Dehydration 1758.2.6 Nutritional Requirements in Xenobiotic Metabolism 175

8.3 Physiological Effects 1768.3.1 Development 1768.3.2 Gender Differences 1788.3.3 Hormones 1808.3.4 Pregnancy 1828.3.5 Disease 1828.3.6 Diurnal Rhythms 182

8.4 Comparative and Genetic Effects 1828.4.1 Variations among Taxonomic Groups 1838.4.2 Selectivity 1888.4.3 Genetic Differences 189

8.5 Chemical Effects 1918.5.1 Inhibition 192

x CONTENTS

8.5.2 Induction 1998.5.3 Biphasic Effects: Inhibition and Induction 207

8.6 Environmental Effects 2078.6.1 Temperature 2078.6.2 Ionizing Radiation 2088.6.3 Light 2088.6.4 Moisture 2088.6.5 Altitude 2088.6.6 Other Stress Factors 209

8.7 Summary and Conclusions 209Bibliography and Suggested Reading 210Sample Questions 211

9. Elimination of Toxicants 213Gerald A. Leblanc

9.1 Introduction 2139.1.1 Size 2139.1.2 Surface Area to Body Mass Ratio 2149.1.3 Compartmentalization 2149.1.4 Lipid Content 2149.1.5 Barriers to the Environment 214

9.2 Transport 2159.3 Renal Elimination 216

9.3.1 Size 2169.3.2 Water Solubility 216

9.4 Hepatic Elimination 2179.4.1 Entero-Hepatic Circulation 2199.4.2 Active Transporters of the Bile Canaliculus 220

9.5 Respiratory Elimination 2209.6 Conclusion 221Bibliography and Suggested Reading 221Sample Questions 222

PART IV TOXIC ACTION 223

10. Acute Toxicity 225Gerald A. Leblanc

10.1 Introduction 22510.2 Acute Exposure and Effect 22510.3 Dose–Response Relationships 22710.4 Nonconventional Dose–Response Relationships 22910.5 Alternative Methods 230

10.5.1 Up-Down Method 23110.5.2 Fixed-Dose Method 23110.5.3 In Vitro Methods 231

CONTENTS xi

10.6 Mechanisms of Acute Toxicity 23110.6.1 Narcosis 23110.6.2 Acetylcholinesterase Inhibition 23210.6.3 Ion Channel Modulators 23210.6.4 Inhibitors of Cellular Respiration 234


11. Chemical Carcinogenesis and Mutagenesis 237Robert C. Smart

11.1 DNA Damage and Mutagenesis 23711.2 General Aspects of Cancer 23911.3 Human Cancer 242

11.3.1 Causes, Incidence, and Mortality Rates of Human Cancer 242

11.3.2 Known Human Carcinogens 24611.3.3 Classifi cation of Human Carcinogens 24811.3.4 Usefulness and Limitations of Mutagenicity Assays

for the Identifi cation of Carcinogens 25011.4 Classes of Agents That Are Associated with Carcinogenesis 251

11.4.1 DNA Damaging Agents 25211.4.2 Epigenetic Agents 252

11.5 General Aspects of Chemical Carcinogenesis 25411.5.1 Initiation-Promotion Model 25611.5.2 Metabolic Activation of Chemical Carcinogens and

DNA Adduct Formation 25711.6 Oncogenes 259

11.6.1 Ras Oncogene 26111.7 Tumor Suppressor Genes 262

11.7.1 p53 Tumor Suppressor Gene 262Bibliography and Suggested Reading 264Sample Questions 264

12. Teratogenesis 265Jill A. Barnes and Ida M. Washington

12.1 Introduction 26512.2 Overview of Embryonic Development 266

12.2.1 Fertilization 26612.2.2 Cleavage Stages 26612.2.3 Determination 26612.2.4 Gastrulation 26712.2.5 Differentiation 26712.2.6 Organogenesis 26712.2.7 Fetal Period 268

xii CONTENTS

12.3 Principles of Teratogenesis 26812.3.1 Wilson’s Principles 26812.3.2 Critical Period 268

12.4 Mechanisms of Teratogenesis 26812.4.1 Genetic Factors 26912.4.2 Teratogens 270

12.5 Future Considerations 272Bibliography and Suggested Reading 272Sample Questions 272

PART V ORGAN TOXICITY 275

13. Hepatotoxicity 277Andrew D. Wallace and Sharon A. Meyer

13.1 Introduction 27713.1.1 Liver Structure 27713.1.2 Liver Function 278

13.2 Susceptibility of the Liver 27913.3 Types of Liver Injury 279

13.3.1 Fatty Liver 28013.3.2 Cholestasis 28113.3.3 Fibrosis and Cirrhosis 28213.3.4 Necrosis 28213.3.5 Apoptosis 28213.3.6 Hepatitis 28313.3.7 Carcinogenesis 283

13.4 Mechanisms of Hepatotoxicity 28313.5 Examples of Hepatotoxicants 285

13.5.1 Carbon Tetrachloride 28513.5.2 Ethanol 28613.5.3 Bromobenzene 28713.5.4 Acetaminophen 28713.5.5 Troglitazone 288

13.6 Metabolic Activation of Hepatotoxicants 288Bibliography and Suggested Reading 289Sample Questions 289

14. Nephrotoxicity 291Joan B. Tarloff and Andrew D. Wallace

14.1 Introduction 29114.1.1 Structural Organization of the Kidney 29114.1.2 Function of the Renal System 292

14.2 Factors Contributing to Nephrotoxicity 292

CONTENTS xiii

14.3 Examples of Nephrotoxicants 29314.3.1 Metals 29514.3.2 Antimicrobial Agents 29514.3.3 Agents that Precipitate in Renal Tubules 29814.3.4 Halogenated Hydrocarbons 29914.3.5 Analgesics 301

14.4 Summary 301Bibliography and Suggested Reading 301Sample Questions 302

15. Toxicology of the Nervous System 303Bonita L. Blake

15.1 Introduction 30315.2 The Nervous System 303

15.2.1 The Neuron 30415.2.2 Neurotransmitters and Their Receptors 30815.2.3 Glial Cells 30915.2.4 The Blood–Brain Barrier 31015.2.5 The Energy-Dependent Nervous System 311

15.3 Toxicant Effects on the Nervous System 31215.3.1 Structural Effects of Toxicants on Neurons 31215.3.2 Toxicant-Mediated Alterations in Synaptic Function 315

15.4 Neurotoxicity Testing 31715.4.1 In Vivo Tests of Animal Exposure 31715.4.2 In Vivo Tests of Human Exposure 31815.4.3 In Vitro Neurochemical and Histopathological

End points 32015.5 Summary 321Bibliography and Suggested Reading 322Sample Questions 322

16. Reproductive System 323Heather Patisaul

16.1 Introduction 32316.1.1 Defi ning Reproductive Toxicity 32316.1.2 Defi ning Endocrine Disruption 324

16.2 The Hypothalamic-Pituitary-Gonadal Axis 32416.3 Male Reproductive Physiology 32616.4 Disruption of Male Reproduction By Toxicants 330

16.4.1 Pesticides 33016.4.2 Metals 33116.4.3 Plastics 332

16.5 Female Reproductive Physiology 33216.5.1 The Ovulatory Cycle 333

xiv CONTENTS

16.6 Disruption of Female Reproduction by Toxicants 33516.6.1 Cigarette Smoke 33616.6.2 Diethylstilbestrol (DES) 33616.6.3 Pesticides 33716.6.4 Plastics 33816.6.5 Phytoestrogens 33816.6.6 Others 341


17. Endocrine Toxicology 345Gerald A. Leblanc

17.1 Introduction 34517.2 Endocrine System 345

17.2.1 Nuclear Receptors 34917.2.2 Membrane-Bound Steroid Hormone Receptors 350

17.3 Endocrine Disruption 35217.3.1 Hormone Receptor Agonists 35217.3.2 Hormone Receptor Antagonists 35517.3.3 Organizational Versus Activational Effects

of Endocrine Toxicants 35617.3.4 Inhibitors of Hormone Synthesis 35617.3.5 Inducers of Hormone Clearance 35717.3.6 Hormone Displacement from Binding Proteins 358

17.4 Incidents of Endocrine Toxicity 35817.4.1 Organizational Toxicity 35817.4.2 Activational Toxicity 35917.4.3 Hypothyroidism 361

17.5 Conclusion 361Bibliography and Suggested Reading 362Sample Questions 362

18. Respiratory Toxicology 363James C. Bonner

18.1 Introduction 36318.2 Anatomy and Function of the Respiratory Tract 363

18.2.1 Upper Respiratory Tract as a Site of Toxicity 36518.2.2 Lower Respiratory Tract as a Site of Toxicity 36618.2.3 Airways of the Lower Respiratory Tract 36718.2.4 Parenchyma of the Lower Respiratory Tract 36918.2.5 Circulatory, Lymphatic, and Nervous System

of the Lung 37118.3 Toxicant-Induced Lung Injury, Remodeling, and Repair 373

18.3.1 Oxidative Stress and Lung Injury 374

CONTENTS xv

18.3.2 Antioxidant Mechanisms in the Lung 37618.3.3 Respiratory Tract Injury from Inhaled Particles

and Fibers 37618.3.4 Particle and Fiber Deposition and Clearance 37718.3.5 Respiratory Tract Injury from Gases and Vapors 379

18.4 Occupational and Environmental Lung Diseases 38018.4.1 Pulmonary Fibrosis 38118.4.2 Asthma 38118.4.3 Hypersensitivity Pneumonitis (HP) 38218.4.4 COPD 38318.4.5 Lung Cancer 384


19. Immune System 387MaryJane K. Selgrade

19.1 Introduction 38719.2 The Immune System 38819.3 Immune Suppression 39119.4 Classifi cation of Immune-Mediated Injury (Hypersensitivity) 39619.5 Effects of Chemicals on Allergic Disease 398

19.5.1 ACD 39819.5.2 Respiratory Allergens 40019.5.3 Adjuvants 40219.5.4 Systemic Hypersensitivity 402

19.6 Other Issues: Autoimmunity and the Developing Immune System 403


PART VI APPLIED TOXICOLOGY 407

20. Toxicity Testing 409Ernest Hodgson and Helen Cunny

20.1 Introduction 40920.2 Experimental Administration of Toxicants 412

20.2.1 Introduction 41220.2.2 Routes of Administration 412

20.3 Chemical and Physical Properties 41420.4 Exposure and Environmental Fate 41420.5 In Vivo Tests 415

20.5.1 Acute Toxicity 41520.5.2 Subchronic Tests 42120.5.3 Chronic Tests 42720.5.4 Special Tests 435

xvi CONTENTS

20.6 In Vitro and Other Short-Term Tests 44220.6.1 Introduction 44220.6.2 Prokaryote Mutagenicity 44220.6.3 Eukaryote Mutagenicity 44420.6.4 DNA Damage and Repair 44720.6.5 Chromosome Aberrations 44820.6.6 Mammalian Cell Transformation 45020.6.7 General Considerations and Testing Sequences 450

20.7 Ecological Effects 45120.7.1 Laboratory Tests 45120.7.2 Simulated Field Tests 45220.7.3 Field Tests 452

20.8 Risk Analysis 45320.9 The Future of Toxicity Testing 453Bibliography and Suggested Reading 454Sample Questions 455

21. Forensic and Clinical Toxicology 457Sharon A. Meyer and Bonita L. Blake

21.1 Introduction 45721.2 Forensic Toxicology 457

21.2.1 Overview 45721.2.2 Evidentiary Requirements 45921.2.3 Sample Type and Chemical Classes Analyzed

in Forensic Toxicology 46021.3 Clinical Toxicology 462

21.3.1 Overview 46221.3.2 Clinical Toxicology and Health Care 46221.3.3 Training and Certifi cation 46321.3.4 Clinical Management of Toxicant Exposure 464

21.4 Analytical Methods in Forensic and Clinical Toxicology 469Bibliography and Suggested Reading 472Sample Questions 473

22. Prevention of Toxicity 475Ernest Hodgson

22.1 Introduction 47522.2 Legislation and Regulation 475

22.2.1 Federal Government 47622.2.2 State Governments 48122.2.3 Legislation and Regulation in Other Countries 482

22.3 Prevention in Different Environments 48222.3.1 Home 48222.3.2 Workplace 48322.3.3 Pollution of Air, Water, and Land 484

CONTENTS xvii

22.4 Education 485Bibliography and Suggested Reading 486Sample Questions 487

23. Human Health Risk Assessment 489Ronald E. Baynes

23.1 Introduction 48923.2 Risk Assessment Methods 490

23.2.1 Hazard Identifi cation 49123.2.2 Exposure Assessment 49223.2.3 Dose Response and Risk Characterization 492

23.3 Noncancer Risk Assessment 49323.3.1 Default Uncertainty and Modifying Factors 49423.3.2 Derivation of Developmental Toxicant RfD 49623.3.3 Determination of RfD and RfC of Naphthalene using

the NOAEL Approach 49623.3.4 Benchmark Dose Approach 49723.3.5 Determination of BMD and BMDL for ETU 49823.3.6 Quantifying Risk for Noncarcinogenic Effects:

Hazard Quotient 49823.3.7 Chemical Mixtures 499

23.4 Cancer Risk Assessment 50023.5 PBPK Modeling 503Bibliography and Suggested Reading 504Sample Questions 505

PART VII ENVIRONMENTAL TOXICOLOGY 507

24. Toxicant Analysis: Analytical Methods and Quality Assurance 509Chris Hofelt

24.1 Introduction 50924.2 Environmental Sample Collection Methods 510

24.2.1 Sampling Schemes 51024.2.2 Environmental Matrices 512

24.3 Analytical Techniques 51424.3.1 Extraction Techniques 51524.3.2 Sample Cleanup and Enrichment 51624.3.3 Analysis 519

24.4 Quantifi cation, QA, and QC 52524.4.1 Quantifi cation Approaches and Techniques 52524.4.2 QA and QC 526


xviii CONTENTS

25. Basics of Environmental Toxicology 531Gerald A. Leblanc and David B. Buchwalter

25.1 Introduction 53125.2 Environmental Persistence 532

25.2.1 Abiotic Degradation 53325.2.2 Biotic Degradation 53325.2.3 Nondegradative Elimination Processes 534

25.3 Bioaccumulation 53525.3.1 Factors That Infl uence Bioaccumulation 538

25.4 Toxicity 53925.4.1 Acute Toxicity 53925.4.2 Mechanisms of Acute Toxicity 54025.4.3 Chronic Toxicity 54125.4.4 Species-Specifi c Chronic Toxicity 54325.4.5 Abiotic and Biotic Interactions 543

25.5 Conclusion 546Bibliography and Suggested Reading 547Sample Questions 547

26. Transport and Fate of Toxicants in the Environment 549Damian Shea

26.1 Introduction 54926.2 Sources of Toxicants to the Environment 55026.3 Transport Processes 553

26.3.1 Advection 55326.3.2 Diffusion 555

26.4 Equilibrium Partitioning 55726.4.1 Air–Water Partitioning 55826.4.2 Octanol–Water Partitioning 55826.4.3 Lipid–Water Partitioning 55926.4.4 Particle–Water Partitioning 559

26.5 Transformation Processes 56026.5.1 Reversible Reactions 56126.5.2 Irreversible Reactions 563

26.6 Environmental Fate Models 567Bibliography and Suggested Reading 569Sample Questions 569

27. Environmental Risk Assessment 571Damian Shea

27.1 Introduction 57127.2 Formulating The Problem 573

27.2.1 Selecting Assessment End Points 57327.2.2 Developing Conceptual Models 576

CONTENTS xix

27.2.3 Selecting Measures 57627.3 Analyzing Exposure and Effects Information 578

27.3.1 Characterizing Exposure 57927.3.2 Characterizing Ecological Effects 580

27.4 Characterizing Risk 58227.4.1 Estimating Risk 58327.4.2 Describing Risk 583

27.5 Managing Risk 587Bibliography and Suggested Reading 588Sample Questions 589

PART VIII NEW APPROACHES IN TOXICOLOGY 591

28. Perspectives on Informatics in Toxicology 593Seth W. Kullman, Carolyn J. Mattingly, Joel N. Meyer, and Andrew Whitehead

28.1 Introduction 59328.2 Transcriptomics 59428.3 Annotation Resources 595

28.3.1 Logistics 59528.3.2 Statistics 59528.3.3 Types of Annotation 596

28.4 Genome Sequencing, Resequencing and Genotyping 59728.5 Epigenomic Profi ling 59828.6 Computational Toxicology 59928.7 Informatics Tools in Toxicology 601Bibliography and Suggested Reading 602Sample Question 605

29. Future Considerations 607Ernest Hodgson

29.1 Introduction 60729.2 Risk Assessment 609

29.2.1 Hazard and Exposure Assessment 60929.2.2 Toxicogenomics 61029.2.3 Proteomics 61029.2.4 Metabolomics 61129.2.5 Systems Biology Approach to Risk Assessment 61129.2.6 Endocrine Disruptors 61229.2.7 Genetically Modifi ed Plants (GMPs) 612

29.3 Risk Management 61329.4 Risk Communication 61329.5 In Vivo Toxicity 61429.6 In Vitro Toxicity 614

xx CONTENTS

29.7 Molecular and Biochemical Toxicology 61429.8 Development of Selective Toxicants 61529.9 Summary and Conclusions 616Bibliography and Suggested Reading 616Sample Questions 617

GLOSSARY 619

INDEX 638

PREFACE TO THE FOURTH EDITION

xxi

There are some excellent general reference works in toxicology, including Casarett and Doull ’ s Toxicology, 6th edition, edited by Curt Klaassen, and the 13 - volume Comprehensive Toxicology, the second edition currently being edited by Charlene McQueen, as well as many specialized monographs on particular topics. However, the scarcity of textbooks designed for teacher and student to use in the classroom setting that impelled us to produce editions 1 through 3 of this work is still apparent and the choice continues to be limited. The authors are, or have been, involved in teaching general toxicology at North Carolina State University and thus have insights into the actual teaching process and in the broader scope of toxicology as well as the subject matter of their areas of specialization.

Rapid advances are occurring in toxicology, particularly in the molecular and integrative aspects, and we hope these are refl ected in this textbook. As an aid to students and teaching faculty, we have added sample questions to each chapter. Answering these questions not only indicates that the material presented has been understood but is, in itself, a learning experience.

At North Carolina State University, we continue to teach a course in general toxicology (TOX801) that is open to graduate students and undergraduate upper-classmen. Our experience leads us to believe that this textbook is suitable, in the junior or senior year, for undergraduate students with some background in chem-istry, biochemistry, and animal physiology. For graduate students, it is intended to lay the foundation for subsequent specialized courses in toxicology, such as those in biochemical and molecular toxicology, environmental toxicology, chemical carci-nogenesis, risk assessment, and so forth.

We share the view that an introductory text must present all of the necessary fundamental information to fulfi ll this purpose, but in as uncomplicated a manner as possible. To enhance readability, references have been omitted from the text, although Suggested Reading or Bibliography is recommended at the end of each chapter.

As with previous editions, the amount of material and the detail with which some of it is presented, is more than is needed for the average general toxicology course. This, however, will permit each instructor to select and emphasize those areas they feel need particular emphasis. The obvious biochemical and molecular bias of some chapters is not accidental; rather, it is based on the philosophy that progress in toxicology continues to depend on further understanding of the fundamental basis of toxic action at the cellular and molecular levels. The depth of coverage of each topic represents that chapter author ’ s judgment of the amount of material appropriate to the beginning level as compared to that appropriate to a more advanced course or text such as Smart and Hodgson, Molecular and Biochemical Toxicology, 4th edition (John Wiley and Sons, 2008).

xxii PREFACE TO THE FOURTH EDITION

Thanks to all of the authors and to the students and faculty of the Department of Environmental and Molecular Toxicology at North Carolina State University. Particular thanks to Jonathan Rose of John Wiley and Sons, who facilitated the project by his hard work, his goodwill and, not least, for his patience.

Raleigh, North Carolina E rnest H odgson March 2010

CONTRIBUTORS

xxiii

Jill A. Barnes, Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Ronald E. Baynes, Center for Chemical Toxicology Research and Pharmacokinetics, North Carolina State University, Raleigh, North Carolina

Bonita L. Blake, Department of Pharmacology and Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

James C. Bonner, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

David B. Buchwalter, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

W. Gregory Cope, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Helen Cunny, National Institute for Environmental Health Sciences, Research Triangle Park, North Carolina

Ernest Hodgson, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Chris Hofelt, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Seth W. Kullman, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Gerald A. LeBlanc, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Carolyn J. Mattingly, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine

Joel N. Meyer, Nicholas School of the Environment, Duke University, Durham, North Carolina

Sharon A. Meyer, Department of Toxicology, University of Louisiana, Monroe, Louisiana

Heather Patisaul, Department of Biology, North Carolina State University, Raleigh, North Carolina

Randy L. Rose, (deceased), Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

xxiv CONTRIBUTORS

MaryJane Selgrade, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina

Damian Shea, Departments of Biology and Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Robert C. Smart, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Joan Tarloff, Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, Philadelphia, Pennsylvania

Andrew D. Wallace, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Ida M. Washington, Department of Comparative Medicine, University of Washington School of Medicine, Seattle, Washington

Andrew Whitehead, Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana

PART I

INTRODUCTION

CHAPTER 1

Introduction to Toxicology

ERNEST HODGSON

Since the publication of the 3rd edition of this textbook (2004) major changes have been initiated in toxicology as the tools of molecular biology, genomics, proteomics, metabolomics, bioinformatics, and systems biology are increasingly brought to bear on the critical areas of mode of action, toxicity testing, and risk analysis. Chapter 2 provides information on new methodology and Part VIII — New Approaches in Toxicology is composed of two chapters of commentary on the current and expected impact of these new methods. While the traditional aspects and subdisciplines of toxicology, as outlined below, are still active and viable, during the next few years all are likely to be impacted and their development accelerated by these new approaches.

1.1 DEFINITION AND SCOPE

Toxicology can be defi ned as that branch of science that deals with poisons, and a poison can be defi ned as any substance that causes a harmful effect when adminis-tered, either by accident or by design, to a living organism. By convention, toxicol-ogy also includes the study of harmful effects caused by physical phenomena, such as radiation of various kinds, noise, and so on. In practice, however, many complica-tions exist beyond these simple defi nitions, both in bringing more precise defi nition to the meaning of poison and to the measurement of toxic effects. Broader defi ni-tions of toxicology, such as “ the study of the detection, occurrence, properties, effects, and regulation of toxic substances, ” although more descriptive, do not resolve the diffi culties. Toxicity itself can rarely, if ever, be defi ned as a single molecular event, but is, rather, a cascade of events starting with exposure, proceed-ing through distribution and metabolism, and ending with interaction with cellular macromolecules (usually DNA or protein) and the expression of a toxic end point (Figure 1.1 ). This sequence may be mitigated by excretion and repair. It is to the complications, and to the science behind them and their resolution, that this textbook is dedicated, particular to the how and why certain substances cause disruptions in biologic systems that result in toxic effects. Taken together, these

A Textbook of Modern Toxicology, Fourth Edition. Edited by Ernest HodgsonCopyright © 2010 John Wiley & Sons, Inc.

3

4 INTRODUCTION TO TOXICOLOGY

diffi culties and their resolution circumscribe the perimeter of the science of toxicology.

The study of toxicology serves society in many ways, not only to protect humans and the environment from the deleterious effects of toxicants, but also to facilitate the development of more selective toxicants such as anticancer and other clinical drugs, pesticides, and so forth.

Poison is a quantitative concept, almost any substance being harmful at some doses but, at the same time, being without harmful effect at some lower dose. Between these two limits, there is a range of possible effects, from subtle long - term chronic toxicity to immediate lethality. Vinyl chloride may be taken as an example. It is a potent hepatotoxicant at high doses, a carcinogen with a long latent period at lower doses, and apparently without effect at very low doses. Clinical drugs are even more poignant examples because, although therapeutic and highly benefi cial at some doses, they are not without deleterious side effects and may be lethal at higher doses. Aspirin (acetylsalicylic acid), for example, is a relatively safe drug at recommended doses and is taken by millions of people worldwide. At the same time, chronic use can cause deleterious effects on the gastric mucosa, and it is fatal at a dose of about 0.2 – 0.5 g/kg. Approximately 15% of reported accidental deaths from poisoning in children result from ingestion of salicylates, particularly aspirin.

The importance of dose is well illustrated by metals that are essential in the diet but are toxic at higher doses. Thus, iron, copper, magnesium, cobalt, manganese, and zinc can be present in the diet at too low a level (defi ciency), at an appropriate level (maintenance), or at too high a level (toxic). The question of dose – response relationships is fundamental to toxicology (see Section 1.4 ).

Toxicant Exposure

Entrance to Body

Skin InhalationIngestion

Absorption into Bloodstream andDistribution to Body Tissues and Organs

Toxicity Storage

Metabolism

Excretion

Figure 1.1 Fate and effect of toxicants in the body.

DEFINITION AND SCOPE 5

The defi nition of a poison, or toxicant, also involves a qualitative biological aspect because a compound, toxic to one species or genetic strain, may be relatively harmless to another. For example, carbon tetrachloride, a potent hepatotoxicant in many species, is relatively harmless to the chicken. Certain strains of rabbit can eat Belladonna with impunity while others cannot. Compounds may be toxic under some circumstances but not others or, perhaps, toxic in combination with another compound but nontoxic alone. The methylenedioxyphenyl insecticide synergists, such as piperonyl butoxide, are of low toxicity to both insects and mammals when administered alone, but are, by virtue of their ability to inhibit xenobiotic - metabolizing enzymes, capable of causing dramatic increases in the toxicity of other compounds.

The measurement of toxicity is also complex. Toxicity may be acute or chronic, and may vary from one organ to another as well as with age, genetics, gender, diet, physiological condition, or the health status of the organism. As opposed to experi-mental animals, which are highly inbred, genetic variation is a most important factor in human toxicity since the human population is highly outbred and shows extensive genetic variation. Even the simplest measure of toxicity, the LD 50 (lethal dose ; the dose required to kill 50% of a population under stated conditions) is highly dependent on the extent to which the above variables are controlled. LD 50 values, as a result, vary markedly from one laboratory to another.

Exposure of humans and other organisms to toxicants may result from many activities: intentional ingestion, occupational exposure, environmental exposure, as well as accidental and intentional (suicidal or homicidal) poisoning. The toxicity of a particular compound may vary with the portal of entry into the body, whether through the alimentary canal, the lungs, or the skin. Experimental methods of administration such as injection may also give highly variable results; thus, the toxic-ity from intravenous (IV), intraperitoneal (IP), intramuscular (IM), or subcutane-ous (SC) injection of a given compound may be quite different. Thus, toxicity may vary as much as 10 - fold with the route of administration. Following exposure, there are multiple possible routes of metabolism, both detoxifying and activating, and multiple possible toxic end points (Figure 1.1 ).

Attempts to defi ne the scope of toxicology, including that which follows, must take into account that the various subdisciplines are not mutually exclusive and are frequently interdependent. Due to overlapping of mechanisms as well as use and chemical classes of toxicants, clear division into subjects of equal extent or importance is not possible.

Many specialized terms are used in the various subdisciplines of toxicology as illustrated in the Dictionary of Toxicology , 2nd edition (Hodgson et al., 1998 ). However, some terms are of particular importance to toxicology in general; these and some more recent terms are defi ned in the glossary to be found at the end of this volume.

Although B through F (following) include subdivisions that encompass essen-tially all of the many aspects of toxicology, there are two new approaches (A, following) that serve to integrate the discipline as a whole.

A. Integrative Approaches 1. Bioinformatics . In the narrow and original meaning, bioinformatics was the

application of information technology to molecular biology. While this is still


the most important aspect of bioinformatics, it is increasingly applied to other fi elds of biology, including molecular and other aspects of toxicology. It is characterized by computationally intensive methodology and includes the design of large databases and the development of techniques for their manip-ulation, including data mining.

2. Systems Biology . Although systems biology has been defi ned in a number of ways, some involving quite simple approaches to limited problems, in the currently most commonly accepted sense, it is an integrative approach to biological structure and function that will be of increasing importance to biology in general and toxicology in particular. In large part, biology has been reductionist throughout its history, studying organs as components of organisms, cells as components of organs, enzymes, nucleic acids, and so on, as components of cells, with the goal of describing function at the molecular level. Systems biology, on the other hand, is holistic and has the objective of discerning interactions between components of biological systems and describing these interactions in rigorous mathematical models. Furthermore, the proponents of systems biology aim to integrate these models at higher and higher levels or organization in order to develop an integrated model of the entire organism.

Clearly, systems biology is in its infancy; however, the ultimate value of having an integrative model that could clarify all of the effects, from the most proximate to the ultimate, of a toxicant on a living organism, will provide enormous benefi ts not only for fundamental studies but in such applied areas as human health risk assessment.

B. Modes of Toxic Action. This includes the consideration, at the fundamental level of organ, cell, and molecular function, of all events leading to toxicity in vivo : uptake, distribution, metabolism, mode of action, and excretion. The term mechanism of toxic action is now more generally used to describe an important molecular event in the cascade of events leading from exposure to toxicity, such as the inhibition of acetylcholinesterase in the toxicity of orga-nophosphorus and carbamate insecticides. Important aspects include the following: 1. Biochemical and molecular toxicology consider events at the biochemical and

molecular levels, including enzymes that metabolize xenobiotics, generation of reactive intermediates, interaction of xenobiotics or their metabolites with macromolecules, gene expression in metabolism and modes of action, signal-ing pathways in toxic action, and so on.

2. Behavioral toxicology deals with the effects of toxicants on animal and human behavior, which is the fi nal integrated expression of nervous function in the intact animal. This involves both the peripheral and central nervous systems, as well as effects mediated by other organ systems, such as the endocrine glands.

3. Nutritional toxicology deals with the effects of diet on the expression of toxicity and with the mechanisms of these effects.

4. Carcinogenesis includes the chemical, biochemical, and molecular events that lead to the large number of effects on cell growth collectively known as cancer.

DEFINITION AND SCOPE 7

5. Teratogenesis includes the chemical, biochemical, and molecular events that lead to deleterious effects on development.

6. Mutagenesis is concerned with toxic effects on the genetic material and the inheritance of these effects.

7. Organ toxicity considers effects at the level of organ function (e.g., neurotox-icity, hepatotoxicity, and nephrotoxicity).

C. Measurement of Toxicants and Toxicity. These important aspects deal primarily with analytical chemistry, bioassay, and applied mathematics, and are designed to provide the methodology to answer certain critically important questions. Is the substance likely to be toxic? What is its chemical identity? How much of it is present? How can we assay its toxic effect, and what is the minimum level at which this toxic effect can be detected? A number of important fi elds are included: 1. Analytical toxicology is a branch of analytical chemistry concerned with the

identifi cation and assay of toxic chemicals and their metabolites in biological and environmental materials.

2. Genomics. The sometimes stated distinction that genomics deals with genomes while molecular biology deals with single genes is unrealistic and unnecessary; it is more appropriate to regard genomics as an aspect of molecular biology that deals not only with genomes and gene expression but also such important aspects as genetic polymorphisms, particularly single nucleotide polymorphisms (SNPs). Techniques, such as microarrays, are now available to examine simultaneously the expression of very large numbers of genes.

3. Proteomics deals with the protein complement of organisms, the entire com-plement being known as the proteome. Thus, while genomics is concerned with gene expression, proteomics examines the products of the expressed genes.

4. Metabolomics is the next step in the sequence from genomics through proteomics and is concerned with the profi le of small molecules produced by the metabolic processes of an organism. Changes in the profi le in response to chemical stress are of importance to both fundamental and applied toxicology.

5. Toxicity testing involves the use of living systems to estimate toxic effects. It covers the gamut from short - term tests for genotoxicity such as the Ames test and cell culture techniques to the use of intact animals for a variety of tests from acute toxicity to lifetime chronic toxicity. Although the term “ bioassay ” is used properly only to describe the use of a living organism to quantitate the amount of a particular toxicant present, it is frequently used to describe any in vivo toxicity test.

6. Toxicologic pathology is that branch of pathology that deals with the effects of toxic agents manifested as changes in subcellular, cellular, tissue, or organ morphology.

7. Structure - activity studies are concerned with the relationship between the chemical and physical properties of a chemical and toxicity and, particularly, the use of such relationships as predictors of toxicity.


8. Biomathematics and statistics relate to many areas of toxicology. They deal with data analysis, the determination of signifi cance, and the formulation of risk estimates and predictive models.

9. Epidemiology , as it applies to toxicology, is of great importance as it deals with the relationship between chemical exposure and human disease in actual populations, rather than in experimental settings.

D. Applied Toxicology. This includes the various aspects of toxicology as they apply in the fi eld or the development of new methodology or new selective toxicants for early application in the fi eld setting . 1. Clinical toxicology is the diagnosis and treatment of human poisoning. 2. Veterinary toxicology is the diagnosis and treatment of poisoning in animals

other than humans, particularly livestock and companion animals, but not excluding feral species. Other important concerns of veterinary toxicology are the possible transmission of toxins to the human population in meat, fi sh, milk, and other foodstuffs, and the care and ethical treatment of experimental animals.

3. Forensic toxicology concerns the medicolegal aspects, including detection of poisons in clinical and other samples.

4. Environmental toxicology is concerned with the movement of toxicants and their metabolites and degradation products in the environment and in food chains, and with the effect of such contaminants on individuals and, especially, populations. Because of the large number of industrial chemicals and possibilities for exposure, as well as the mosaic of overlapping laws that govern such exposure, this area of applied toxicology is well developed.

5. Industrial toxicology is a specifi c area of environmental toxicology that deals with the work environment and constitutes a signifi cant part of industrial hygiene .

E. Chemical Use Classes. This includes the toxicology aspects of the development of new chemicals for commercial use. In some of these use classes, toxicity, at least to some organisms, is a desirable trait; in others, it is an undesirable side effect. Use classes are not composed entirely of synthetic chemicals; many natural products are isolated and are used for commercial and other purposes and must be subjected to the same toxicity testing as that required for synthetic chemicals. Examples of such natural products include the insecticide, pyrethrin, the clinical drug, digitalis, and the drug of abuse, cocaine. 1. Agricultural chemicals include many compounds, such as insecticides, herbi-

cides, fungicides, and rodenticides, in which toxicity to the target organism is a desired quality whereas toxicity to “ nontarget species ” is to be avoided. Development of such selectively toxic chemicals is one of the applied roles of comparative toxicology.

2. Clinical drugs are properly the province of pharmaceutical chemistry and pharmacology. However, toxic side effects and testing for them clearly fall within the science of toxicology.

3. Drugs of abuse are chemicals taken for psychological or other effects and may cause dependence and toxicity. Many of these are illegal but some are of clinical signifi cance when used correctly.

RELATIONSHIP TO OTHER SCIENCES 9

4. Food additives are of concern to toxicologists only when they are toxic or being tested for possible toxicity.

5. Industrial chemicals are so numerous that testing them for toxicity or control-ling exposure to those known to be toxic is a large area of toxicological activity.

6. Naturally occurring substances include many phytotoxins, mycotoxins, miner-als, and so on, all occurring in the environment. The recently expanded and now extensive use of herbal “ remedies ” and dietary supplements has become a cause of concern for toxicologists and regulators. Not only is their effi cacy frequently dubious, but their potential toxicity is also largely unknown.

7. Combustion products are not properly a use class but are a large and impor-tant class of toxicants, generated primarily from fuels and other industrial chemicals.

F. Regulatory Toxicology. These aspects, concerned with the formulation of laws, and regulations authorized by laws, are intended to minimize the effect of toxic chemicals on human health and the environment. 1. Legal aspects are the formulation of laws and regulations and their enforce-

ment. In the United States, enforcement falls under such government agen-cies as the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and the Occupational Safety and Health Administration (OSHA). Similar government agencies exist in many other countries.

2. Risk assessment is the defi nition of risks, potential risks, and the risk – benefi t equations necessary for the regulation of toxic substances. Risk assessment is logically followed by risk communication and risk management. Risk assess-ment, risk communication, and risk management are frequently referred to as risk analysis .

1.2 RELATIONSHIP TO OTHER SCIENCES

Toxicology is a highly eclectic science and human activity drawing from, and con-tributing to, a broad spectrum of other sciences and human activities. At one end of the spectrum are those sciences that contribute their methods and philosophical concepts to serve the needs of toxicologists, either in research or in the application of toxicology to human affairs. At the other end of the spectrum are those sciences to which toxicology contributes.

In the fi rst group, chemistry, biochemistry, pathology, physiology, epidemiology, immunology, ecology, and biomathematics have long been important while molecu-lar biology has, in the last two or three decades, contributed to dramatic advances in toxicology.

In the group of sciences to which toxicology contributes signifi cantly are such aspects of medicine as forensic medicine, clinical toxicology, pharmacy, and pharmacology, public health, and industrial hygiene. Toxicology also contributes in an important way to veterinary medicine, and to such aspects of agriculture as the development and safe use of agricultural chemicals. The contributions of toxicology to environmental studies have become increasingly important in recent years.


Clearly, toxicology is preeminently an applied science, dedicated to the enhance-ment of the quality of life and the protection of the environment. It is also much more. Frequently, the perturbation of normal life processes by toxic chemicals enables us to learn more about the life processes themselves. The use of dinitro-phenol and other uncoupling agents to study oxidative phosphorylation and the use of α - amanitin to study RNA polymerases are but two of many examples. The fi eld of toxicology has expanded enormously in recent decades, both in numbers of toxicologists and in accumulated knowledge. This expansion has brought a change from a primarily descriptive science to one which utilizes an extensive range of methodology to study the mechanisms involved in toxic events.

1.3 A BRIEF HISTORY OF TOXICOLOGY

Much of the early history of toxicology has been lost, and in much that has survived, toxicology is of almost incidental importance in manuscripts dealing primarily with medicine. Some, however, deal more specifi cally with toxic action or with the use of poisons for judicial execution, suicide, or political assassination. Regardless of the paucity of the early record, and given the need for people to avoid toxic animals and plants, toxicology must be one of the oldest practical sciences.

The Egyptian papyrus, Ebers , dating from about 1500 BC, must rank as the earli-est surviving pharmacopeia, and the surviving medical works of Hippocrates, Aristotle, and Theophrastus, published during the period 400 – 250 BC, all include some mention of poisons. The early Greek poet Nicander treats, in two poetic works, animal toxins (Therica) and antidotes to plant and animal toxins (Alexipharmica ). The earliest surviving attempt to classify plants according to their toxic and therapeutic effects is that of Dioscorides, a Greek employed by the Roman emperor Nero about 50 AD.

There appear to have been few advances in either medicine or toxicology between the time of Galen (131 – 200 AD) and that of Paracelsus (1493 – 1541). It was the latter who, despite frequent confusion between fact and mysticism, laid the groundwork for the later development of modern toxicology by recognizing the importance of the dose – response relationship. His famous statement “ All substances are poisons; there is none that is not a poison. The right dose differentiates a poison and a remedy ” succinctly summarizes that concept. His belief in the value of experimenta-tion was also a break with earlier tradition.

There were some important developments during the eighteenth century. Probably the best known is the publication of Ramazini ’ s Diseases of Workers in 1700 which led to his recognition as the father of occupational medicine. The correlation between the occupation of chimney sweeps and scrotal cancer by Percival Pott in 1775 is almost as well - known although it was foreshadowed by Hill ’ s correlation of nasal cancer and snuff use in 1761.

Orfi la, a Spaniard working at the University of Paris in the early nineteenth century, is generally regarded as the father of modern toxicology. He clearly identi-fi ed toxicology as a separate science and, in 1815, published the fi rst book devoted exclusively to toxicology. An English translation in 1817 was entitled A General System of Toxicology or, A Treatise on Poisons, Found in the Mineral, Vegetable

DOSE–RESPONSE RELATIONSHIPS 11

and Animal Kingdoms, Considered in Their Relations with Physiology, Pathology and Medical Jurisprudence. Workers of the late nineteenth century who produced treatises on toxicology include Christian, Kobert, and Lewin. The recognition of the site of action of curare by Claude Bernard (1813 – 1878) began the modern study of the mechanisms of toxic action. Since then, advances have been numerous — too numerous to list in detail. They have increased our knowledge of the chemistry of poisons, the treatment of poisoning, the analysis of toxicants and toxicity, as well as modes of toxic action and detoxication processes, and specifi c molecular events in the poisoning process.

With the publication of her controversial book, The Silent Spring , in 1962, Rachel Carson became an important infl uence in initiating the modern era of environmen-tal toxicology. Her book emphasized stopping the widespread, indiscriminate use of pesticides and other chemicals and advocated use patterns based on sound ecology. Although sometimes inaccurate and with arguments often based on frankly anecdotal evidence, her book is often credited as the catalyst leading to the establishment of the U.S. EPA and she is regarded by many as the mother of the environmental movement.

It is clear, however, that since the 1960s, toxicology has entered a phase of rapid development and has changed from a science that was largely descriptive to one in which the importance of mechanisms of toxic action is generally recognized. Since the 1970s, with increased emphasis on the use of the techniques of molecular biology, the pace of change has increased even further, and signifi cant advances have been made in many areas, including chemical carcinogenesis and xenobiotic metabolism, among many others.

1.4 DOSE – RESPONSE RELATIONSHIPS

As mentioned previously, toxicity is a relative event that depends not only on the toxic properties of the chemical and the dose administered but also on individual and interspecifi c variation in the metabolic processing of the chemical. The fi rst recognition of the relationship between the dose of a compound and the response elicited has been attributed to Paracelsus (see Section 1.3 ). It is noteworthy that his statement includes not only that all substances can be toxic at some dose, but that “ the right dose differentiates a poison from a remedy, ” a concept that is the basis for pharmaceutical therapy.

A typical dose – response curve is shown in Figure 1.2 , in which the percentage of organisms or systems responding to a chemical is plotted against the dose. For many chemicals and effects, there will be a dose below where no effect or response is observed. This is known as the threshold dose . This concept is of signifi cance because it implies that a no observed effect level (NOEL) can be determined and that this value can be used to determine the safe intake for food additives and contaminants such as pesticides. Although this is generally accepted for most types of chemicals and toxic effects, for chemical carcinogens acting by a genotoxic mechanism, the shape of the curve is controversial, and for regulatory purposes, their effect is assumed to be a no - threshold phenomenon. Dose – response relationships are discussed in more detail in Chapter 10 — Acute Toxicity and Chapter 20 — Toxicity Testing .


1.5 SOURCES OF TOXIC COMPOUNDS

Given the enormous number of toxicants, it is diffi cult to classify them, either chemi-cally, by function, or by mode of action since many of them would fall into several classes. Some are natural products, many are synthetic organic chemicals of use to society, while some are byproducts of industrial processes and waste disposal. It is useful, however, to categorize them according to the expected routes of exposure or according to their uses.

A. Exposure Classes. Exposure classes include toxicants in food, air, water, and soil as well as toxicants characteristic of domestic and occupational settings. Toxicant use classes are described in detail in Chapter 3 .

B. Use Classes. Use classes include drugs of abuse, therapeutic drugs, agricultural chemicals, food additives and contaminants, metals, solvents, combustion prod-ucts, cosmetics, and toxins. Some of these, such as combustion products, are the products of use processes rather than being use classes. All of these groups of chemicals are discussed in detail in Chapter 4 .

1.6 MOVEMENT OF TOXICANTS IN THE ENVIRONMENT

Chemicals released into the environment rarely remain in the form, or at the location, of release. For example, agricultural chemicals used as sprays may drift from the point of application as air contaminants or enter run - off water as water contaminants. Many of these chemicals are susceptible to fungal or bacterial degradation and are rapidly detoxifi ed, frequently being broken down to products that can enter the carbon, nitrogen, and oxygen cycles. Other agricultural chemicals, particularly halogenated organic compounds, are recalcitrant to a greater or lesser degree to metabolism by microorganisms and persist in soil and water as contami-nants; they may enter biologic food chains and move to higher trophic levels or persist in processed crops as food contaminants. This same scenario is applicable to

100

75

50

25

0

% R

espo

nse

Dose

Figure 1.2 A typical dose – response curve.

SUGGESTED READING 13

any toxicant released into the environment either for a specifi c use or as a result of industrial processes, combustion, and so on. Chemicals released into the environ-ment are also susceptible to chemical degradation, a process often stimulated by ultraviolet light.

Although most transport between inanimate phases of the environment results in wider dissemination, but, at the same time, dilution of the toxicant in question, transfer between living creatures may result in increased concentration or bioaccu-mulation. Lipid - soluble toxicants are readily taken up by organisms following expo-sure in air, water, or soil. Unless rapidly metabolized, they persist in the tissues long enough to be transferred to the next trophic level. At each level, the lipophilic toxicant tends to be retained while the bulk of the food is digested, utilized, and excreted, thus increasing the toxicant concentration. At some point in the chain, the toxicant can become deleterious, particularly if the organism at that level is more susceptible than those at the level preceding it. Thus, the eggshell thinning in certain raptorial birds was almost certainly due to the uptake of DDT (1,1,1 - trichloro - 2,2 - bis(4 - chlorophenyl) ethane) and DDE (1,1 - dichloro - 2,2 - bis(4 - chlorophenyl) ethane) and their particular susceptibility to this type of toxicity. Simplifi ed food chains are shown in Figure 1.3 .

It is clear that such transport can occur through both aquatic and terrestrial food chains, although in the former, higher members of the chains, such as fi sh, can accumulate large amounts of toxicants directly from the medium. This accumulation occurs because of the large area of gill fi laments, their intimate contact with the water, and the high fl ow rate of water over them. Given these characteristics and a toxicant with a high partition coeffi cient between lipid membranes and water, considerable uptake is inevitable.

These and all other environmental aspects of toxicology are discussed in Part VII.

BIBLIOGRAPHY AND SUGGESTED READING

Hodgson , E. , R. B. Mailman , and J. E. Chambers , eds. Dictionary of Toxicology , 2nd ed . London : Macmillan , 1998 , 504 pp.

Klaassen , C. D. , ed. Casarett and Doull ’ s Toxicology: The Basic Science of Poisons , 6th ed . New York : McGraw - Hill , 2001 , 1236 pp.

Smart , R. C. and E. Hodgson , eds. Molecular and Biochemical Toxicology , 4th ed. Hoboken, NJ : Wiley , 2008 , 901 pp.

Wexler , P. , ed. Encyclopedia of Toxicology , 2nd ed . Oxford, UK : Elsevier , 2005 , 4 volumes.

Soil InvertebratesTerrestrial Vertebrates

Terrestrial Plants Herbivorous Animals

Predatory Birds or MammalsPredatory Invertebrates

Predatory Birds or Mammals

SoilResidues

Figure 1.3 Examples of simplifi ed food chains.


SAMPLE QUESTIONS

1. Briefl y defi ne the following terms: a. Toxicology b. Poison c. Genomics d. Proteomics e. Metabolomics

2. Toxicity has been described as a cascade of events initiated by exposure to a harmful chemical. Name the principal steps in this cascade.

3. Name and defi ne three important chemical use classes.

CHAPTER 2

Introduction to Biochemical and Molecular Methods in Toxicology

ERNEST HODGSON , GERALD A. LEBLANC , SHARON A. MEYER , and ROBERT C. SMART

2.1 INTRODUCTION

This chapter is not designed to summarize biochemical methods long used in toxicology such as colorimetric and radiometric methods for the investigation of xenobiotic metabolism, either in vivo or in vitro , but rather to give a brief summary of the methods of molecular, biochemical, and cellular biology that have become, more recently, of critical importance in toxicological research. Areas of methodol-ogy that have achieved prominence since the publication of the 3rd edition of this textbook (Hodgson, 2004 ) include proteomics (Section 2.5 , below), metabolomics (Section 2.6 , below), and bioinformatics (Section 2.7 , below). The current chapter owes much to Chapters 2 through 8 of Molecular and Biochemical Toxicology (Smart and Hodgson, 4th edition, 2008; see Bibliography), and the reader is referred to these chapters for additional information.

2.2 CELL CULTURE TECHNIQUES

While scientists have had the ability to culture many unicellular organisms for some time, recent advances in the culture of cells from multicellular organisms have played a pivotal role in recent advances in toxicology. Cells can be isolated and either maintained in a viable state for enough time to conduct informative experi-ments or, in some cases, can be propagated in culture. The advantages of cultured cells are that they can provide living systems for the investigation of toxicity that are simplifi ed relative to the intact organism or they can be used as replacements for whole animal toxicity testing if the toxic end point can be validated. Human cells play an important role in the extrapolation of toxic effects, discovered in experimental animals, to humans. Cultured cells, either from humans or other mammals, are utilized in many of the molecular methods mentioned below. There

A Textbook of Modern Toxicology, Fourth Edition. Edited by Ernest HodgsonCopyright © 2010 John Wiley & Sons, Inc.

15

16 BIOCHEMICAL AND MOLECULAR METHODS IN TOXICOLOGY

are, however, limitations in the use of cellular methods. It has not been possible to culture many cell types, and of those that have been cultured, the loss of differenti-ated cell function is a common problem. Extrapolation of fi ndings to the intact animal is often problematical, and the use of undefi ned media constituents such as serum, often essential for cell viability, may have unwanted or undefi ned effects on cell function and toxicant bioavailability.

Studies have been carried out on cells isolated from tissues and maintained in suspension culture or on cells that have formed monolayers.

2.2.1 Suspension Cell Culture

Circulating blood cells or cells easily obtained by lavage such as peritoneal and alveolar macrophages can normally survive in suspension culture when provided with a suitable nutrient medium. Cells from organized solid organs or tissues must be separated from the tissue and, if possible, separated into cell types, before being suspended in such a medium.

Cell association within organs depends on protein complex formation, which in turn is Ca 2+ - dependent. Consequently, dissociation media generally contain a pro-teolytic enzyme and the Ca 2+ chelator EDTA (ethylenediaminetetraacetic acid). There are a number of methods available to separate cell types from the mixture of dispersed cells, the commonest being centrifugation without a density gradient, wherein cells are separated by size, or centrifugation through a density gradient wherein cells are separated on the basis of their buoyant density.

Cells in suspension may be maintained for a limited period of time in defi ned media or for longer periods in nutrient, but less well - defi ned, media. In either case, these cultures are often used for studies of xenobiotic metabolism.

2.2.2 Monolayer Cell Culture

Proliferation of most cells in culture requires attachment to a substrate and occurs until limited by cell - to - cell contact, resulting in the formation of a cellular mono-layer. The substrate provided for attachment is usually polystyrene modifi ed to carry a charge. The medium for continued maintenance and growth contains salts and glucose, usually with a bicarbonate buffer. Because of the bicarbonate buffering system, these cultures are maintained in a 5 – 10% CO 2 atmosphere in a temperature and humidity controlled incubator. Many cells require serum for optimal growth, inducing considerable variability into the experimental system. Since the factors provided by serum are numerous and complex, defi ned serum substitutes are not always successful. The factors provided by serum include proteins such as growth factors, insulin, and transferrin (to provide available iron), small organic molecules such as ethanolamine, and pyruvate and inorganic ions, such as selenium.

2.2.3 Indicators of Toxicity in Cultured Cells

Routine observation of cultured cells is usually carried out by phase contrast micros-copy, utilizing the inverted phase contrast microscope. More recently, more detailed observations have become possible utilizing fl uorescent tags and inverted fl uores-cent microscopes. Fluorescent tags currently in use permit the assessment of oxidant

CELL CULTURE TECHNIQUES 17

status and mitochondrial function as well as the intracellular concentration of sulf-hydryl groups, Ca 2+ , H + , Na + , and K + .

Toxicity to cultured cells may be the result either of inadequacies in the culture or the toxicity effects of the chemical being investigated. Short - term toxicity is usually evaluated by examination of end points that indicate effects on cellular organelles such as leakage of cell constituents into the medium, uptake of dyes into the cell, and the formation of surface “ blebs. ” This is illustrated in Figure 2.1 .

Longer - term assessments of cell toxicity are highly dependent upon the relevant toxic end point. They may include measurement of growth competence, apoptosis, and/or necrosis, incorporation of radioactive precursors into essential cellular con-stituents such as RNA, DNA, and protein and specialized cellular functions. Some examples of the use of cultured cell lines in the study of toxicity effects are shown in Table 2.1 .

2.2.4 Use of Stem Cells

Although the use of human stem cells is still controversial, their use in biomedical research is increasing. At the same time, stem cells from surrogate animals have long been used in biomedical, including toxicological, research. Probably the best example is the use of cultured mouse embryonic stem cells for generation of “ knock - out ” mice. Such mice have been widely used in the study of xenobiotic - metabolizing enzymes (XMEs ), nuclear receptors, and the like.

The value of cultured stem cells in toxicology lies in their ability to provide a continuous source of cells that can be manipulated to provide a desired mature cell

DYE INFLUX MEMBRANE LEAKAGE

Trypan bluepropidium iodide

Cytoplasmic LDH, Cr2−

Plasma membrane

neutralred

Lysosomes↓ Neutral red

uptake

↓

↓

↓↓

BLEB

MitochondriaMTT ReductionRhodamine 123

retention

EndoplasmicreticulumProtein

synthesis

Synthesis

NucleusDNA, RNA

H+

ATP/A

DP

Nucleus Cortical m

icrofilaments

Mitoch

ondria

Lysosome

Figure 2.1 Idealized diagram of a cell to illustrate parameters often used to measure cyto-toxicity and the corresponding affected subcellular organelle. From A Textbook of Modern Toxicology , 3rd ed., ed. E. Hodgson. New York: Wiley, 2004.

TABLE 2.1 Application of Human Cell Lines Retaining Differentiated Properties for the Study of Toxic Effects and a Comparison with Primary Human Hepatocytes

Cell Type Source Differentiated Cell Type

Toxicant Measured End Point

Cell lines SK - N - SH Human

neuroblastoma Neuron Anesthetic N 2 O Depressed cholinergic Ca 2+

signaling HepG2 Human

hepatoblastoma Hepatocyte Cyclophosphamide

(antineoplastic) Cytochrome P450 (CYP) -

dependent genotoxicity Rifampicin (PXR ligand) Inhibition of bile acid synthesis

Caco - 2 Human colon adenocarcinoma

Intestinal epithelial cell

Arsenic Transepithelial leakiness

Primary hepatocyte Fipronil, fi pronil sulfoxide Adenylate kinase release, Induction of caspase 3/7 Induction of CYP isoforms

Deltamethrin (pyrethroid) Adenylate kinase release, Induction of caspase 3/7 Induction of CYP isoforms

Permethrin (pyrethroid) Adenylate kinase release, Induction of caspase 3/7 Induction of CYP isoforms

DEET (N,N - diethyl - meta - toluamide) (repellent )

Adenylate kinase release, Induction of caspase 3/7 Induction of CYP isoforms

Chlorpyrifos (OP) Adenylate kinase release, Induction of caspase 3/7 Induction of CYP isoforms

PXR, pregnane X receptor; OP, organophosphorus .

18

MOLECULAR TECHNIQUES 19

type. This could alleviate the use of surrogate animals in toxicity testing and provide metabolically competent human cell types.

2.2.5 Cell Culture Models as “ Alternative ” Toxicity Tests

Due primarily to the fact that the cell represents an excellent intermediate level of biological organization between the intact organism and the cellular organelle or enzyme/receptor levels, the primary use of cell culture models to date has been in mechanistic studies of chemical toxicity. At present, however, much effort is being placed on development of cell culture models as replacements for surrogate animals in toxicity. This arises not only from ethical concerns over animal use but also for economy of time and expense. Further, the use of human - derived cell lines may be an advantage in studies related to human health assessment.

Although diffi culties are often encountered, particularly in agreement between the cell culture method and in vivo results as well as quantitative relationships between toxicants of related chemical structure or mode of toxic action, it appears that cell culture methods will be useful as early screens in tiered protocols for product safety testing.

Another emerging application of cell culture toxicity testing techniques is the development of cell lines engineered for a particular function, often for high - throughput screening protocols. An excellent example is in area of testing for endocrine disruptors and the recent mandate that chemicals in commerce be tested for endocrine disrupting activity. This involves the development of cell lines engi-neered to contain a vector with a reporter gene whose expression is responsive to activation of a cotransfected steroid hormone receptor. A similar approach is being adopted for the detection of dioxin - like compounds through their interaction with the aryl hydrocarbon receptor (Ah receptor ).

2.3 MOLECULAR TECHNIQUES

Recombinant DNA techniques, including molecular cloning, have provided recent dramatic advances in many areas of both fundamental and applied biology, toxicol-ogy not excepted. Responses to toxicants often involve changes in gene expression and the microarray techniques enable the simultaneous examination of the global level of expression of thousands of genes in a single experiment. The completion of the human genome project now permits toxic effects in humans to be investigated and will facilitate extrapolation from experimental animals. The human genome will also provide the essential genetic background information for studies of polymor-phisms in xenobiotic - metabolizing and other enzymes. Such polymorphisms have already been shown to be very important in individual sensitivity to clinical drugs and in the defi nition of populations and/or individuals at increased risk from par-ticular toxicants. Identifi cation of carcinogen - induced mutations, particularly in oncogenes and tumor - suppressor genes, are important in chemical carcinogenesis. The ability to develop “ knock - out ” and “ knock - in ” animals that lack a particular gene or express an altered gene in place of the wild - type gene, respectively, as well as knockdown of specifi c genes in cell culture are proving important in toxicological studies. Polymerase chain reaction (PCR) is an extremely versatile technique that


can be used for many applications including gene cloning, gene mutagenesis, and quantitative gene expression analysis.

Gene structure and any of the processes involved in DNA expression, including transcription, mRNA processing, and translation and protein synthesis (Figure 2.2 ), can all be examined by molecular techniques. In toxicology, this may include toxic effects on these processes or the role of the processes in the mechanism of toxic action.

2.3.1 Molecular Cloning

The basic principle of molecular cloning is the insertion of a DNA segment into a suitable vector. The vector is an autonomously replicating DNA molecule, and the inserted DNA segment may be as large as a gene or as small as a few nucleotides. The vector containing the DNA is inserted into a cell such as a bacterium, where it can be replicated many times and either the DNA or the expressed protein subsequently isolated (Figure 2.3 ).

2.3.2 c DNA and Genomic Libraries

cDNA or genomic libraries are collections of DNA fragments incorporated into a recombinant vector and transformed into an appropriate host cell. In the case of cDNA libraries, the cDNAs complementary to all of the mRNAs in the tissue or cell sample are synthesized in a procedure using reverse transcriptase, before incor-poration into the vector. With genomic DNA libraries the genomic DNA is digested,

Promoter region containingproximal and distal elementsand enhancers

EXON EXON EXONINTRON INTRON

5′

5′

3′DNA sense strand

Cap siteSplicejunctions

Splicejunctions

Translation initiation codon

Polyadenylation signal

Translation termination codon

TRANSCRIPTION

PROCESSING

TRANSLATION

Amino terminusProtein

mRNA

hnRNA

Met..........Phe.....Stop Carboxy terminus

..ATG.. GT

..AUG.. GU

..AUG... ..UUU....UAA...AAUAA AAAAAAAA 3′

AG...UUU... GU AG ..UAA....AAUAA AAAAAAA

AG..TTT... ..TAA.....AATAAGT AG

Figure 2.2 Transcription, mRNA processing, and translation. DNA sense strand is desig-nated by bold lines, hnRNA and mRNA by thinner lines. Exons are shown as rectangles and introns as the intervening spaces between exons. From A Textbook of Modern Toxicology , 3rd ed., ed. E. Hodgson. New York: Wiley, 2004.

MOLECULAR TECHNIQUES 21

before cloning into the vector, with a restriction enzyme to produce an overlapping set of DNA fragments of some 12 – 20 kb.

These libraries have been used in many screening procedures, including gene identifi cation and gene regulation. Today, with availability of genomic information/annotation for numerous species including mouse, rat, and human, direct bioinfor-matic analysis of such information allows for PCR approaches for the cloning of genes, promoter regions, and mRNA (cDNA). In fact, most applications that used cDNA and genomic libraries have been superceded by other methods, particularly those based on PCR.

2.3.3 Northern and Southern Blot Analysis

Northern analysis is usually used to identify and quantitate specifi c mRNAs in a sample. Southern analysis is used to determine whether or not a gene of interest is present as well as its copy number. Other uses for Southern analysis include iden-tifying restriction fragment length polymorphisms and changes in heterozygosity.

In both Southern and Northern analyses, restriction digested DNA fragments or RNA, respectively, are separated by size when electrophoresed on agarose gel. The separated molecules are transferred, by electroblotting or capillary blotting, onto a nylon or nitrocellulose membrane. The immobilized RNA or DNA is reacted with a radiolabeled, chemiluminescent, or fl uorescent probe that is complementary to the DNA/RNA of interest, unbound probe is washed off and the membrane exposed,

CTT

AA AATTC

GG

Eco-RI

Eco-RI

CTT

AAG

GA

ATT CTTAA

G

GAATTC

AATTCG

GCTTAA

Transform into E. coliand select with ampicillin

Bacterial coloniescontaining plasmid DNA

AmpO-RI O-RI

O-RI

R Amp R

Amp R

Figure 2.3 Molecular cloning using a plasmid vector. From A Textbook of Modern Toxicology , 3rd ed., ed. E. Hodgson. New York: Wiley, 2004.


in the case of radioactive probes, to radioautographic fi lm to visualize the sample of interest.

2.3.4 PCR

PCR is a powerful technique that can, starting with amounts of DNA as small as those found in single cells, amplify the DNA until large amounts are available for many different kinds of research. Twenty to forty cycles of PCR can provide up to 10 5 times the original DNA sample.

It is necessary to know the fl anking sequence of the DNA of interest in order to construct appropriate primers. These primers are complementary to the sequence at each end of the DNA sequence to be amplifi ed. The DNA is incubated in a thermal cycler with thermostable DNA polymerase, all four deoxyribonucleotide triphosphates (dNTP), and the primers. The incubation temperature is raised to separate the DNA strands, lowered to permit annealing of the primers to the complementary regions of the DNA, and then raised to permit the polymerase to synthesize DNA. This cycle is then repeated up to 40 times. The PCR technique has been used for many types of toxicological investigation including uncovering polymorphisms in XMEs, cloning genes for functional studies as well as promoter regions of genes for gene regulation studies.

2.3.5 Evaluation of Gene Expression, Regulation, and Function

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to deter-mine levels of a particular mRNA, nuclear run - on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specifi c elements in the promoter region responsible for the control of expression, and the electrophoretic mobility shift assay (EMSA) is used to measure binding of a transcription factor to its specifi c DNA consensus sequence . High - throughput reporter gene assays are currently used to examine molecular pathways altered by toxicants. These assays employ specifi c regulatory promoter elements that respond to specifi c types of stressors/inputs; for example, estrogenic agents, reactive oxygen stress, and dioxin - related agents are engineered upstream of a reporter gene (i.e., luciferase), and cell lines containing these constructs can be treated with the toxicant of interest and reporter output quantifi ed .

Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Microarrays are based on the principle that any gene being expressed at any point in time is giving rise to a specifi c, corresponding mRNA. The microarray itself consists of spots of DNA (c . 200 μ ) bound to a suitable matrix. The mRNAs in the biological sample in question bind to the corresponding DNA and can be visualized by techniques involving dyes. Given the complexity of the data obtained (often thousands of genes are evaluated on a single microarray), special techniques have been developed for array scanning, data extraction, and statistical analysis. A typical microarray experi-ment is illustrated in Figure 2.4 .

Real - time reverse transcriptase – polymerase chain reaction (RT - PCR) is com-monly used to amplify and quantitate mRNAs of interest. In fact, this technique is

IMMUNOCHEMICAL TECHNIQUES 23

replacing the Northern technique described above as the preferred technique to measure changes in gene expression and the mRNA level. Gene function in cultured cells can be investigated by the forced expression of the gene product in a suitable expression system or through the use of small interfering RNAs (siRNAs), where the expression of the gene of interest can be knocked down in cultured cells. Gene function can also be studied in vivo through the creation of transgenic mice which overexpress the gene of interest or knock - out mice in which the gene in question has been functionally deleted or knock - in mice where an altered gene (i.e., serine is replaced by alanine to study the role of posttranslational modifi cations involving phosphorylation) is expressed in place of the wild - type gene.

A general, but more detailed and specifi c, account of these methods may be found in Smart (2008) and Oleksiak (2008) (see Bibliography).

2.4 IMMUNOCHEMICAL TECHNIQUES

Most of the recently developed methods for the detection, characterization, and quantitation of proteins (Leblanc, 2008 ) are immunoassays based on the fact that proteins are antigens, compounds that can be recognized by an antibody. It is also true that, by combining small molecules (haptens) with a larger carrier molecule such as a protein, these methods can be extended to small molecules of interest

Treatment

(a)

(b)

Treatment

RNA extraction

RNA labeling

Array hybridization

Array scanning

Data extraction

Data transformation

Data normalization

Statistical analyses

Biological meaning

Biologicalreplicates

Array and spotreplicates

Technical replicates

Wet lab

Dry lab

Dye replicates Cy3 Cy5Cy3 Cy5

Cy3 Cy5 Cy3 Cy5Cy3 Cy5

Cy3 Cy5

Figure 2.4 Schematic of a microarray experiment. (a) Experimental design incorporating both biological and technical replication. There are three treated mice and three control mice, providing biological replication. RNA from each mouse is labeled with each dye and hybridized more than once, providing technical replication. (b) Outline of the wet and dry laboratory steps involved in a microarray experiment. From Molecular and Biochemical Toxicology , 4th ed., eds. R.C. Smart and E. Hodgson. Hoboken, NJ: Wiley, 2008.


since antibodies can be produced that recognize epitopes (specifi c sites on the antigen recognized by the antibody) that include the hapten.

The antibodies used may be polyclonal or monoclonal, each with characteristics fi tting them for use in particular immunochemical methods. Injection of a mammal with a foreign protein (immunogen) gives rise to an immune reaction that includes the generation of antibodies from B lymphocytes. Each B lymphocyte gives rise to only a single antibody type that recognizes a single epitope on the antigen. However, since these antibodies are derived from many different B lymphocytes, the mixture of antibodies can recognize and bind to many different epitopes on the antigen. This mixture of antibodies can be isolated from the serum of the treated animal and is known, collectively, as polyclonal antibodies . Howev

A TEXTBOOK OF - Action Modulerswikimariner.actionmodulers.com/wiki/images/8/80/Modern... · 2017-11-17 · PART III TOXICANT PROCESSING IN VIVO 77 5. Absorption and Distribution of

Documents