Molecular and Biochemical Toxicology E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 1756

Veröffentlichungsjahr: 2013

Beschreibung

An essential resource for graduate students, academic andindustrial toxicologists, and environmental health scientists andprofessionals Over the course of thirty years and three editions,Introduction to Biochemical Toxicology has been an importantsource for coverage of the ongoing quest to define the biochemical,cellular, and molecular events induced by toxicants at the cellularand organismic levels. Now, as the principles and methods ofmolecular and cellular biology as well as genomic sciences play anever increasing role in mechanistic toxicology, significant changeshave been made to the book, resulting in this important newedition-now titled Molecular and Biochemical Toxicology,Fourth Edition. Much more than an introductory text, this crucial new editionhas been completely revised to provide timely and thorough coverageof the underlying biochemical, molecular, and cellular mechanismsthrough which toxicants produce their adverse effects.Toxicological issues are covered from the molecule to the cell tothe organ level. Complex methods used in toxicology are alsodescribed in a straightforward, easy-to-understand style.Additional features of this new edition include: * New chapters that explore the interface between toxicology andgenomic sciences, including: bioinformatics, proteomics,metabolomics, and toxicogenomics * Increased emphasis on structure, mechanism, and regulation ofxenobiotic metabolizing enzymes, toxicogenetics, and xenobiotictransporters * Additional new chapters on: molecular epidemiology and geneticsusceptibility, DNA damage and mutagenesis, DNA repair, mechanismsof cell death, mitochondrial dysfunction, metals, reproductivetoxicology, developmental toxicology, and reactiveoxygen/metabolites and toxicity Molecular and Biochemical Toxicology, Fourth Editionguides graduate students, toxicologists, and environmental healthprofessionals through the principles of molecular and biochemicaltoxicology and the complex mechanisms of toxicity. Whether it'sused in the classroom or in industry, research, or academia, thisbook is essential for anyone interested in understanding themolecular mechanisms through which toxicants produce adverseeffects.

Leseprobe

Contents

Cover

Half Title page

Title page

Copyright page

Preface

Contributors

Chapter 1: Molecular and Biochemical Toxicology: Definition and Scope

1.1 Introduction

1.2 Toxicology

1.3 Biochemical Toxicology

1.4 Cellular Toxicology

1.5 Molecular Toxicology

1.6 Proteomics and Metabolomics

1.7 Conclusions

Suggested Reading

Chapter 2: Overview of Molecular Techniques in Toxicology: Genes and Transgenes

2.1 Applicability of Molecular Techniques to Toxicology

2.2 Overview of the Genetic Code and Flow of Genetic Information

2.3 Molecular Cloning

2.4 Southern and Northern Blot Analyses

2.5 Polymerase Chain Reaction (PCR)

2.6 Some Methods to Evaluate Gene Expression and Regulation

2.7 Methods to Evaluate Gene Function

Suggested Reading

Chapter 3: Toxicogenomics

3.1 Introduction

3.2 A Primer of Genomics

3.3 Tools and Approaches

3.4 Genomes

3.5 Functional Genomics

3.6 Conclusions

Suggested Reading

Chapter 4: Proteomics

4.1 Introduction to Proteomics

4.2 Properties of Proteins

4.3 Fields of Proteomic Research

4.4 Mass Spectrometers and Protein Identification

4.5 Proteomic Platforms

4.6 Proteomes and Subproteomes: Expectations and Reality

4.7 Summary

Suggested Reading

Chapter 5: Metabolomics

5.1 Introduction

5.2 Methods

5.3 Diagnostics and Functional Genomics

5.4 Metabolomics and Toxicology

Suggested Reading

Chapter 6: Bioinformatics

6.1 Introduction

6.2 Obtaining the Genbank Record of a Known Gene

6.3 Sequence Comparison

6.4 Database Searching with BLAST

6.5 Extensions to Multiple Sequences

6.6 Genetic Mapping

6.7 Conclusion

Suggested Reading

Chapter 7: Immunochemical Techniques In Toxicology

7.1 Introduction

7.2 Definitions

7.3 Immunogens and Antigens

7.4 Polyclonal Antibodies

7.5 Monoclonal Antibodies

7.6 Immunoassays

7.7 Conclusions

Suggested Reading

Chapter 8: Cellular Techniques

8.1 Introduction

8.2 Cellular Studies in Intact Tissue

8.3 Studies with Dispersed, Isolated Cells

8.4 Monolayer Cell Culture

8.5 Observation of Cultured Cells

8.6 Indicators of Toxicity

8.7 Artifacts and Confounders

8.8 Replacement of Animal Testing with Cell Culture Models

8.9 Conclusions

Suggested Reading

Chapter 9: Structure, Mechanism, and Regulation of Cytochromes P450

9.1 Introduction

9.2 Complexity of the Cytochrome P450 Gene Superfamily

9.3 Cytochrome P450 Structure

9.4 Mechanisms of P450 Catalysis

9.5 Cytochrome P450 Regulation

9.6 Transgenic Animal Models

9.7 Reactive Oxygen Species

9.8 Posttranslation Modification of P450s

9.9 Summary

Suggested Reading

Chapter 10: Phase 1 Metabolism of Toxicants and Metabolic Interactions

10.1 Introduction

10.2 Microsomal Monooxygenations: General Background

10.3 Nonmicrosomal Oxidations

10.4 Cooxidation by Prostaglandin Synthetase

10.5 Reduction Reactions

10.6 Hydrolysis

10.7 Epoxide Hydration

10.8 DDT-Dehydrochlorinase

10.9 Interactions Involving Xenobiotic Metabolizing Enzymes

Suggested Reading

Chapter 11: Phase I—Toxicogenetics

11.1 Introduction

11.2 Polymorphisms in CYP Isoforms

11.3 Polymorphisms in Alcohol Dehydrogenase

11.4 Polymorphisms in Aldehyde Dehydrogenase

11.5 Polymorphisms in Flavin-Containing Monooxygenases

11.6 Polymorphisms in Epoxide Hydrolase

11.7 Polymorphisms in Serum Cholinesterase

11.8 Polymorphisms in Paraoxonase (PON1)

11.9 Polymorphisms: Mechanistic Classification

11.10 Polymorphisms and Drug Metabolism

11.11 Methods Used for the Study of Polymorphisms

11.12 Epidemiology

Suggested Reading

Chapter 12: Phase II—Conjugation of Toxicants

12.1 Introduction

12.2 Conjugation Reactions

12.3 Glycosides

12.4 Sulfation

12.5 Methylation

12.6 Acylation

12.7 Glutathione S-Tranferases

12.8 Cysteine S-Conjugate β-Lyase

12.9 Lipophilic Conjugation

12.10 Phase II Activation

12.11 Phase III Elimination

12.12 Conclusions

Suggested Reading

Chapter 13: Regulation and Polymorphisms in Phase II Genes

13.1 Introduction

13.2 Roles of Phase II Genes and Polymorphisms

13.3 The Antioxidant Responsive Element and Phase II Gene Regulation

13.4 Summary

Suggested Reading

Chapter 14: Developmental Effects on Xenobiotic Metabolism

14.1 Introduction

14.2 Xenobiotic Metabolism During Development

14.3 Effects of Pregnancy on Maternal Xenobiotic Metabolism

14.4 Developmental Effects on Xenobiotic Metabolism in Nonmammalian Species

14.5 Cycles in Development

Suggested Reading

Chapter 15: Cellular Transport and Elimination

15.1 Transport as a Determinant of Xenobiotic Action

15.2 Factors Affecting Membrane/Tissue Permeability

15.3 Xenobiotic Transporters

15.4 Altered Xenobiotic Transport

Suggested Reading

Chapter 16: Mechanisms of Cell Death

16.1 Introduction

16.2 How Cells/Tissues React to “Stress”

16.3 Cell Injury and Cell Death

16.4 Morphology of Cell Injury and Cell Death

16.5 Apoptosis

Acknowledgments

Suggested Reading

Chapter 17: Mitochondrial Dysfunction

17.1 Introduction

17.2 Mitochondrial Function

17.3 Mitochondrial Apotosis/Necrosis

17.4 Toxicant-Induced Mitochondrial Apoptosis/Necrosis

Suggested Reading

Chapter 18: Glutathione-Dependent Mechanisms in Chemically Induced Cell Injury and Cellular Protection Mechanisms

18.1 Introduction

18.2 Glutathione-Dependent Conjugation of Chemicals

18.3 GSH-Dependent Bioactivation of Chemicals

18.4 Oxidative Stress

18.5 Glutathione-Dependent Cellular Defense Systems

18.6 Glutathione/Glutathionylation Dependent Signaling Systems and Antioxidant Defense

18.7 Conclusions

Acknowledgement

Suggested Reading

Chapter 19: Toxicant–Receptor Interactions: Fundamental Principles

19.1 Definition of a Receptor

19.2 Receptor Superfamilies

19.3 The Study of Receptor-Toxicant Interactions

19.4 Relationship of Receptor Occupancy to Functional Effects

Suggested Reading

Chapter 20: Reactive Oxygen/Reactive Metabolites and Toxicity

20.1 Introduction

20.2 Enzymes Involved in Bioactivation

20.3 Stability of Reactive Metabolites

20.4 Factors Affecting Activation and Toxicity

20.5 Reactive Oxygen Species and Toxicity

Suggested Reading

Chapter 21: Metals

21.1 Introduction

21.2 Understanding Metal Ion Reactions in Biological Systems

21.3 Modes of Metal Toxicity

21.4 Metals and Oxidative Stress

21.5 Metallothioneins

21.6 Examples of Toxic Metals

21.7 Metals and Cancer

Acknowledgments

Suggested Reading

Chapter 22: DNA Damage and Mutagenesis

22.1 Introduction

22.2 Endogenous DNA Damage

22.3 Environmental DNA Damage

22.4 Concepts of Mutagenesis

22.5 Mechanisms of DNA Damage-Induced Mutagenesis

Suggested Reading

Chapter 23: DNA Repair

23.1 Introducton

23.2 Direct Reversal of Base Damage

23.3 Base Excision Repair

23.4 Nucleotide Excision Repair

23.5 Mismatch Repair

23.6 Recombinational Repair

23.7 DNA Repair and Chromatin Structure

23.8 DNA Damage and Cell Cycle Checkpoints

23.9 Summary

Suggested Reading

Chapter 24: Carcinogenesis

24.1 Introduction and Historical Perspective

24.2 Human Cancer

24.3 Categorization of Agents Associated with Carcinogenesis

24.4 Somatic Mutation Theory

24.5 Epigenetic Mechanism of Tumorigenesis

24.6 Multistage Tumorigenesis

24.7 Tumor Viruses

24.8 Cellular Oncogenes

24.9 Tumor Suppressor Genes

24.10 Mutator Phenotype/DNA Stability Genes

24.11 Interaction of Oncogenes and Tumor Suppressor Genes

24.12 Genetically Modified Mouse Models

24.13 Conclusions

Suggested Reading

Chapter 25: Genetic Toxicology

25.1 Introduction and Historical Perspective

25.2 Genetic Toxicology and Risk Assessment (General Considerations)

25.3 Genotoxicity Assays

25.4 Use of Mechanistic Data in Cancer and Genetic Risk Assessments (Specific Considerations)

25.5 New Research Directions

25.6 Conclusions

Acknowledgments

Suggested Reading

Chapter 26: Molecular Epidemiology and Genetic Susceptibility

26.1 Introduction

26.2 Basic Concepts in Epidemiology

26.3 Biomarkers Used in Molecular Epidemiology

26.4 Biomarkers of Genetic Susceptibility

26.5 Summary and Concluding Remarks

Suggested Readings

Chapter 27: Respiratory Toxicity

27.1 Introduction

27.2 Anatomy and Function of the Respiratory Tract

27.3 Toxicant-Induced Lung Injury, Remodeling, and Repair

27.4 Occupational and Environmental Lung Diseases

Suggested Reading

Chapter 28: Hepatotoxicity

28.1 Introduction

28.2 Liver Organization and Cellular Components

28.3 Types of Chemically Induced Lesions

28.4 Mechanisms of Chemically Induced Hepatotoxicity

28.5 Interactions

28.6 Detection and Prediction of Hepatotoxicity

28.7 Compounds Causing Liver Injury

28.8 Conclusion

Suggested Reading

Chapter 29: Biochemical Mechanisms of Renal Toxicity

29.1 Introduction

29.2 Fundamental Aspects of Renal Physiology

29.3 Factors Contributing to Nephrotoxicity

29.4 Assessment of Nephrotoxicity

29.5 Site-Specific Nephrotoxicity

29.6 Summary

Suggested Reading

Chapter 30: Biochemical Toxicology of the Peripheral Nervous System

30.1 Introduction

30.2 Specialized Aspects of Neuronal Metabolism

30.3 Specialized Aspects of Schwann Cell Metabolism

30.4 Toxic Neuropathies

30.5 Conclusion

Acknowledgments

Suggested Reading

Chapter 31: Biochemical Toxicology of the Central Nervous System

31.1 Introduction

31.2 CNS Sites of Toxic Action

31.3 Factors Affecting Neurotoxicant Susceptibility

31.4 Mechanisms of Neurotoxicity and Neuroprotection

31.5 The Dynamic Nervous System: Adaptability, Plasticity, and Repair

Suggested Reading

Chapter 32: Immunotoxicity

32.1 Introduction

32.2 Organization of the Immune System

32.3 Immunotoxicology

32.4 Mechanisms of Immune Suppression

32.5 Mechanisms Associated with Hypersensitivity

32.6 Mechanisms Associated with Autoimmune Disease

Suggested Reading

Chapter 33: Reproductive Toxicology

33.1 Introduction

33.2 General Principles of Reproductive Toxicology

33.3 Sexual Differentiation

33.4 Neuroendocrine Regulation of Reproduction

33.5 Male Reproductive System

33.6 Female Reproductive System

33.7 General Categories of Reproductive Toxicants

33.8 Summary

Suggested Reading

Chapter 34: Developmental Toxicology

34.1 Introduction

34.2 Overview of Development

34.3 Wilson’s Principles of Teratology

34.4 Selected Examples of Developmental Toxicants

34.5 Summary

Suggested Reading

Chapter 35: Dermatotoxicology

35.1 Introduction

35.2 Functions of Skin

35.3 Epidermis

35.4 Anatomical Factors to Consider in Model Selection

35.5 Percutaneous Absorption and Penetration

35.6 Dermatotoxicity

35.7 Dermal Toxicity of Nanoparticles

35.8 Conclusion

Suggested Reading

Index

MOLECULAR AND BIOCHEMICAL TOXICOLOGY

Copyright © 2008 by John Wiley & Sons, Inc. All rights reserved

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

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Library of Congress Cataloging-in-Publication Data:

Molecular and biochemical toxicology / [edited by] Robert C. Smart, Ernest Hodgson. - 4th ed.p.; cm.Rev. ed of: Introduction to biochemical toxicology. 2001.Includes bibliographical references and index.ISBN 978-0-470-10211-4 (cloth)1. Biochemical toxicology. I. Smart, Robert C., 1954- II. Hodgson, Ernest, 1932- III. Introduction to biochemical toxicology. [DNLM: 1. Poisoning–metabolism. 2. Poisons-metabolism. 3. Molecular Biology-methods. 4. Toxicology-methods. QV 600 M7178 2008]RA1219.5.I58 2008615.9-dc222007044577

PREFACE

This is the 4th edition of a series that was initiated in 1980 as Introduction to Biochemical Toxicology, edited by Ernest Hodgson and Frank E. Guthrie and based on a course at North Carolina State University that has now been offered for almost 30 years. The 2nd and 3rd editions, with the same title, were edited by Hodgson and Levi and by Hodgson and Smart. The changes in both scope and specific content are greater between this edition and the previous one and, in fact, are so extensive as to require several important changes. First, to recognize the changing roles of the co-editors, they are now listed as Smart and Hodgson. Second, the title has been changed to Molecular and Biochemical Toxicology.

The incorporation of the principles and methods of molecular biology into mechanistic toxicology has continued apace and is reflected not only in the change in title but also in the addition of new chapters and the integration of molecular material into most, if not all, of the chapters represented in the 3rd edition and extensively revised for the 4th. The section on Methodology (Chapters 2–8) now contains additional chapters on Toxicogenomics, Proteomics, Metabolomics, and Bioinformatics, and the section on Toxicant Processing (Chapters 9–15) has been reorganized to better emphasize the role of polymorphisms in xenobiotic-metabolizing enzymes, transporters, and so on. The section on Mechanisms (Chapters 16–26) now contains chapters on Cell Death and separate chapters on DNA Damage and DNA Repair. The final section on Target Organs (Chapters 27–35), although containing essentially the same chapters as the 3rd edition, has several new authors and all chapters are extensively revised.

We believe that both instructors and advanced students will continue to find this series a usable, integral component of their graduate curriculum, with the broad scope providing a background that will enable all instructors to select material to suit their particular needs. Many thanks to all of the contributing authors and to all at John Wiley & Sons who made this edition possible.

Raleigh, North Carolina

ROBERT C. SMARTERNEST HODGSON

CONTRIBUTORS

Bonita L. Blake, Departments of Pharmacology and Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

James C. Bonner, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Thomas W. Bouldin, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Christal C. Bowman, Immunotoxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

David B. Buchwalter, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Taehyeon M. Cho, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

John F. Couse, Taconic Inc., Albany Operations, Rensselaer, New York 12144

Edward L. Croom, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Helen C. Cunny, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Parikshit C. Das, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Nigel Deighton, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Susan Elmore, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Sarah J. Ewing, Penn State Erie, The Behrend College, Erie, PA 16563

Dori R. Germolec, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Jeffry F. Goodrum, (retired) Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Ernest Hodgson, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Mac Law, Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27695

Gerald A. LeBlanc, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Kari D. Loomis, Functional Genomics Program, North Carolina State University, Raleigh, North Carolina 27695

Robert W. Luebke, Immunotoxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Ruth M. Lunn, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Richard B. Mailman, Departments of Psychiatry, Pharmacology, Neurology and Medicinal Chemistry, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Elizabeth L. MacKenzie, Education and Training Systems International, Chapel Hill, North Carolina 27514

Isabel Mellon, Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536

B. Alex Merrick, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Sharon A. Meyer, College of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana 71209

David S. Miller, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Nancy A. Monteiro-Riviere, Center for Chemical Toxicology Research and Pharmacokinetics, North Carolina State University, Raleigh, North Carolina 27606

Dahlia M. Nielsen, Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695

Ninomiya-Tsuji, Jun, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Marjorie F. Oleksiak, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, Florida, 33149

R. Julian Preston, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Donald J. Reed, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331

Martin J J. Ronis, Department of Pharmacology and Toxicology, Arkansas Children’s Nutrition Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72202

Randy L. Rose, (deceased) Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

MaryJane K. Selgrade, Immunotoxicology Branch, U. S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

John M. Seubert, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton AB, Canada

Robert C. Smart, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633.

Ralph J. Smialowicz, (retired) Immunotoxicology Branch, U. S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Mariana C. Stern, Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90089

Eric A. Stone, Department of Statistics, North Carolina State University, Raleigh, North Carolina 27695

Joan B. Tarloff, Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, Philadelphia, Pennsylvania 10104

Arrel D. Toews, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Yoshiaki Tsuji, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Andrew D. Wallace, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Zhigang Wang, Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536

Marsha D. Ward, Immunotoxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Darryl C. Zeldin, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

CHAPTER 1

Molecular and Biochemical Toxicology: Definition and Scope

ERNEST HODGSON

Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

ROBERT C. SMART

Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633

1.1 INTRODUCTION

After the publication of the previous edition, toxicology saw a dramatic increase in the application of the principles and methods of molecular biology. Biochemical and molecular toxicology are concerned with the definition, at the molecular and cellular levels, of the cascade of events that is initiated by exposure to a toxicant and culminates in the expression of a toxic endpoint. Molecular techniques have provided a wealth of mechanistic information about the role of gene function in the interaction of xenobiotics and living organisms. The development of “knockout” mice with genes of interest deleted, along with the development of “humanized mice” with human genes inserted into their genome, has proven extremely valuable in investigations of toxicant metabolism and modes of toxic action. This edition, the fourth (retitled Molecular and Biochemical Toxicology), reflects this by the inclusion of chapters on molecular methods and the inclusion, in essentially every chapter, of molecular studies and approaches currently used in understanding the metabolic processing and mode of toxic action of xenobiotics.

Toxicology can be defined as the branch of science dealing with poisons. Having said that, attempts to define all of the various parameters lead to difficulties. The first difficulty is seen in the definition of a poison. Broadly speaking, a poison is any substance causing harmful effects in an organism to which it is administered, either deliberately or by accident. Clearly, this effect is dose-related inasmuch as any substance, at a low enough dose, is without effect, while many, if not most, substances have deleterious effects at some higher dose. Much of toxicology deals with compounds exogenous to the normal metabolism of the organism, with such compounds being referred to as xenobiotics. However, many endogenous compounds, including metabolic intermediates such as glutamate, or hormones such as thyroxine, are toxic when administered in unnaturally high doses. Similarly, trace nutrients such as selenium, which are essential in the diet at low concentrations, are frequently toxic at higher levels. Such effects are properly included in toxicology, while the endogenous generation of high levels of metabolic intermediates due to disease or metabolic defect is not, although the effects on the organism may be similar.

The expression of toxicity, hence the assessment of toxic effects, is another parameter of considerable complexity. Acute toxicity, usually measured as mortality and expressed as the lethal dose or concentration required to kill 50% of an exposed population under defined conditions (LD50 or LC50), is probably the simplest measure of toxicity. Nevertheless, it varies with age, gender, diet, the physiological condition of the animals, environmental conditions, and the method of administration. Chronic toxicity may be manifested in a variety of ways, including cancer, cataracts, peptic ulcers, and reproductive effects, to name only a few. Furthermore, chemicals may have different effects at different doses. For example, vinyl chloride is a potent hepatotoxicant at high doses and is a carcinogen with a very long latent period at low doses. Considerable variation also exists in the toxic effects of the same chemical administered to different animal species, or even to the same animal when administered via different routes. Malathion, for example, has relatively low toxicity to mammals, but is toxic enough to insects to be a widely used commercial insecticide.

1.2 TOXICOLOGY

Toxicology is clearly related to two of the applied biologies; medicine and agriculture. In medicine, clinical diagnosis and treatment of poisoning as well as the management of toxic side effects of clinical drugs are areas of significance. In agriculture, the development of selective biocides such as insecticides, herbicides, and fungicides is important, and their nontarget effects are of considerable public health significance. Toxicology may also be considered an area of fundamental science because the adaptation of organisms to toxic environments has important implications for ecology and evolution.

The tools of chemistry, biochemistry, and molecular biology are the primary tools of toxicology, and progress in toxicology is closely linked to the development of new methodology in these sciences. Those of chemistry provide analytical methods for toxicants and their metabolites, particularly for forensic toxicology, residue analysis, and toxicant metabolism; those of biochemistry provide methods for the investigation of metabolism and modes of toxic action; and those of molecular biology provide methods for investigations of the roles of genes and gene expression in toxicity.

1.3 BIOCHEMICAL TOXICOLOGY

Biochemical toxicology deals with processes that occur at the cellular and molecular levels when toxic chemicals interact with living organisms. Defining these interactions is fundamental to our understanding of toxic effects, both acute and chronic, and is essential for the development of new therapies, for the determination of toxic hazards, and for the development of new clinical drugs for medicine and biocides for agriculture.

The poisoning process may be thought of as a cascade of more or less distinct events. While biochemical and molecular toxicology are involved in all of these, their involvement in exposure analysis is restricted to the discovery and use of biomarkers of exposure (see Chapter 26). Following exposure, uptake involves the biochemistry of cell membranes and distribution, or transport processes within the body (Chapters 15 and 35). Metabolism, which may take place at portals of entry or, following distribution, in other organs, particularly the liver, may either detoxify toxicants or activate them to reactive metabolites more toxic than the parent chemical (see Chapters 9 through 14). Chemicals with intrinsic toxicity or reactive metabolites are involved in various modes of toxic action, usually initiated by interactions with macromolecules such as proteins and DNA. The study of modes of toxic action is a critically important area of toxicology (see Chapters 16 through 26). The final phase of detoxication, namely excretion (see Chapters 15 and 29), is studied at the cellular, organ, and intact organism levels.

Many of these aspects are studied at the organ level (discussed in Chapters 27 through 35), including portals of entry, respiratory toxicology, hepatotoxicology, nephrotoxicology, toxicology of the peripheral and central nervous systems, immunotoxicity, reproductive and developmental toxicity, and dermatotoxicity.

1.4 CELLULAR TOXICOLOGY

The culture of cells isolated from living organisms has been known since the early years of the twentieth century. By the 1950s the development of standardized culture media and the development of immortalized cell lines increased the utility of cultured cells in many areas of experimental biology, including toxicology. The use of cell culture in toxicological research is an established and useful approach for a number of reasons, including its use in investigating toxic effects on intact cellular systems in a situation less complex than that in the intact organism and its potential utility for routine toxicity testing systems for regulatory evaluations.

Some cells, such as hepatocytes, must be used in primary culture since they will not divide in culture and are relatively short-lived, while other cell lines are capable of division and can, in suitable media, be maintained indefinitely. In other cases, cells have been “immortalized” by fusion with tumor cells and thereafter retain the ability to divide in culture while, at the same time, maintaining many of the properties of the original nontumor cells. All of the various approaches to the use of cultured cells in biochemical and molecular toxicology are summarized in Chapter 8. The relatively recent union of the techniques of cell and molecular biology has been enormously productive for experimental toxicology since cells can be used for the expression of genetic constructs, reproduction of recombinant enzymes, and so on.

1.5 MOLECULAR TOXICOLOGY

The field of molecular biology is usually held to have begun with the description of the double helical structure of DNA by Watson and Crick in 1953, followed by the elucidation of the genetic code in the 1960s. In the subsequent half-century the techniques of molecular biology have expanded exponentially as has its importance in many, if not most, fields of biology. The success of the human genome project has given rise to an entire field devoted to the description of the complete genomes of organisms at all levels in the evolutionary tree. An overview of molecular techniques is presented in Chapter 2, and a review of toxicogenomics is presented in Chapter 3.

The techniques that have proven most valuable in toxicology include those of molecular cloning, the polymerase chain reaction, and the production of genetically modified mice. Microarrays, used to evaluate gene expression under various conditions, including exposure to toxicants, are becoming more important and, in concert with other molecular techniques, are being considered as potentially useful in such applied areas as hazard assessment and risk analysis.

Bioinformatics, which deals with the maintenance, analysis, and integration of genomic data, is discussed in Chapter 6.

1.6 PROTEOMICS AND METABOLOMICS

Since molecular biology is often held to be restricted to events involving nucleic acids, mention must be made of (a) proteomics, the analysis of all proteins in a sample of biological material, and (b) metabolomics, the analysis of all metabolites in a sample of biological material. These fields are discussed in Chapters 4 and 5.

1.7 CONCLUSIONS

The preceding brief description of the nature and scope of biochemical and molecular toxicology should make clear that the study of toxic action is a many-faceted subject, covering all aspects from the initial environmental contact with a toxicant to its toxic endpoints and to its ultimate excretion back into the environment. A considerable amount of material is summarized in the chapters following, but many essentials still remain to be discovered.

SUGGESTED READING

Alberts, B., Roberts, K., Lewis, J., Raff, M., Walter, P., and Johnson, A. Molecular Biology of the Cell, 4th ed., Garland Publishing, New York, 2002.

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. Current Protocols in Molecular Biology, John Wiley & Sons, Hoboken NJ, 1987–2007.

Bus, J. S., Costa, L. G., Hodgson, E., Lawrence, D. A., and Reed, D. J. Current Protocols in Toxicology, John Wiley & Sons, Hoboken, NJ, 1999-2007.

Hodgson, E. (Ed.). A Textbook of Modern Toxicology, 3rd ed., John Wiley & Sons, Hoboken NJ, 2004.

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

Watson, J. D., Baker, Tania A., Bell, S. P., Gann, A., Levine, M., and Losick, R. Molecular Biology of the Gene, 5th ed., Benjamin Cummings, Menlo Park, CA, 2003.

CHAPTER 2

Overview of Molecular Techniques in Toxicology: Genes and Transgenes

ROBERT C. SMART

Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633

2.1 APPLICABILITY OF MOLECULAR TECHNIQUES TO TOXICOLOGY

Molecular cloning and techniques involving the manipulation of DNA and RNA have revolutionized the biological sciences. These techniques have allowed for the elucidation of gene function in complex biological processes such as cell growth, differentiation, development, and cancer as well as chemical toxicity. In a much broader sense, these techniques have allowed for the successful completion of the Human, Mouse, and Rat Genome Projects. Genomic information and bioinformatic technologies derived from these projects will have a major impact on our understanding of chemical-induced toxicity as it relates to mechanisms, species differences, individual susceptibility, and the use of appropriate animal models for hazard characterization.

Molecular techniques have wide applicability in understanding the mechanisms and the responses of an organism to xenobiotic insult. For example, the response of a host organism to a xenobiotic often results in adaptive responses involving alterations in gene expression. Using various molecular approaches, such genes can be identified and their roles elucidated. Expression of these genes could be used as biomarkers of exposure. Chemical-induced alterations in gene expression and pathways can be elucidated using DNA microarrays where the expression of hundreds to thousands of genes can be studied in a single experiment. Additional examples where molecular biology can be applied to toxicology include the identification of chemical carcinogen-induced mutations in oncogenes and tumor suppressor genes. In certain cases, the mutation spectra in onogene/tumor suppressor gene in human tumors can be used as a molecular fingerprint to aid in the identification of the responsible carcinogen. This approach is currently utilized in the field of Molecular Epidemiology. Genetic susceptibility to xenobiotic insult can be the result of certain genetic polymorphisms. Molecular techniques can be used to identify these genetic polymorphisms and to characterize their toxicological significance. Molecular approaches can also be employed to understand species and sex differences to toxicant responses as they relate to differences in gene structure, function, and expression.

The introduction of a gene of interest into a cell or animal is a powerful approach to characterize the function of a gene in a toxicological response. For example, this approach can be used to express human xenobiotic metabolizing genes in recipient cells in culture. Such ectopically expressed genes can facilitate the determination of substrate specificity and the role of individual enzymes in the production and detoxification of toxic metabolites as well as species differences. The ability to generate transgenic animals and gene knockout and knockin animals allows for study of gene function in vivo. The creation of transgenic, knockout, and knockin mice provide a powerful approach in mechanistic toxicological studies and in the development of in vivo models designed to be more predictive of human toxicity. This chapter is intended to provide a conceptual overview/primer of molecular techniques/approaches. For more comprehensive and detailed information the reader is referred to the textbook . For technical information and protocols the reader is referred to the following laboratory manuals: and