Wiley InterScience: Reference Work: Current Protocols in Toxicology Current Protocols in Toxicology Copyright © 2005 by John Wiley & Sons, Inc All Rights Reserved ISBN: 0-471-24106-7 Last time updated: September 2005 Current Protocols in Toxicology is a "best-practices" collection of lab protocols for accurate, efficient assessments of toxicity in whole organisms, organs and tissues, cells, and biochemical pathways Continuously updated since its initial publication in May 1999, this quarterly-updated two-volume set - provides the latest models and methods from molecular biology, cell biology, biochemistry, and genetics, plus sophisticated toxicological procedures from leading laboratories - offers expert guidelines for evaluating the effects of substances on human physiology and metabolism - provides valuable reference information in three appendices, including stock solutions and equipment, commonly used techniques, and using information Edited by: Lucio G Costa (University of Washington); Ernest Hodgson (North Carolina State University); David A Lawrence (Wadsworth Center); Terence R Ozolins (Pfizer, Inc.); Donald J Reed (Oregon State University); William F Greenlee, Advisory Editor (CIIT); Past Editor-in-Chief: Mahin Maines; Past Editors: I Glenn Sipes, Shigeru Sassa Series Editor: Kathy Morgan Editors EDITORIAL BOARD Lucio G Costa University of Washington Seattle, Washington Ernest Hodgson North Carolina State University Raleigh, North Carolina David A Lawrence Wadsworth Center Albany, New York Donald J Reed Oregon State University Corvallis, Oregon ADVISORY EDITOR William F Greenlee CIIT Centers for Health Research Research Triangle Park, North Carolina PAST EDITOR-IN-CHIEF Mahin D Maines University of Rochester School of Medicine Rochester, New York PAST EDITORS Shigeru Sassa Rockefeller University New York, New York I Glenn Sipes University of Arizona Tucson, Arizona Chapter Toxicological Models Introduction Unit 1.1 Nonhuman Primates as Animal Models for Toxicology Research Unit 1.2 Statistical Approaches to the Design of Toxicology Studies Unit 1.3 Transgenic Animals in Toxicology Unit 1.4 DNA Microarrays: An Overview of Technologies and Applications to Toxicology Unit 1.5 The Use of Fish-Derived Cell Lines for Investigation of Environmental Contaminants Unit 1.6 Sea Urchin Embryos and Larvae as Biosensors for Neurotoxicants Unit 1.7 Zebrafish: An Animal Model for Toxicological Studies Unit 1.8 Preclinical Models of Parkinson's Disease Chapter Assessment of Cell Toxicity Introduction Unit 2.1 Current Concepts in Cell Toxicity Unit 2.2 Determination of Apoptosis and Necrosis Unit 2.3 Detection of Covalent Binding Unit 2.4 Measurement of Lipid Peroxidation Unit 2.5 Measurements of Intracellular Free Calcium Concentration in Biological Systems Unit 2.6 In Vitro Methods for Detecting Cytotoxicity Unit 2.7 In Situ Hybridization Histochemistry Unit 2.8 Confocal Microscopy Unit 2.9 Measurement of Expression of the HSP70 Protein Family Unit 2.10 Analysis of Mitochondrial Dysfunction During Cell Death Chapter Genetic Toxicology: Mutagenesis and Adduct Formation Introduction Unit 3.1 The Salmonella (Ames) Test for Mutagenicity Unit 3.2 Measurement of a Malondialdehyde-DNA Adduct Unit 3.3 Mutagenesis Assays in Mammalian Cells Unit 3.4 Cell Transformation Assays Unit 3.5 Assays for DNA Damage Unit 3.6 Detecting Epigenetic Changes: DNA Methylation Unit 3.7 Assays for Detecting Chromosomal Aberrations Unit 3.8 Methods for Measuring DNA Adducts and Abasic Sites I: Isolation, Purification, and Analysis of DNA Adducts in Intact DNA Unit 3.9 Methods for Measuring DNA Adducts and Abasic Sites II: Methods for Measurement of DNA Adducts Chapter Techniques for Analysis of Chemical Biotransformation Introduction Unit 4.1 Measurement of Cytochrome P-450 Unit 4.2 Purification of Cytochrome P-450 Enzymes Unit 4.3 Measurements of UDP- Glucuronosyltransferase (UGT) Activities Unit 4.4 Detection of Metabolites Using High-Performance Liquid Chromatography and Mass Spectrometry Unit 4.5 Measurement of Aryl and Alcohol Sulfotransferase Activity Unit 4.6 Measuring the Activity of Arylamine N-Acetyltransferase (NAT) Unit 4.7 Measurement of Carboxylesterase (CES) Activities Unit 4.8 Analysis of the Aryl Hydrocarbon Receptor (AhR) Signal Transduction Pathway Unit 4.9 Measurements of Flavin-Containing Monooxygenase (FMO) Activities Unit 4.10 Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolases and Phosphoric Triester Hydrolases Unit 4.11 Techniques for Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl-CoA:Amino Acid N-Acyltransferase: The Amino Acid Conjugation Pathway Unit 4.12 Determination of Paraoxonase Status and Genotypes at Specific Polymorphic Sites Unit 4.13 Human Cytochrome P450: Metabolism of Testosterone by CYP3A4 and Inhibition by Ketoconazole Unit 4.14 Biotransformation Studies Using Rat Proximal Tubule Cells Unit 4.15 TaqMan Real Time—Polymerase Chain Reaction Methods for Determination of Nucleotide Polymorphisms in Human N-Acetyltransferase-1 (NAT1) and -2 (NAT2) Unit 4.16 Evaluation of the Cytochrome b5/Cytochrome b5 Reductase Pathway Unit 4.17 Measurement of Xenobiotic Carbonyl Reduction in Human Liver Fractions Chapter Toxicokinetics Introduction Unit 5.1 Measurement of Bioavailability: Measurement of Absorption Through Skin In Vitro Unit 5.2 Measurement of Bioavailability: Measuring Absorption Through Skin In Vivo in Rats and Humans Unit 5.3 Measurement of Disposition Half-Life, Clearance, and Residence Times Unit 5.4 Isolated Perfused Porcine Skin Flap Unit 5.5 Porcine Skin Flow-Through Diffusion Cell System Unit 5.6 Toxicant Transport by P-Glycoprotein Unit 5.7 Collection of Bile and Urine Samples for Determining the Urinary and Hepatobiliary Disposition of Xenobiotics in Mice Chapter The Glutathione Pathway Introduction Unit 6.1 Overview of Glutathione Function and Metabolism Unit 6.2 Measurement of Glutathione and Glutathione Disulfide Unit 6.3 Measurement of Glutathione Transport Unit 6.4 Measurement of Glutathione Transferases Unit 6.5 HPLC-Based Assays for Enzymes of Glutathione Biosynthesis Unit 6.6 -Glutamyl Transpeptidase Activity Assay Unit 6.7 Oxidant-Induced Regulation of Glutathione Synthesis Unit 6.8 Measurement of Glutathione Conjugates Unit 6.9 Coenzyme A and Coenzyme A-Glutathione Mixed Disulfide Measurements by HPLC Chapter Assessment of the Activity of Antioxidant Enzymes Introduction Unit 7.1 Analysis of Glutathione-Related Enzymes Unit 7.2 Measurement of Glutathione Reductase Activity Unit 7.3 Analysis of Superoxide Dismutase Activity Unit 7.4 Measurement of Thioredoxin and Thioredoxin Reductase Unit 7.5 Measurement of MnSOD and CuZnSOD Activity in Mammalian Tissue Homogenates Unit 7.6 Measurement of Ascorbic Acid and Dehydroascorbic Acid in Biological Samples Chapter Heme Synthesis Pathway Introduction Unit 8.1 The Heme Biosynthesis Pathway and Clinical Manifestations of Abnormal Function Unit 8.2 Measurement of ALA Synthase Activity Unit 8.3 Measurement of Heme Concentration Unit 8.4 Measurement of Uroporphyrinogen Decarboxylase Activity Unit 8.5 Measurement of Protoporphyrinogen Oxidase Activity Unit 8.6 Measurement of -Aminolevulinate Dehydratase Activity Unit 8.7 Measurement of Ferrochelatase Activity Unit 8.8 Measurement of Erythrocyte Protoporphyrin Concentration by Double Extraction and Spectrofluorometry Unit 8.9 HPLC Methods for Analysis of Porphyrins in Biological Media Chapter Heme Degradation Pathway Introduction Unit 9.1 Overview of Heme Degradation Pathway Unit 9.2 Detection of Heme Oxygenase Activity by Measurement of CO Unit 9.3 Detection of Heme Oxygenase and Proteins and Bilirubin Formation Unit 9.4 Detection of Biliverdin Reductase Activity Unit 9.5 Histochemical Analysis of Heme Degradation Enzymes Unit 9.6 An HPLC Method to Detect Heme Oxygenase Activity Unit 9.7 Functional Analysis of the Heme Oxygenase-1 Gene Promoter Unit 9.8 Quantitation of Human Heme Oxygenase (HO-1) Copies by Competitive RT-PCR Unit 9.9 Purification and Characterization of Heme Oxygenase Chapter 10 The Nitric Oxide/Guanylate Cyclase Pathway Introduction Unit 10.1 Overview of the Pathway and Functions of Nitric Oxide Unit 10.2 Assay of Tissue Activity of Nitric Oxide Synthase Unit 10.3 Detection of Nitrosated Proteins Unit 10.4 Fluorometric Techniques for the Detection of Nitric Oxide and Metabolites Unit 10.5 Measurement of cGMP and Soluble Guanylyl Cyclase Activity Unit 10.6 Histochemical Analysis of Nitric Oxide Synthase by NADPH Diaphorase Staining Unit 10.7 Immunocytochemical Analysis of Cyclic Nucleotides Unit 10.8 Methods for Distinguishing Nitrosative and Oxidative Chemistry of Reactive Nitrogen Oxide Species Derived from Nitric Oxide Unit 10.9 Inducible Nitric Oxide Synthase Expression Chapter 11 Neurotoxicology Introduction Unit 11.1 Overview of Neurotoxicology Unit 11.2 Neurobehavioral Screening in Rodents Unit 11.3 Assessment of Spatial Memory Unit 11.4 Advanced Behavioral Testing in Rodents: Assessment of Cognitive Function in Animals Unit 11.5 Testing for Organophosphate-Induced Delayed Polyneuropathy Unit 11.6 Risk Assessment and Neurotoxicology Unit 11.7 Neurobehavioral Testing in Humans Unit 11.8 Mouse Models of Global Cerebral Ischemia Unit 11.9 Mouse Models of Focal Cerebral Ischemia Unit 11.10 Principles of Electrophysiology: An Overview Unit 11.11 Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices Unit 11.12 Whole-Cell Patch-Clamp Electrophysiology of Voltage-Sensitive Channels Unit 11.13 Detection and Assessment of Xenobiotic-Induced Sensory Neuropathy Unit 11.14 Methods to Produce Brain Hyperthermia Chapter 12 Biochemical and Molecular Neurotoxicology Introduction Unit 12.1 Biochemical Approaches to Studying Neurotoxicity Unit 12.2 Development of an In Vitro Blood-Brain Barrier Unit 12.3 Culturing Rat Hippocampal Neurons Unit 12.4 Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures Unit 12.5 Analytical Cytology: Applications to Neurotoxicology Unit 12.6 Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator Unit 12.7 Isolation of Cerebellar Granule Cells from Neonatal Rats Unit 12.8 Measurement of Glial Fibrillary Acidic Protein Unit 12.9 Aggregating Neural Cell Cultures Unit 12.10 Coculturing Neurons and Glial Cells Unit 12.11 Determining the Ability of Xenobiotic Metals to Bind a Specific Protein Domain by Electrophoresis Unit 12.12 Morphological Measurement of Neurotoxic Injury in the Peripheral Nervous System: Preparation of Material for Light and Transmission Electron Microscopic Evaluation Chapter 13 Teratology Introduction Unit 13.1 Overview of Teratology Unit 13.2 Rat Embryo Cultures for In Vitro Teratology Unit 13.3 Micromass Cultures in Teratology Unit 13.4 Using Chicken Embryos for Teratology Studies Unit 13.5 In Vivo Assessment of Prenatal Developmental Toxicity in Rodents Unit 13.6 Organ Culture of Midfacial Tissue and Secondary Palate Unit 13.7 Overview of Behavioral Teratology Unit 13.8 Statistical Analysis of Behavioral Data Chapter 14 Hepatotoxicology Introduction Unit 14.1 Overview of Hepatotoxicity Unit 14.2 Preparation of Hepatocytes Unit 14.3 Small Animal Models of Hemorrhagic Shock—Induced Liver Dysfunction Unit 14.4 Isolation of Liver Kupffer Cells Unit 14.5 Measurement of Hepatobiliary Transport Chapter 15 Gene Targeting Introduction Unit 15.1 Embryonic Stem (ES) Cell Culture Basics Unit 15.2 Genotyping Embryonic Stem (ES) Cells Unit 15.3 Aggregation Chimeras (ES Cell—Embryo) Unit 15.4 Reporter Genes to Detect Cre Excision in Mice Chapter 16 Male Reproductive Toxicology Introduction Unit 16.1 In Vivo Models for Male Reproductive Toxicology Unit 16.2 Guidelines for Mating Rodents Unit 16.3 Histopathology of the Male Reproductive System I: Techniques Unit 16.4 Histopathology of the Male Reproductive System II: Interpretation Unit 16.5 Monitoring Endocrine Function in Males: Using Intra-Atrial Cannulas to Monitor Plasma Hormonal Dynamics in Toxicology Experiments Unit 16.6 Epididymal Sperm Count Unit 16.7 Performing a Testicular Spermatid Head Count Unit 16.8 Transgenerational (In Utero/Lactational) Exposure to Investigate the Effects of Endocrine Disrupting Compounds (EDCs) in Rats Chapter 17 Oxidative Stress Introduction Unit 17.1 Formation and Functions of Protein Sulfenic Acids Unit 17.2 Measurement of Protein Sulfenic Acid Content Unit 17.3 Fluorescence Microplate Reader Measurement of Tissue Susceptibility to Lipid Peroxidation Unit 17.4 In Situ Localization of Nonenzymatic Peroxidase-Like Activity of Tissue-Bound Transition Metals Unit 17.5 F2-Isoprostanes as Markers of Oxidant Stress: An Overview Unit 17.6 Quantification of F2-Isoprostanes by Gas Chromatography/Mass Spectrometry as a Measure of Oxidant Stress Unit 17.7 Immuno-Spin Trapping: Detection of Protein-Centered Radicals Chapter 18 Immunotoxicology Introduction Unit 18.1 Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment Unit 18.2 Local Lymph Node Assays Unit 18.3 Murine Asthma Models Unit 18.4 Use of Bronchoalveolar Lavage to Detect Lung Injury Unit 18.5 Measuring Lymphocyte Transcription Factor Activity by ELISA Unit 18.6 Measuring the Activity of Cytolytic Lymphocytes Unit 18.7 Solid-Phase Immunoassays Unit 18.8 Immune Cell Phenotyping Using Flow Cytometry Unit 18.9 In Vitro Model for Modulation of Helper T Cell Differentiation and Activation Appendix Using Information 1A Safe Use of Radioisotopes 1B Transgenic and Gene-Targeted Mouse Lines for Toxicology Studies Appendix Laboratory Stock Solutions and Equipment 2A Common Stock Solutions and Buffers 2B Standard Laboratory Equipment Appendix Commonly Used Techniques 3A Molecular Biology Techniques 3B Techniques for Mammalian Cell Tissue Culture 3C Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization 3D Detection and Quantitation of Radiolabeled Proteins in Gels and Blots 3E Northern Blot Analysis of RNA 3F One-Dimensional SDS Gel Electrophoresis of Proteins 3G Spectrophotometric Determination of Protein Concentration 3H Dialysis and Concentration of Protein Solutions 3I The Colorimetric Detection and Quantitation of Total Protein Appendix Suppliers Selected Suppliers of Reagents and Equipment FOREWORD T oxicological research is driven by the need to understand and assess the human and ecological risks of exposure to chemicals and other toxicants as well as by interest in using toxic agents to elucidate basic biological and pathobiological processes The level of research activity in this field is higher, the rate of change in knowledge more rapid, and interest in applying scientific information to societally important issues is greater than ever before These are exciting and challenging times to be working in toxicology The ongoing ferment builds on the extraordinary advances being made in the understanding of biological systems at the molecular level This fundamental knowledge provides the opportunity for greatly enhanced insight into how chemicals and other stressors may damage biological structures and processes, influence the rate of biological repair, and lead to reversible or irreversible diseases or to a return to health Society increasingly calls on the scientific community for the knowledge needed both to reevaluate the health hazards of existing products and technologies and to evaluate the prospective hazards of new ones Such information is used to develop guidelines and regulations designed to ensure that these new products and technologies not harm people or the environment Acquiring sound, reproducible scientific data that can be integrated with existing information to advance the knowledge of toxicants and living systems requires rigorous adherence to the scientific method This means intelligent, thoughtful individuals identifying important needs, formulating testable hypotheses, designing experiments to test them, meticulously conducting these experiments, carefully reviewing and interpreting data, and ultimately presenting this information to scientific peers, including publishing it in the peer-reviewed literature Current Protocols in Toxicology is a clear and well-documented compendium of the most important methods in the field—proven approaches developed by leading researchers—for the benefit of other experimentalists, from students to seasoned investigators Since toxicology by its nature is multidisciplinary, other titles in the Current Protocols series may also provide relevant methods Although review of the literature cited for each procedure can give added insight into the underlying theory and breadth of applications, the protocols have been carefully designed to provide clear, step-by-step descriptions that can easily be followed even by the relatively inexperienced Regular updates to Current Protocols in Toxicology manual will help ensure an awareness of changes in previously documented methods and of methods newly developed Use of these protocols will avoid unnecessary duplication of effort in development and validation when the methods are applied without modification, and will speed up the development of more refined methods that will further advance the field of toxicology and, in turn, may have a place in future updates Roger O McClellan Chemical Industry Institute of Toxicology Research Triangle Park, North Carolina Current Protocols in Toxicology Contributed by Roger O McClellan Current Protocols in Toxicology (1999) Copyright © 1999 by John Wiley & Sons, Inc i PREFACE T he span of research in toxicology has been expanding and diversifying precipitously in recent years One cause for this is the ongoing increase in industrial activity and in the generation of toxic compounds that then find their way into the environment Another is the intensifying public awareness of the health effects of chemical exposure The expansion of the field can be observed by attending any major scientific event dedicated to toxicology—such as the annual meetings of the Society of Toxicology, whose attendance has tripled in the course of the 1990s Examining the meeting program for one of these events provides a very good feel for the broad scope of toxicology For those who have attended such meetings periodically over the past few years, the dynamic nature of the field and its explosive growth is obvious: there is simply more in-depth research going on every year This is in contrast to toxicology’s early years, when the field was dominated by research involving gross assessment of organisms’ responses to toxic chemicals More recent times have witnessed the emergence of applications of state-of-the-art technology to the study of toxicity responses in organisms and living cells, along with phenomenal advancement in molecular and biochemical techniques, which increasingly are finding their way into toxicology research laboratories A growing number of presentations at toxicology meetings constitute bridges between basic toxicology research and approaches to improving human health and environmental quality It is this changing and expanding face of toxicology and its methodologies that represented the greatest challenge in assembling Current Protocols in Toxicology We have attempted to include those methods that are presently central to modern toxicology and that we expect will remain valuable tomorrow Like the field of toxicology, with its quarterly supplements this book will continue to expand in scope, to include more topics and methods as the field advances Because toxicological questions may be addressed using methods deriving from a wide variety of disciplines, other titles in the Current Protocols series may also provide methods that can be applied in your research Molecular biology techniques, in particular, are integral to toxicological investigation Such techniques are included where appropriate within units in this book; however, where these protocols are located may not be readily apparent from the table of contents To help you find them, Table A.3A.1 in APPENDIX 3A provides a listing of specific techniques and where they can be found, either in this book or in related Current Protocols manuals In addition, protocols for a number of basic techniques will be added to APPENDIX in future supplements Although mastery of the techniques in this manual will enable readers to pursue research in toxicology, the manual is not intended to be a substitute for graduate-level courses or a comprehensive textbook in the field An inevitable hazard of manual writing is that protocols may become obsolete as the field expands and new techniques are developed To safeguard this manual from inexorable obsolescence (and perhaps pleasantly surprise the users of the manual!), we provide quarterly supplements to provide protocols that utilize new innovations and technologies in the field The updatable formats—looseleaf binder, CD-ROM, Intranet, and online Internet—easily accommodate the addition of this new material Current Protocols in Toxicology Contributed by Mahin D Maines, Lucio G Costa, Donald J Reed, Shigeru Sassa, and I Glenn Sipes Current Protocols in Toxicology (1999) iii-vi Copyright © 1999 by John Wiley & Sons, Inc iii ... Carolina Current Protocols in Toxicology Contributed by Roger O McClellan Current Protocols in Toxicology (1999) Copyright © 1999 by John Wiley & Sons, Inc i PREFACE T he span of research in toxicology. .. Wiley InterScience: Reference Work: Current Protocols in Toxicology Current Protocols in Toxicology Copyright © 2005 by John Wiley & Sons, Inc All Rights Reserved ISBN:... carefully reviewing and interpreting data, and ultimately presenting this information to scientific peers, including publishing it in the peer-reviewed literature Current Protocols in Toxicology is