Current Protocols in Toxicology

What's New in Current Protocols in Toxicology
Supplement 29, September 2006

UNIT 4.20 NMR Analysis of Thiodiglycol Oxidation by Mammalian Alcohol Dehydrogenases
UNIT 7.8 Measurement of the Vitamin E Metabolites, Carboxyethyk Hydroxychromans (CEHCs), in Biological Samples
UNIT 18.11 Analysis of Immunotoxicity by Enumeration of Antibody-Producing B Cells
UNIT 18.12 The Reporter Antigen Popliteal Lymph Node Assay

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.

Updated every three months in all formats, CPTox is constantly evolving to keep pace with the very latest discoveries and developments. A year of these quarterly updates is included in the initial CPTox purchase price. That's 520 pages of new (71%) and revised (29%) content on average every year since the initial publication of the work in May 1999! Presently two volumes in its looseleaf print version, CPTox...

* 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: James S. Bus (The Dow Chemical Company); Lucio G. Costa (University of Washington); Ernest Hodgson (North Carolina State University); David A. Lawrence (Wadsworth Center); Donald J. Reed (Oregon State University); Advisory Editor: William F. Greenlee; Past Editor-in-Chief: Mahin Maines; Past Editors: I. Glenn Sipes, Shigeru Sassa

Series Editor: Kathy Morgan


Chapter 1 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 2 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
Unit 2.11 Single-Cell Analysis of Lipid Rafts in Lymphocytes and in T Cell—Containing Immunoconjugates

Chapter 3 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
Unit 3.10 Analysis of Mismatch Repair in Human Nuclear Extracts

Chapter 4 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 1 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
Unit 4.18 Aldehyde Dehydrogenases: Measurement of Activities and Protein Levels
Unit 4.19 Measurement of Human Cytochrome P4501A2 (CYP1A2) Activity In Vitro
Unit 4.20 NMR Analysis of Thiodiglycol Oxidation by Mammalian Alcohol Dehydrogenases

Chapter 5 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 6 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
Unit 6.10 Overview of Protein Glutathionylation
Unit 6.11 Measurement of Protein Glutathionylation

Chapter 7 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
Unit 7.7 Catalase Activity Assays
Unit 7.8 Measurement of the Vitamin E Metabolites, Carboxyethyl Hydroxychromans (CEHCs), in Biological Samples

Chapter 8 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 9 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 1 and 2 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
Unit 13.9 Methods in the Analysis of Maternal Behavior in the Rodent
Unit 13.10 Scoring of Social Interactions and Play in Mice During Adolescence
Unit 13.11 Scoring Learning and Memory in Developing Rodents

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
Unit 16.9 Hershberger Assay to Investigate the Effects of Endocrine-Disrupting Compounds with Androgenic or Antiandrogenic Activity in Castrate-Immature Male 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
Unit 17.8 Detection of Stress Proteins as Biomarkers of Oxidative Stress

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
Unit 18.10 Host Resistance Model to an Intracellular Pathogen
Unit 18.11 Analysis of Immunotoxicity by Enumeration of Antibody-Producing B Cells
Unit 18.12 The Reporter Antigen Popliteal Lymph Node Assay

Appendix 1 Using Information
1A Safe Use of Radioisotopes
1B Transgenic and Gene-Targeted Mouse Lines for Toxicology Studies

Appendix 2 Laboratory Stock Solutions and Equipment
2A Common Stock Solutions and Buffers
2B Standard Laboratory Equipment

Appendix 3 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