Current Protocols in Toxicology
Table of Contents

Foreword by Roger O. McClellan
Preface
Contributors

1 Toxicological Models
1.1 Nonhuman Primates as Animal Models for Toxicology Research
1.2 Statistical Approaches to the Design of Toxicology Studies
1.3 Transgenic Animals in Toxicology
1.4 DNA Microarrays: An Overview of Technologies and Applications to Toxicology
1.5 The Use of Fish-Derived Cell Lines for Investigation of Environmental Contaminants
1.6 Sea Urchin Embryos and Larvae as Biosensors for Neurotoxicants
1.7 Zebrafish: An Animal Model for Toxicological Studies (New, August 2003)
1.8 Preclinical Models of Parkinson's Disease

2 Assessment of Cell Toxicity
2.1 Current Concepts in Cell Toxicity
2.2 Determination of Apoptosis and Necrosis
2.3 Detection of Covalent Binding
2.4 Measurement of Lipid Peroxidation
2.5 Measurements of Intracellular Free Calcium Concentration in Biological Systems
2.6 In Vitro Methods for Detecting Cytotoxicity
2.7 In Situ Hybridization
2.8 Confocal Microscopy
2.9 Measurement of Expression of the HSP70 Protein Family
2.10 Analysis of Mitochondrial Dysfunction During Cell Death

3 Genetic Toxicology: Mutagenesis and Adduct Formation
3.1 The Salmonella (Ames) Test for Mutagenicity
3.2 Measurement of a Malondialdehyde-DNA Adduct
3.3 Mutagenesis Assays in Mammalian Cells
3.4 Cell Transformation Assays
3.5 Assays for DNA Damage
3.6 Detecting Epigenetic Changes: DNA Methylation
3.7 Assays for Detecting Chromosomal Aberrations
3.8 Methods for Measuring DNA Adducts and Abasic Sites: I. Isolation, Purification, and Analysis of DNA Adducts in Intact DNA
3.9 Methods for Measuring DNA Adducts and Abasic Sites: II. Methods for Measurement of DNA Adducts

4 Techniques for Analysis of Chemical Biotransformation
4.1 Measurement of Cytochrome P-450
4.2 Purification of Cytochrome P-450 Enzymes
4.3 Measurements of UDP-Glucuronosyltransferase (UGT) Activities
4.4 Detection of Metabolites Using High-Performance Liquid Chromatography and Mass Spectrometry
4.5 Measurement of Aryl and Alcohol Sulfotransferase Activity
4.6 Measuring the Activity of Arylamine N-Acetyltransferase
4.7 Measurement of Carboxylesterase (CES) Activities
4.8 Analysis of the Aryl Hydrocarbon Receptor (AhR) Signal Transduction Pathway
4.9 Measurements of Flavin-Containing Monooxygenase (FMO) Activities
4.10 Assays for the Classification of Two Types of Esterases
4.11 Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl-CoA:Amino Acid N-Acyltransferase
4.12 Determination of Paraoxonase 1 Status and Genotypes at Specific Polymorphic Sites
4.13 Human Cytochrome P450: Metabolism of Testosterone by CYP3A4 and Inhibition by Ketoconazole
4.14 Biotransformation Studies Using Rat Proximal Tubule Cells
4.15 TaqMan Real Time—Polymerase Chain Reaction Methods for Determination of Nucleotide Polymorphisms in Human N-Acetyltransferase-1 (NAT1) and -2 (NAT2)

5 Toxicokinetics
5.1 Measurement of Bioavailability: Measurement of Absorption Through Skin In Vitro
5.2 Measurement of Bioavailability: Measuring Absorption Through Skin In Vivo in Rats and Humans
5.3 Measurement of Disposition Half-Life, Clearance, and Residence Times
5.4 Isolated Perfused Porcine Skin Flap
5.5 Porcine Skin Flow-Through Diffusion Cell System
5.6 Toxicant Transport by P-Glycoprotein
5.7 Collection of Bile and Urine Samples for Determining the Urinary and Hepatobiliary Disposition of Xenobiotics in Mice

6 The Glutathione Pathway
6.1 Overview of Glutathione Function and Metabolism
6.2 Measurement of Glutathione and Glutathione Disulfide
6.3 Measurement of Glutathione Transport
6.4 Measurement of Glutathione Transferases
6.5 HPLC-Based Assays for Enzymes of Glutathione Biosynthesis
6.6 g-Glutamyl Transpeptidase Activity Assay
6.7 Oxidant-Induced Regulation of Glutathione Synthesis
6.8 Measurement of Glutathione Conjugates
6.9 Coenzyme A and Coenzyme A-Glutathione Mixed Disulfide Measurements by HPLC

7 Assessment of the Activity of Antioxidant Enzymes
7.1 Analysis of Glutathione-Related Enzymes
7.2 Measurement of Glutathione Reductase Activity
7.3 Analysis of Superoxide Dismutase Activity
7.4 Measurement of Thioredoxin and Thioredoxin Reductase
7.5 Measurement of MnSOD and CuZnSOD Activity in Mammalian Tissue Homogenates
7.6 Measurement of Ascorbic Acid and Dehydroascorbic Acid in Biological Samples

8 Heme Synthesis Pathway
8.1 The Heme Biosynthesis Pathways and Clinical Manifestations of Abnormal Function
8.2 Measurement of ALA Synthase Activity
8.3 Measurement of Heme Concentration
8.4 Measurement of Uroporphyrinogen Decarboxylase Activity
8.5 Measurement of Protoporphyrinogen Oxidase Activity
8.6 Measurement of d-Aminolevulinate Dehydratase Activity
8.7 Measurement of Ferrochelatase Activity
8.8 Measurement of Erythrocyte Protoporphyrin Concentration
8.9 HPLC Methods for Analysis of Porphyrins in Biological Media

9 Heme Degradation Pathway
9.1 Overview of Heme Degradation Pathway
9.2 Detection of Heme Oxygenase Activity by Measurement of CO
9.3 Detection of Heme Oxygenase 1 and 2 Proteins and Bilirubin Formation
9.4 Detection of Biliverdin Reductase Activity
9.5 Histochemical Analysis of Heme Degradation Enzymes
9.6 An HPLC Method to Detect Heme Oxygenase Activity
9.7 Functional Analysis of the Heme Oxygenase-1 Gene Promoter
9.8 Quantitation of Human Heme Oxygenase (HO-1) Copies by Competitive RT-PCR
9.9 Purification and Characterization of Heme Oxygenase

10 The Nitric Oxide/Guanylate Cyclase Pathway
10.1 Overview of the Pathway and Functions of Nitric Oxide
10.2 Assay of Tissue Activity of Nitric Oxide Synthase
10.3 Detection of Nitrosated Proteins
10.4 Fluorometric Techniques for the Detection of Nitric Oxide and Metabolites
10.5 Measurement of cGMP and Soluble Guanylyl Cyclase Activity
10.6 Histochemical Analysis of Nitric Oxide Synthase by NADPH Diaphorase Staining
10.7 Immunocytochemical Analysis of Cyclic Nucleotides
10.8 Methods for Distinguishing Nitrosative and Oxidative Chemistry
10.9 Inducible Nitric Oxide Synthase Expression

11 Neurotoxicology
11.1 Overview of Neurotoxicology
11.2 Neurobehavioral Screening in Rodents
11.3 Assessment of Spatial Memory
11.4 Advanced Behavioral testing in Rodents: Assessment of Cognitive Function in Animals
11.5 Testing for Organophosphate-Induced Delayed Polyneuropathy
11.6 Risk Assessment and Neurotoxicology
11.7 Neurobehavioral Testing in Humans
11.8 Mouse Models of Global Cerebral Ischemia
11.9 Mouse Models of Focal Cerebral Ischemia
11.10 Principles of Electrophysiology: An Overview (New, August 2003)
11.11 Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices (New, August 2003)
11.12 Whole-Cell Patch-Clamp Electrophysiology of Voltage-Sensitive Channels (New, August 2003)
11.13 Detection and Assessment of Xenobiotic-Induced Sensory Neuropathy

12 Biochemical and Molecular Neurotoxicology
12.1 Biochemical Approaches to Studying Neurotoxicity
12.2 Development of an In Vitro Blood-Brain Barrier
12.3 Culturing Rat Hippocampal Neurons
12.4 Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures
12.5 Analytical Cytology: Applications to Neurotoxicology
12.6 Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator
12.7 Isolation of Cerebellar Granule Cells from Neonatal Rats
12.8 Measurement of Glial Fibrillary Acidic Protein
12.9 Aggregating Neural Cell Cultures
12.10 Coculturing Neurons and Glial Cells
12.11 Determining the Ability of Xenobiotic Metals to Bind a Specific Protein Domain by Electrophoresis
12.12 Morphological Measurement of Neurotoxic Injury in the Peripheral Nervous System: Preparation of Material for Light and Transmission Electron Microscopic Evaluation

13 Teratology
13.1 Overview of Teratology
13.2 Rat Embryo Cultures for In Vitro Teratology
13.3 Micromass Cultures in Teratology
13.4 Using Chicken Embryos for Teratology Studies
13.5 In Vivo Assessment of Prenatal Developmental Toxicity in Rodents )
13.6 Organ Culture of Midfacial Tissue and Secondary Palate

14 Hepatotoxicity
14.1 Overview of Hepatotoxicity
14.2 Preparation of Hepatocytes
14.3 Small Animal Models of Hemorrhagic Shock-Induced Liver Dysfunction
14.4 Isolation of Liver Kupffer Cells
14.5 Measurement of Hepatobiliary Transport

15 Computation and Theoretical Methods
15.1 Embryonic Stem (ES) Cell Culture Basics
15.2 Genotyping Embryonic Stem (ES) Cells
15.3 Aggregation Chimeras (ES Cell–Embryo)
15.4 Reporter Genes to Detect Cre Excision in Mice

16 Male Reproductive Toxicology
16.1 In Vivo Models for Male Reproductive Toxicology
16.2 Guidelines for Mating Rodents
16.3 Histopathology of the Male Reproductive System I: Techniques
16.4 Histopathology of the Male Reproductive System II: Interpretation
16.5 Monitoring Endocrine Function in Males
16.6 Epididymal Sperm Count
16.7 Performing a Testicular Spermatid Head Count
16.8 Transgenerational (In Utero/Lactational) Exposure to Investigate the Effects of Endocrine Disrupting Compounds (EDCs) in Rats

17 Oxidative Stress

17.1 Formation and Functions of Protein Sulfenic Acids
17.2 Measurement of Protein Sulfenic Acid Content
17.3 Fluorescence Microplate Reader Measurement of Tissue Susceptibility to Lipid Peroxidation

17.4 In Situ Localization of Nonenzymatic Peroxidase-Like Activity of Tissue-Bound Transition Metals

Chapter 18 Immunotoxicology
Introduction)
18.1 Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment)
18.2 Local Lymph Node Assays
18.3 Murine Asthma Models
18.4 Use of Bronchoalveolar Lavage to Detect Lung Injury
18.5 Measuring Lymphocyte Transcription Factor Activity by ELISA
18.6 Measuring the Activity of Cytolytic Lymphocytes Appendices
A.1 USEFUL INFORMATION
A. Safe Use of Radioisotopes
B. Transgenic and Gene-Targeted Mouse Lines for Toxicology Studies

A.2 LABORATORY STOCK SOLUTIONS AND EQUIPMENT
A. Common Stock Solutions and Buffers
B. Standard Laboratory Equipment

A.3 COMMONLY USED TECHNIQUES
A. Molecular Biology Techniques
B. Techniques for Mammalian Cell Tissue Culture
C. Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
D. Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
E. Northern Blot Analysis of RNA
F. One-Dimensional SDS Gel Electrophoresis of Proteins
G. Spectrophotometric and Colorimetric Determination of Protein Concentration
H. Dialysis and Concentration of Protein Solutions

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