Current Protocols in Toxicology

Cover Page......Page 2
Edition Info......Page 3
Editors......Page 4
Contents......Page 5
Foreword......Page 9
Preface......Page 10
Protocols......Page 11
Reader Response......Page 12
ACKNOWLEDGMENTS......Page 13
INTRODUCTION......Page 14
1.1 Nonhuman Primates as Animal Models for Toxicology Research......Page 16
1.2 Statistical Approaches to the Design of Toxicology Studies......Page 25
1.3 Transgenic Animals in Toxicology......Page 43
1.4 DNA Microarrays: An Overview of Technologies and Applications to Toxicology......Page 69
1.5 The Use of Fish-Derived Cell Lines for Investigation of Environmental Contaminants......Page 85
1.6 Sea Urchin Embryos and Larvae as Biosensors for Neurotoxicants......Page 102
1.7 Zebrafish: An Animal Model for Toxicological Studies......Page 126
1.8 Preclinical Models of Parkinson’s Disease......Page 144
INTRODUCTION......Page 175
2.1 Current Concepts in Cell Toxicity......Page 177
2.2 Determination of Apoptosis and Necrosis......Page 193
2.3 Detection of Covalent Binding......Page 227
2.4 Measurement of Lipid Peroxidation......Page 262
2.5 Measurements of Intracellular Free Calcium Concentration in Biological Systems......Page 275
2.6 In Vitro Methods for Detecting Cytotoxicity......Page 327
2.7 In Situ Hybridization Histochemistry......Page 354
2.8 Confocal Microscopy......Page 367
2.9 Measurement of Expression of the HSP70 Protein Family......Page 388
2.10 Analysis of Mitochondrial Dysfunction During Cell Death......Page 428
INTRODUCTION......Page 455
3.1 The Salmonella (Ames) Test for Mutagenicity......Page 459
3.2 Measurement of a Malondialdehyde-DNA Adduct......Page 488
3.3 Mutagenesis Assays in Mammalian Cells......Page 504
3.4 Cell Transformation Assays......Page 511
3.5 Assays for DNA Damage......Page 520
3.6 Detecting Epigenetic Changes: DNA Methylation......Page 556
3.7 Assays for Detecting Chromosomal Aberrations......Page 564
3.8 Methods for Measuring DNA Adducts and Abasic Sites I: Isolation, Purification, and Analysis of DNA Adducts in Intact DNA......Page 580
3.9 Methods for Measuring DNA Adducts and Abasic Sites II: Methods for Measurement of DNA Adducts......Page 598
INTRODUCTION......Page 633
4.1 Measurement of Cytochrome P-450......Page 637
4.2 Purification of Cytochrome P-450 Enzymes......Page 651
4.3 Measurements of UDP-Glucuronosyltransferase Glucuronosyltransferase......Page 663
4.4 Detection of Metabolites Using High-Performance Liquid Chromatography and Mass Spectrometry......Page 678
4.5 Measurement of Aryl and Alcohol Sulfotransferase Activity......Page 696
4.6 Measuring the Activity of Arylamine N-Acetyltransferase (NAT)......Page 705
4.7 Measurement of Carboxylesterase (CES) Activities......Page 718
4.8 Analysis of the Aryl Hydrocarbon Receptor (AhR) Signal Transduction Pathway......Page 732
4.9 Measurements of Flavin-Containing Monooxygenase (FMO) Activities......Page 777
4.10 Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolases and Phosphoric Triester Hydrolases......Page 788
4.11 Techniques for Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl-CoA:Amino Acid N-Acyltransferase: The Amino Acid Conjugation Pathway......Page 802
4.12 Determination of Paraoxonase 1 Status and Genotypes at Specific Polymorphic Sites......Page 818
4.13 Human Cytochrome P450: Metabolism of Testosterone by CYP3A4 and Inhibition by Ketoconazole......Page 837
4.14 Biotransformation Studies Using Rat Proximal Tubule Cells......Page 846
4.15 TaqMan Real Time–Polymerase Chain Reaction Methods for Determination of Nucleotide Polymorphisms in Human N-Acetyltransferase-1 (NAT1) and -2 (NAT2)......Page 862
4.16 Evaluation of the Cytochrome b5/ Cytochrome b5 Reductase Pathway......Page 873
4.17 Measurement of Xenobiotic Carbonyl Reduction in Human Liver Fractions......Page 890
INTRODUCTION......Page 915
5.1 Measurement of Bioavailability: Measurement of Absorption Through Skin In Vitro......Page 917
5.2 Measurement of Bioavailability: Measuring Absorption Through Skin In Vivo in Rats and Humans......Page 931
5.3 Measurement of Disposition Half-Life, Clearance, and Residence Times......Page 941
5.4 Isolated Perfused Porcine Skin Flap......Page 970
5.5 Porcine Skin Flow-Through Diffusion Cell System......Page 979
5.6 Toxicant Transport by P-Glycoprotein......Page 987
5.7 Collection of Bile and Urine Samples for Determining the Urinary and Hepatobiliary Disposition of Xenobiotics in Mice......Page 1000
INTRODUCTION......Page 1007
6.1 Overview of Glutathione Function and Metabolism......Page 1010
6.2 Measurement of Glutathione and Glutathione Disulfide......Page 1028
6.3 Measurement of Glutathione Transport......Page 1042
6.4 Measurement of Glutathione Transferases......Page 1056
6.5 HPLC-Based Assays for Enzymes of Glutathione Biosynthesis......Page 1066
6.6 γ-Glutamyl Transpeptidase Activity Assay......Page 1080
6.7 Oxidant-Induced Regulation of Glutathione Synthesis......Page 1088
6.8 Measurement of Glutathione Conjugates......Page 1109
6.9 Coenzyme A and Coenzyme A-Glutathione Mixed Disulfide Measurements by HPLC......Page 1133
INTRODUCTION......Page 1141
7.1 Analysis of Glutathione-Related Enzymes......Page 1143
7.2 Measurement of Glutathione Reductase Activity......Page 1175
7.3 Analysis of Superoxide Dismutase Activity......Page 1179
7.4 Measurement of Thioredoxin and Thioredoxin Reductase......Page 1188
7.5 Measurement of MnSOD and CuZnSOD Activity in Mammalian Tissue Homogenates......Page 1202
7.6 Measurement of Ascorbic Acid and Dehydroascorbic Acid in Biological Samples......Page 1213
INTRODUCTION......Page 1228
8.1 The Heme Biosynthesis Pathway and Clinical Manifestations of Abnormal Function......Page 1231
8.2 Measurement of ALA Synthase Activity......Page 1241
8.3 Measurement of Heme Concentration......Page 1252
8.4 Measurement of Uroporphyrinogen Decarboxylase Activity......Page 1259
8.5 Measurement of Protoporphyrinogen Oxidase Activity......Page 1272
8.6 Measurement of δ-Aminolevulinate Dehydratase Activity......Page 1285
8.7 Measurement of Ferrochelatase Activity......Page 1296
8.8 Measurement of Erythrocyte Protoporphyrin Dehydratase Activity Spectrofluorometry......Page 1304
8.9 HPLC Methods for Analysis of Porphyrins in Biological Media......Page 1316
INTRODUCTION......Page 1334
9.1 Overview of Heme Degradation Pathway......Page 1336
9.2 Detection of Heme Oxygenase Activity by Measurement of CO......Page 1341
9.3 Detection of Heme Oxygenase 1 and 2 Proteins and Bilirubin Formation......Page 1351
9.4 Detection of Biliverdin Reductase Activity......Page 1360
9.5 Histochemical Analysis of Heme Degradation Enzymes......Page 1370
9.6 An HPLC Method to Detect Heme Oxygenase Activity......Page 1392
9.7 Functional Analysis of the Heme Oxygenase-1 Gene Promoter......Page 1406
9.8 Quantitation of Human Heme Oxygenase (HO-1) Copies by Competitive RT-PCR......Page 1427
9.9 Purification and Characterization of Heme Oxygenase......Page 1437
INTRODUCTION......Page 1454
10.1 Overview of the Pathway and Functions of Nitric Oxide......Page 1456
10.2 Assay of Tissue Activity of Nitric Oxide Synthase......Page 1462
10.3 Detection of Nitrosated Proteins......Page 1475
10.4 Fluorometric Techniques for the Detection of Nitric Oxide and Metabolites......Page 1483
10.5 Measurement of cGMP and Soluble Guanylyl Cyclase Activity......Page 1495
10.6 Histochemical Analysis of Nitric Oxide Synthase by NADPH Diaphorase Staining......Page 1503
10.7 Immunocytochemical Analysis of Cyclic Nucleotides......Page 1507
10.8 Methods for Distinguishing Nitrosative and Oxidative Chemistry of Reactive Nitrogen Oxide Species Derived from Nitric Oxide......Page 1524
10.9 Inducible Nitric Oxide Synthase Expression......Page 1536
INTRODUCTION......Page 1552
11.1 Overview of Neurotoxicology......Page 1554
11.2 Neurobehavioral Screening in Rodents......Page 1562
11.3 Assessment of Spatial Memory......Page 1578
11.4 Advanced Behavioral Testing in Rodents: Assessment of Cognitive Function in Animals......Page 1596
11.5 Testing for Organophosphate-Induced Delayed Polyneuropathy......Page 1630
11.6 Risk Assessment and Neurotoxicology......Page 1644
11.7 Neurobehavioral Testing in Humans......Page 1651
11.8 Mouse Models of Global Cerebral Ischemia......Page 1668
11.9 Mouse Models of Focal Cerebral Ischemia......Page 1692
11.10 Principles of Electrophysiology: An Overview......Page 1718
11.11 Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices......Page 1729
11.12 Whole-Cell Patch-Clamp Electrophysiology of Voltage-Sensitive Channels......Page 1767
11.13 Detection and Assessment of Xenobiotic-Induced Sensory Neuropathy......Page 1781
11.14 Methods to Produce Brain Hyperthermia......Page 1789
INTRODUCTION......Page 1815
12.1 Biochemical Approaches to Studying Neurotoxicity......Page 1817
12.2 Development of an In Vitro Blood-Brain Barrier......Page 1824
12.3 Culturing Rat Hippocampal Neurons......Page 1835
12.4 Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures......Page 1852
12.5 Analytical Cytology: Applications to Neurotoxicology......Page 1867
12.6 Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator......Page 1883
12.7 Isolation of Cerebellar Granule Cells from Neonatal Rats......Page 1902
12.8 Measurement of Glial Fibrillary Acidic Protein......Page 1912
12.9 Aggregating Neural Cell Cultures......Page 1924
12.10 Coculturing Neurons and Glial Cells......Page 1941
12.11 Determining the Ability of Xenobiotic Metals to Bind a Specific Protein Domain by Electrophoresis......Page 1958
12.12 Morphological Measurement of Neurotoxic Injury in the Peripheral Nervous System: Preparation of Material for Light and Transmission Electron Microscopic Evaluation......Page 1966
INTRODUCTION......Page 1985
13.1 Overview of Teratology......Page 1986
13.2 Rat Embryo Cultures for In Vitro Teratology......Page 1994
13.3 Micromass Cultures in Teratology......Page 2006
13.4 Using Chicken Embryos for Teratology Studies......Page 2020
13.5 In Vivo Assessment of Prenatal Developmental Toxicity in Rodents......Page 2039
13.6 Organ Culture of Midfacial Tissue and Secondary Palate......Page 2064
13.7 Overview of Behavioral Teratology......Page 2075
13.8 Statistical Analysis of Behavioral Data......Page 2081
INTRODUCTION......Page 2110
14.1 Overview of Hepatotoxicity......Page 2111
14.2 Preparation of Hepatocytes......Page 2117
14.3 Small Animal Models of Hemorrhagic Shock-Induced Liver Dysfunction......Page 2130
14.4 Isolation of Liver Kupffer Cells......Page 2138
14.5 Measurement of Hepatobiliary Transport......Page 2150
INTRODUCTION......Page 2167
15.1 Embryonic Stem (ES) Cell Culture Basics......Page 2169
15.2 Genotyping Embryonic Stem (ES) Cells......Page 2184
15.3 Aggregation Chimeras (ES Cell–Embryo)......Page 2194
15.4 Reporter Genes to Detect Cre Excision in Mice......Page 2206
INTRODUCTION......Page 2217
16.1 In Vivo Models for Male Reproductive Toxicology......Page 2219
16.2 Guidelines for Mating Rodents......Page 2234
16.3 Histopathology of the Male Reproductive System I: Techniques......Page 2245
16.4 Histopathology of the Male Reproductive System II: Interpretation......Page 2263
16.5 Monitoring Endocrine Function in Males: Using Intra-Atrial Cannulas to Monitor Plasma Hormonal Dynamics in Toxicology Experiments......Page 2277
16.6 Epididymal Sperm Count......Page 2294
16.7 Performing a Testicular Spermatid Head Count......Page 2299
16.8 Transgenerational (In Utero/Lactational) Exposure to Investigate the Effects of Endocrine Disrupting Compounds (EDCs) in Rats......Page 2305
INTRODUCTION......Page 2321
17.1 Formation and Functions of Protein Sulfenic Acids......Page 2323
17.2 Measurement of Protein Sulfenic Acid Content......Page 2338
17.3 Fluorescence Microplate Reader Measurement of Tissue Susceptibility to Lipid Peroxidation......Page 2358
17.4 In Situ Localization of Nonenzymatic Peroxidase-Like Activity of Tissue-Bound Transition Metals......Page 2368
INTRODUCTION......Page 2413
18.1 Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment......Page 2416
18.2 Local Lymph Node Assays......Page 2436
18.3 Murine Asthma Models......Page 2448
18.4 Use of Bronchoalveolar Lavage to Detect Lung Injury......Page 2469
18.5 Measuring Lymphocyte Transcription Factor Activity by ELISA......Page 2479
18.6 Measuring the Activity of Cytolytic Lymphocytes......Page 2486
18.7 Solid-Phase Immunoassays......Page 2513
18.8 Immune Cell Phenotyping Using Flow Cytometry......Page 2532
1A Safe Use of Radioisotopes......Page 2566
1B Transgenic and Gene-Targeted Mouse Lines for Toxicology Studies......Page 2579
2A Common Stock Solutions and Buffers......Page 2590
2B Standard Laboratory Equipment......Page 2596
3A Molecular Biology Techniques......Page 2598
3B Techniques for Mammalian Cell Tissue Culture......Page 2601
3C Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization......Page 2616
3D Detection and Quantitation of Radiolabeled Proteins in Gels and Blots......Page 2630
3E Northern Blot Analysis of RNA......Page 2640
3F One-Dimensional SDS Gel Electrophoresis of Proteins......Page 2653
3G Spectrophotometric Determination of Protein Concentration......Page 2687
3H Dialysis and Concentration of Protein Solutions......Page 2694
3I The Colorimetric Detection and Quantitation of Total Protein......Page 2699
SELECTED SUPPLIERS OF REAGENTS AND EQUIPMENT......Page 2727