Current Protocols in Toxicology

Current Protocols in Toxicology

Online ISBN: 9780471140856

DOI: 10.1002/0471140856

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  1. Preface
  2. Foreword
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Toxicological Models
    1. Introduction
    2. UNIT 1.1 Nonhuman Primates as Animal Models for Toxicology Research
    3. UNIT 1.2 Statistical Approaches to the Design of Toxicology Studies
    4. UNIT 1.3 Transgenic Animals in Toxicology
    5. UNIT 1.4 DNA Microarrays: An Overview of Technologies and Applications to Toxicology
    6. UNIT 1.5 The Use of Fish-Derived Cell Lines for Investigation of Environmental Contaminants: An Update Following OECD's Fish Toxicity Testing Framework No. 171
    7. UNIT 1.6 Sea Urchin Embryos and Larvae as Biosensors for Neurotoxicants
    8. UNIT 1.7 Zebrafish: An Animal Model for Toxicological Studies
    9. UNIT 1.8 Preclinical Models of Parkinson's Disease
    10. UNIT 1.9 The Nematode C. elegans as an Animal Model to Explore Toxicology In Vivo: Solid and Axenic Growth Culture Conditions and Compound Exposure Parameters
    11. UNIT 1.10 Use of Medaka in Toxicity Testing
    12. UNIT 1.11 Minipigs: Applications in Toxicology
    13. UNIT 1.12 Developmental Toxicity Assays Using the Drosophila Model
    14. UNIT 1.13 Using the Cytosensor Microphysiometer to Assess Ocular Toxicity
  5. Chapter 2 Assessment of Cell Toxicity
    1. Introduction
    2. UNIT 2.1 Current Concepts in Cell Toxicity
    3. UNIT 2.2 Determination of Apoptosis and Necrosis
    4. UNIT 2.3 Detection of Covalent Binding
    5. UNIT 2.4 Measurement of Lipid Peroxidation
    6. UNIT 2.5 Measurements of Intracellular Free Calcium Concentration in Biological Systems
    7. UNIT 2.6 In Vitro Methods for Detecting Cytotoxicity
    8. UNIT 2.7 In Situ Hybridization Histochemistry
    9. UNIT 2.8 Confocal Microscopy
    10. UNIT 2.9 Measurement of Expression of the HSP70 Protein Family
    11. UNIT 2.10 Analysis of Mitochondrial Dysfunction During Cell Death
    12. UNIT 2.11 Single-Cell Analysis of Lipid Rafts in Lymphocytes and in T Cell–Containing Immunoconjugates
    13. UNIT 2.12 Detection of the Mitochondrial Apoptosis-Induced Channel (MAC) and Its Regulation by Bcl-2 Family Proteins
    14. UNIT 2.13 Mouse Models for Human Hemato-Lymphopoiesis
    15. UNIT 2.14 Cross-Species Genetic Toxicity Assessment Accomplished by Flow Cytometric Analysis of Blood
    16. UNIT 2.15 Current Concepts in Drug-Induced Mitochondrial Toxicity
    17. UNIT 2.16 Fluorescent pH and Oxygen Probes of the Assessment of Mitochondrial Toxicity in Isolated Mitochondria and Whole Cells
    18. UNIT 2.17 Assessment of Gap Junctional Intercellular Communication
    19. UNIT 2.18 Role of Integrative Signaling Through Gap Junctions in Toxicology
    20. UNIT 2.19 Fluorescence Correlation Spectroscopy: Detecting and Interpreting the Mobility of Transmembrane Proteins In Vivo
    21. UNIT 2.20 Assessment of Mitochondrial Toxicity in HepG2 Cells Cultured in High-Glucose- or Galactose-Containing Media
    22. UNIT 2.21 Measuring Intracellular hsp70 in Leukocytes by Flow Cytometry
    23. UNIT 2.22 Murine Embryonic Stem Cell Derivation, In Vitro Pluripotency Characterization, and In Vivo Teratoma Formation
    24. UNIT 2.23 High-Throughput, Cell-Based Screens to Identify Small-Molecule Inhibitors of Ricin Toxin and Related Category B Ribosome Inactivating Proteins (RIPs)
    25. UNIT 2.24 Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay
  6. Chapter 3 Genetic Toxicology: Mutagenesis and Adduct Formation
    1. Introduction
    2. UNIT 3.1 The Salmonella (Ames) Test for Mutagenicity
    3. UNIT 3.2 Measurement of a Malondialdehyde-DNA Adduct
    4. UNIT 3.3 Mutagenesis Assays in Mammalian Cells
    5. UNIT 3.4 Cell Transformation Assays
    6. UNIT 3.5 Assays for DNA Damage
    7. UNIT 3.6 Detecting Epigenetic Changes: DNA Methylation
    8. UNIT 3.7 Assays for Detecting Chromosomal Aberrations
    9. UNIT 3.8 Methods for Measuring DNA Adducts and Abasic Sites I: Isolation, Purification, and Analysis of DNA Adducts in Intact DNA
    10. UNIT 3.9 Methods for Measuring DNA Adducts and Abasic Sites II: Methods for Measurement of DNA Adducts
    11. UNIT 3.10 Analysis of Mismatch Repair in Human Nuclear Extracts
    12. UNIT 3.11 High-Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA-Encoded Protein Levels in Eukaryotic Cells
    13. UNIT 3.12 Alkaline Comet Assay for Assessing DNA Damage in Individual Cells
    14. UNIT 3.13 Use of Ciliogenesis to Detect Aneugens: The Role of Primary Cilia
  7. Chapter 4 Techniques for Analysis of Chemical Biotransformation
    1. Introduction
    2. UNIT 4.1 Measurement of Cytochrome P-450
    3. UNIT 4.2 Purification of Cytochrome P-450 Enzymes
    4. UNIT 4.3 Measurements of UDP- Glucuronosyltransferase (UGT) Activities
    5. UNIT 4.4 Detection of Metabolites Using High-Performance Liquid Chromatography and Mass Spectrometry
    6. UNIT 4.5 Measurement of Aryl and Alcohol Sulfotransferase Activity
    7. UNIT 4.6 Measuring the Activity of Arylamine N-Acetyltransferase (NAT)
    8. UNIT 4.7 Measurement of Carboxylesterase (CES) Activities
    9. UNIT 4.8 Analysis of the Aryl Hydrocarbon Receptor (AhR) Signal Transduction Pathway
    10. UNIT 4.9 Measurements of Flavin-Containing Monooxygenase (FMO) Activities
    11. UNIT 4.10 Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolases and Phosphoric Triester Hydrolases
    12. 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
    13. UNIT 4.12 Determination of Paraoxonase 1 Status and Genotypes at Specific Polymorphic Sites
    14. UNIT 4.13 Human Cytochrome P450: Metabolism of Testosterone by CYP3A4 and Inhibition by Ketoconazole
    15. UNIT 4.14 Biotransformation Studies Using Rat Proximal Tubule Cells
    16. UNIT 4.15 TaqMan Real Time–Polymerase Chain Reaction Methods for Determination of Nucleotide Polymorphisms in Human N-Acetyltransferase-1 (NAT1) and -2 (NAT2)
    17. UNIT 4.16 Evaluation of the Cytochrome b5/Cytochrome b5 Reductase Pathway
    18. UNIT 4.17 Measurement of Xenobiotic Carbonyl Reduction in Human Liver Fractions
    19. UNIT 4.18 Aldehyde Dehydrogenases: Measurement of Activities and Protein Levels
    20. UNIT 4.19 Measurement of Human Cytochrome P4501A2 (CYP1A2) Activity In Vitro
    21. UNIT 4.20 NMR Analysis of Thiodiglycol Oxidation by Mammalian Alcohol Dehydrogenases
    22. UNIT 4.21 Monoamine Oxidase Assays
    23. UNIT 4.22 Biochemical and Genetic Analysis of NAD(P)H:Quinone Oxidoreductase 1 (NQO1)
    24. UNIT 4.23 Measurement of Soluble Epoxide Hydrolase (sEH) Activity
    25. UNIT 4.24 Enzymatic Activity of Human Carboxylesterases
    26. UNIT 4.25 In Vitro Evaluation of Metabolic Drug-Drug Interactions: A Descriptive and Critical Commentary
    27. UNIT 4.26 Assays for S-Adenosylmethionine (AdoMet/SAM)-Dependent Methyltransferases
    28. UNIT 4.27 Measurement of In Vitro Cytochrome P450 2B6 Activity
    29. UNIT 4.28 Azoreductase in Staphylococcus aureus
    30. UNIT 4.29 Measurement of Phenylalanine Monooxygenase (PAH) Activities
    31. UNIT 4.30 Co-Oxidation by Cyclooxygenases
    32. UNIT 4.31 An Overview of Arsenic Metabolism and Toxicity
    33. UNIT 4.32 In Vitro Assays of Inorganic Arsenic Methylation
    34. UNIT 4.33 Analysis of Arsenical Metabolites in Biological Samples
    35. UNIT 4.34 Purification of Arsenic (+3 Oxidation State) Methyltransferase from Rat Liver Cytosol
    36. UNIT 4.35 Manipulation of Expression of Arsenic (+3 Oxidation State) Methyltransferase in Cultured Cells
    37. UNIT 4.36 Measurement of Cysteine S-Conjugate β-Lyase Activity
    38. UNIT 4.37 Aldehyde Reduction by Cytochrome P450
    39. UNIT 4.38 CYP1B1 Detection
    40. UNIT 4.39 Simultaneous, Untargeted Metabolic Profiling of Polar and Nonpolar Metabolites by LC-Q-TOF Mass Spectrometry
    41. UNIT 4.40 Multi-Parametric Analysis of Cell Death Pathways Using Live-Cell Microscopy
    42. UNIT 4.41 Metabolism of Xenobiotics by Aldehyde Oxidase
    43. UNIT 4.42 Determination of Metabolic Stability Using Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss)
  8. Chapter 5 Toxicokinetics
    1. Introduction
    2. UNIT 5.1 Measurement of Bioavailability: Measurement of Absorption Through Skin In Vitro
    3. UNIT 5.2 Measurement of Bioavailability: Measuring Absorption Through Skin In Vivo in Rats and Humans
    4. UNIT 5.3 Measurement of Disposition Half-Life, Clearance, and Residence Times
    5. UNIT 5.4 Isolated Perfused Porcine Skin Flap
    6. UNIT 5.5 Porcine Skin Flow-Through Diffusion Cell System
    7. UNIT 5.6 Toxicant Transport by P-Glycoprotein
    8. UNIT 5.7 Collection of Bile and Urine Samples for Determining the Urinary and Hepatobiliary Disposition of Xenobiotics in Mice
    9. UNIT 5.8 Determination of ADME and Bioavailability Following Intravenous, Oral, and Dermal Routes of Exposure
  9. Chapter 6 The Glutathione Pathway
    1. Introduction
    2. UNIT 6.1 Overview of Glutathione Function and Metabolism
    3. UNIT 6.2 Measurement of Glutathione and Glutathione Disulfide
    4. UNIT 6.3 Measurement of Glutathione Transport
    5. UNIT 6.4 Measurement of Glutathione Transferases
    6. UNIT 6.5 HPLC-Based Assays for Enzymes of Glutathione Biosynthesis
    7. UNIT 6.6 γ-Glutamyl Transpeptidase Activity Assay
    8. UNIT 6.7 Oxidant-Induced Regulation of Glutathione Synthesis
    9. UNIT 6.8 Measurement of Glutathione Conjugates
    10. UNIT 6.9 Coenzyme A and Coenzyme A-Glutathione Mixed Disulfide Measurements by HPLC
    11. UNIT 6.10 Overview of Protein Glutathionylation
    12. UNIT 6.11 Measurement of Protein Glutathionylation
    13. UNIT 6.12 Glutathione-Dependent Bioactivation
    14. UNIT 6.13 Methods for Measuring Cysteine S-Conjugate β-Lyase Activity
    15. UNIT 6.14 Measurement of Aminoacylases
    16. UNIT 6.15 Measurement of Cysteine Dioxygenase Activity and Protein Abundance
    17. UNIT 6.16 Glutamate Cysteine Ligase (GCL) Transgenic and Gene-Targeted Mice for Controlling Glutathione Synthesis
    18. UNIT 6.17 Methods to Detect Protein Glutathionylation
    19. UNIT 6.18 Quantification of Glutathione in Caenorhabditis elegans
  10. Chapter 7 Assessment of the Activity of Antioxidant Enzymes
    1. Introduction
    2. UNIT 7.1 Analysis of Glutathione-Related Enzymes
    3. UNIT 7.2 Measurement of Glutathione Reductase Activity
    4. UNIT 7.3 Analysis of Superoxide Dismutase Activity
    5. UNIT 7.4 Measurement of Thioredoxin and Thioredoxin Reductase
    6. UNIT 7.5 Measurement of MnSOD and CuZnSOD Activity in Mammalian Tissue Homogenates
    7. UNIT 7.6 Measurement of Ascorbic Acid and Dehydroascorbic Acid in Biological Samples
    8. UNIT 7.7 Catalase Activity Assays
    9. UNIT 7.8 Measurement of the Vitamin E Metabolites, Carboxyethyl Hydroxychromans (CEHCs), in Biological Samples
    10. UNIT 7.9 Overview of Peroxiredoxins in Oxidant Defense and Redox Regulation
    11. UNIT 7.10 Measurement of Peroxiredoxin Activity
  11. Chapter 8 Heme Synthesis Pathway
    1. Introduction
    2. UNIT 8.1 The Heme Biosynthesis Pathway and Clinical Manifestations of Abnormal Function
    3. UNIT 8.2 Measurement of ALA Synthase Activity
    4. UNIT 8.3 Measurement of Heme Concentration
    5. UNIT 8.4 Measurement of Uroporphyrinogen Decarboxylase Activity
    6. UNIT 8.5 Measurement of Protoporphyrinogen Oxidase Activity
    7. UNIT 8.6 Measurement of δ-Aminolevulinate Dehydratase Activity
    8. UNIT 8.7 Measurement of Ferrochelatase Activity
    9. UNIT 8.8 Measurement of Erythrocyte Protoporphyrin Concentration by Double Extraction and Spectrofluorometry
    10. UNIT 8.9 HPLC Methods for Analysis of Porphyrins in Biological Media
  12. Chapter 9 Heme Degradation Pathway
    1. Introduction
    2. UNIT 9.1 Overview of Heme Degradation Pathway
    3. UNIT 9.2 Detection of Heme Oxygenase Activity by Measurement of CO
    4. UNIT 9.3 Detection of Heme Oxygenase 1 and 2 Proteins and Bilirubin Formation
    5. UNIT 9.4 Detection of Biliverdin Reductase Activity
    6. UNIT 9.5 Histochemical Analysis of Heme Degradation Enzymes
    7. UNIT 9.6 An HPLC Method to Detect Heme Oxygenase Activity
    8. UNIT 9.7 Functional Analysis of the Heme Oxygenase-1 Gene Promoter
    9. UNIT 9.8 Quantitation of Human Heme Oxygenase (HO-1) Copies by Competitive RT-PCR
    10. UNIT 9.9 Purification and Characterization of Heme Oxygenase
  13. Chapter 10 The Nitric Oxide/Guanylate Cyclase Pathway
    1. Introduction
    2. UNIT 10.1 Overview of the Pathway and Functions of Nitric Oxide
    3. UNIT 10.2 Assay of Tissue Activity of Nitric Oxide Synthase
    4. UNIT 10.3 Detection of Nitrosated Proteins
    5. UNIT 10.4 Fluorometric Techniques for the Detection of Nitric Oxide and Metabolites
    6. UNIT 10.5 Measurement of cGMP and Soluble Guanylyl Cyclase Activity
    7. UNIT 10.6 Histochemical Analysis of Nitric Oxide Synthase by NADPH Diaphorase Staining
    8. UNIT 10.7 Immunocytochemical Analysis of Cyclic Nucleotides
    9. UNIT 10.8 Methods for Distinguishing Nitrosative and Oxidative Chemistry of Reactive Nitrogen Oxide Species Derived from Nitric Oxide
    10. UNIT 10.9 Inducible Nitric Oxide Synthase Expression
  14. Chapter 11 Neurotoxicology
    1. Introduction
    2. UNIT 11.1 Overview of Neurotoxicology
    3. UNIT 11.2 Neurobehavioral Screening in Rodents
    4. UNIT 11.3 Assessment of Spatial Memory
    5. UNIT 11.4 Advanced Behavioral Testing in Rodents: Assessment of Cognitive Function in Animals
    6. UNIT 11.5 Testing for Organophosphate-Induced Delayed Polyneuropathy
    7. UNIT 11.6 Risk Assessment and Neurotoxicology
    8. UNIT 11.7 Neurobehavioral Testing in Humans
    9. UNIT 11.8 Mouse Models of Global Cerebral Ischemia
    10. UNIT 11.9 Mouse Models of Focal Cerebral Ischemia
    11. UNIT 11.10 Principles of Electrophysiology: An Overview
    12. UNIT 11.11 Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices
    13. UNIT 11.12 Whole-Cell Patch-Clamp Electrophysiology of Voltage-Sensitive Channels
    14. UNIT 11.13 Detection and Assessment of Xenobiotic-Induced Sensory Neuropathy
    15. UNIT 11.14 Methods to Produce Brain Hyperthermia
    16. UNIT 11.15 Experimental Models of Hypoxic-Ischemic Encephalopathy: Hypoxia-Ischemia in the Immature Rat
    17. UNIT 11.16 Morphological Assessment of Neurite Outgrowth in Hippocampal Neuron-Astrocyte Co-Cultures
    18. UNIT 11.17 F3-Isoprostanes as a Measure of in vivo Oxidative Damage in Caenorhabditis elegans
    19. UNIT 11.18 Methods to Measure Olfactory Behavior in Mice
    20. UNIT 11.19 High-Resolution Multi-Photon Imaging of Morphological Structures of Caenorhabditis elegans
    21. UNIT 11.20 RNASeq in C. elegans Following Manganese Exposure
    22. UNIT 11.21 A Simple Light Stimulation of Caenorhabditis elegans
  15. Chapter 12 Biochemical and Molecular Neurotoxicology
    1. Introduction
    2. UNIT 12.1 Biochemical Approaches to Studying Neurotoxicity
    3. UNIT 12.2 Development of an In Vitro Blood-Brain Barrier
    4. UNIT 12.3 Culturing Rat Hippocampal Neurons
    5. UNIT 12.4 Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures
    6. UNIT 12.5 Analytical Cytology: Applications to Neurotoxicology
    7. UNIT 12.6 Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator
    8. UNIT 12.7 Isolation of Cerebellar Granule Cells from Neonatal Rats
    9. UNIT 12.8 Measurement of Glial Fibrillary Acidic Protein
    10. UNIT 12.9 Aggregating Neural Cell Cultures
    11. UNIT 12.10 Coculturing Neurons and Glial Cells
    12. UNIT 12.11 Determining the Ability of Xenobiotic Metals to Bind a Specific Protein Domain by Electrophoresis
    13. UNIT 12.12 Morphological Measurement of Neurotoxic Injury in the Peripheral Nervous System: Preparation of Material for Light and Transmission Electron Microscopic Evaluation
    14. UNIT 12.13 Determination of Cholinesterase in Blood and Tissue
    15. UNIT 12.14 Measurement of Isoprostanes as Markers of Oxidative Stress in Neuronal Tissue
    16. UNIT 12.15 Application of Single-Cell Microfluorimetry to Neurotoxicology Assays
    17. UNIT 12.16 Morphometric Analysis in Neurodegenerative Disorders
    18. UNIT 12.17 Neonatal Rat Primary Microglia: Isolation, Culturing, and Selected Applications
    19. UNIT 12.18 Cellular fura-2 Manganese Extraction Assay (CFMEA)
    20. UNIT 12.19 Methods to Assess Neuroinflammation
    21. UNIT 12.20 Assessment of Adult Neurogenesis in Mice
    22. UNIT 12.21 Comparative Human and Rat “Neurosphere Assay” for Developmental Neurotoxicity Testing
    23. UNIT 12.22 Synaptic Structure Quantification in Cultured Neurons
  16. Chapter 13 Teratology
    1. Introduction
    2. UNIT 13.1 Overview of Teratology
    3. UNIT 13.2 Rat Embryo Cultures for In Vitro Teratology
    4. UNIT 13.3 Micromass Cultures in Teratology
    5. UNIT 13.4 Using Chicken Embryos for Teratology Studies
    6. UNIT 13.5 In Vivo Assessment of Prenatal Developmental Toxicity in Rodents
    7. UNIT 13.6 Organ Culture of Midfacial Tissue and Secondary Palate
    8. UNIT 13.7 Overview of Behavioral Teratology
    9. UNIT 13.8 Statistical Analysis of Behavioral Data
    10. UNIT 13.9 Methods in the Analysis of Maternal Behavior in the Rodent
    11. UNIT 13.10 Scoring of Social Interactions and Play in Mice During Adolescence
    12. UNIT 13.11 Scoring Learning and Memory in Developing Rodents
    13. UNIT 13.12 Analysis of Ultrasonic Vocalizations Emitted by Infant Rodents
  17. Chapter 14 Hepatotoxicology
    1. Introduction
    2. UNIT 14.1 Overview of Hepatotoxicity
    3. UNIT 14.2 Preparation of Hepatocytes
    4. UNIT 14.3 Small Animal Models of Hemorrhagic Shock–Induced Liver Dysfunction
    5. UNIT 14.4 Isolation of Liver Kupffer Cells
    6. UNIT 14.5 Measurement of Hepatobiliary Transport
    7. UNIT 14.6 Measuring Covalent Binding in Hepatotoxicity
    8. UNIT 14.7 A Rat Primary Hepatocyte Culture Model for Aging Studies
    9. UNIT 14.8 Assessment of Mitochondrial Dysfunction Arising from Treatment with Hepatotoxicants
    10. UNIT 14.9 Assessing Cell Fusion and Cytokinesis Failure as Mechanisms of Clone 9 Hepatocyte Multinucleation In Vitro
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      UNIT 14.10 Assessment of Metabolic Stability Using the Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction
    12. UNIT 14.11 High-Throughput Gene Silencing and mRNA Expression Analysis in Hepatocyte Sandwich Cultures
    13. UNIT 14.12 The History and Use of Human Hepatocytes for the Treatment of Liver Diseases: The First 100 Patients
    14. UNIT 14.13 The Use of Induced Pluripotent Stem Cells for the Study and Treatment of Liver Diseases
  18. Chapter 15 Gene Targeting
    1. Introduction
    2. UNIT 15.1 Embryonic Stem (ES) Cell Culture Basics
    3. UNIT 15.2 Genotyping Embryonic Stem (ES) Cells
    4. UNIT 15.3 Aggregation Chimeras (ES Cell–Embryo)
    5. UNIT 15.4 Reporter Genes to Detect Cre Excision in Mice
  19. Chapter 16 Male Reproductive Toxicology
    1. Introduction
    2. UNIT 16.1 In Vivo Models for Male Reproductive Toxicology
    3. UNIT 16.2 Guidelines for Mating Rodents
    4. UNIT 16.3 Histopathology of the Male Reproductive System I: Techniques
    5. UNIT 16.4 Histopathology of the Male Reproductive System II: Interpretation
    6. UNIT 16.5 Monitoring Endocrine Function in Males: Using Intra-Atrial Cannulas to Monitor Plasma Hormonal Dynamics in Toxicology Experiments
    7. UNIT 16.6 Epididymal Sperm Count
    8. UNIT 16.7 Performing a Testicular Spermatid Head Count
    9. UNIT 16.8 Transgenerational (In Utero/Lactational) Exposure to Investigate the Effects of Endocrine Disrupting Compounds (EDCs) in Rats
    10. UNIT 16.9 Hershberger Assay to Investigate the Effects of Endocrine-Disrupting Compounds with Androgenic or Antiandrogenic Activity in Castrate-Immature Male Rats
    11. UNIT 16.10 Preparation of Rodent Testis Co-Cultures
  20. Chapter 17 Oxidative Stress
    1. Introduction
    2. UNIT 17.1 Formation and Functions of Protein Sulfenic Acids
    3. UNIT 17.2 Measurement of Protein Sulfenic Acid Content
    4. UNIT 17.3 Fluorescence Microplate Reader Measurement of Tissue Susceptibility to Lipid Peroxidation
    5. UNIT 17.4 In Situ Localization of Nonenzymatic Peroxidase-Like Activity of Tissue-Bound Transition Metals
    6. UNIT 17.5 F2-Isoprostanes as Markers of Oxidant Stress: An Overview
    7. UNIT 17.6 Quantification of F2-Isoprostanes by Gas Chromatography/Mass Spectrometry as a Measure of Oxidant Stress
    8. UNIT 17.7 Immuno-Spin Trapping: Detection of Protein-Centered Radicals
    9. UNIT 17.8 Detection of Stress Proteins as Biomarkers of Oxidative Stress
    10. UNIT 17.9 Identification and Characterization of Oxylipid-Protein and Peptide Conjugates by Mass Spectrometry
    11. UNIT 17.10 Complex I Assay in Mitochondrial Preparations from CNS
    12. UNIT 17.11 Measuring Electrophile Stress
    13. UNIT 17.12 Thioredoxin Redox Western Analysis
    14. UNIT 17.13 Transcription Factor Nrf2: Examination of Nuclear Protein Levels by Immunoblotting and Promoter Response Element Binding by Chromatin Immunoprecipitation (ChIP)
    15. UNIT 17.14 LC-MS/MS Quantitation of Mercapturic Acid Conjugates of Lipid Peroxidation Products as Markers of Oxidative Stress
    16. UNIT 17.15 High-Throughput, Multiplexed Analysis of 3-Nitrotyrosine in Individual Proteins
    17. UNIT 17.16 Measurement of Arginine Metabolites: Regulators of Nitric Oxide Metabolism
    18. UNIT 17.17 Detection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5′-Cyclo-2′-deoxyadenosine by 32P-Postlabeling Assay
  21. Chapter 18 Immunotoxicology
    1. Introduction
    2. UNIT 18.1 Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment
    3. UNIT 18.2 Local Lymph Node Assays
    4. UNIT 18.3 Murine Asthma Models
    5. UNIT 18.4 Use of Bronchoalveolar Lavage to Detect Lung Injury
    6. UNIT 18.5 Measuring Lymphocyte Transcription Factor Activity by ELISA
    7. UNIT 18.6 Measuring the Activity of Cytolytic Lymphocytes
    8. UNIT 18.7 Solid-Phase Immunoassays
    9. UNIT 18.8 Immune Cell Phenotyping Using Flow Cytometry
    10. UNIT 18.9 Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells
    11. UNIT 18.10 Host Resistance Model to an Intracellular Pathogen
    12. UNIT 18.11 Analysis of Immunotoxicity by Enumeration of Antibody-Producing B Cells
    13. UNIT 18.12 The Reporter Antigen Popliteal Lymph Node Assay
    14. UNIT 18.13 Fluorescence Microscopy and Flow Cytometric Analysis of Peyer's Patches and Intestinal Immune Cells
    15. UNIT 18.14 Analysis of Modulation of Immunoglobulin Gene Expression
    16. UNIT 18.15 Developmental Immunotoxicity (DIT): Assays for Evaluating Effects of Exogenous Agents on Development of the Immune System
    17. UNIT 18.16 Mining the Salivary Proteome with Grating-Coupled Surface Plasmon Resonance Imaging and Surface Plasmon Coupled Emission Microarrays
    18. UNIT 18.17 In Vitro Assays of Chemotaxis as a Window into Mechanisms of Toxicant-Induced Immunomodulation
    19. UNIT 18.18 Investigating the Effects of Particulate Matter on House Dust Mite and Ovalbumin Allergic Airway Inflammation in Mice
  22. Chapter 19 Regulatory Testing
    1. Introduction
    2. UNIT 19.1 Toxicology Testing in Drug Discovery and Development
    3. UNIT 19.2 Toxicity Testing in Chemical Safety Evaluation
  23. Chapter 20 Alternative Methodologies in Toxicology
    1. Introduction
    2. UNIT 20.1 Overview of Alternative Methodologies in Toxicology
    3. UNIT 20.2 CFU-GM Assay for Evaluation of Drug Myelotoxic Activity
    4. UNIT 20.3 The Syrian Hamster Embryo (SHE) Low pH Cell Transformation Assay
    5. UNIT 20.4 Neutral Red Uptake Cytotoxicity Tests for Estimating Starting Doses for Acute Oral Toxicity Tests
    6. UNIT 20.5 CFU-MK Assay for Acute Thrombocytopenia
    7. UNIT 20.6 Caco-2 Cells as a Model for Intestinal Absorption
    8. UNIT 20.7 The Local Lymph Node Assay (LLNA)
    9. UNIT 20.8 The NCTC 2544 IL-18 Assay for the In Vitro Identification of Contact Allergens
    10. UNIT 20.9 microRNA Profiling as Tool for Developmental Neurotoxicity Testing (DNT)
    11. UNIT 20.10 Caenorhabditis elegans as a Model for Toxic Effects of Nanoparticles: Lethality, Growth, and Reproduction
    12. UNIT 20.11 PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage
  24. Chapter 21 Gastrointestinal Toxicology
    1. Introduction
    2. UNIT 21.1 Overview of Gastrointestinal Toxicology
    3. UNIT 21.2 Methods to Measure Gastric Mucosal Lesions in the Rat
    4. UNIT 21.3 Assessment of Enteral Bacteria
    5. UNIT 21.4 Assessment of Ischemic Vascular Damage
    6. UNIT 21.5 Measurement of Gastric Acid Secretion in the Anaesthetized Rat
    7. UNIT 21.6 Endogenous Gastric Mediators: Patho-Physiological Role and Measurements
    8. UNIT 21.7 Measurement of Small Intestinal Damage
    9. UNIT 21.8 Assessment of Gastrointestinal Motility Using Three Different Assays In Vitro
    10. UNIT 21.9 Assessment of Gastrointestinal Propulsive Activity Using Three Different Models of Peristalsis In Vivo in the Mouse
    11. UNIT 21.10 Evaluation of Gastric Ulcerogenic and Healing Impairment Effects of Bisphosphonates: Adverse Gastric Reactions of Bisphosphonate
    12. UNIT 21.11 Assessment of Intestinal Peristalsis in Vitro
    13. UNIT 21.12 In Vivo Measurement of Intragastric Pressure with a Rubber Balloon in the Anesthetized Rat
  25. Chapter 22 Stem Cells in Toxicology
    1. Introduction
    2. UNIT 22.1 Using Chemotherapeutic Drugs to Trigger Hematopoietic Stem Cell Activation and Determine Hematological Toxicity
    3. UNIT 22.2 Isolation and Applications of Prostate Side Population Cells Based on Dye Cycle Violet Efflux
    4. UNIT 22.3 Preparation of Epithelial and Mesenchymal Stem Cells from Murine Mammary Gland
    5. UNIT 22.4 Multielectrode Array (MEA) Assay for Profiling Electrophysiological Drug Effects in Human Stem Cell-Derived Cardiomyocytes
  26. Chapter 23 Drug Transporters
    1. Introduction
    2. UNIT 23.1 Measuring Altered Disposition of Xenobiotics in Experimental Models of Liver Disease
    3. UNIT 23.2 Use of Zinc Finger Nuclease Technology to Knock Out Efflux Transporters in C2BBe1 Cells
    4. UNIT 23.3 Renal Transporters in Drug Disposition, Drug-Drug Interactions, and Nephrotoxicity
    5. UNIT 23.4 The Vesicular Transport Assay: Validated In Vitro Methods to Study Drug-Mediated Inhibition of Canalicular Efflux Transporters ABCB11/BSEP and ABCC2/MRP2
    6. UNIT 23.5 Membrane Vesicle ABC Transporter Assays for Drug Safety Assessment
    7. UNIT 23.6 Assessment of Drug Transporter Function Using Fluorescent Cell Imaging
    8. UNIT 23.7 Current Concepts in Drug-Induced Bile Salt Export Pump (BSEP) Interference
    9. UNIT 23.8 Pre-Plated Cell Lines for ADMETox Applications in the Pharmaceutical Industry
  27. Chapter 24 Respiratory Toxicology
    1. UNIT 24.1 In Situ Measurement of Vapor Uptake in the Rodent Upper Respiratory Tract
    2. UNIT 24.2 Isolation of Endothelial Cells from Mouse Lung
    3. UNIT 24.3 How to Measure Alterations in Alveolar Barrier Function as a Marker of Lung Injury
    4. UNIT 24.4 Inhalation Toxicology Methods: The Generation and Characterization of Exposure Atmospheres and Inhalational Exposures
  28. Chapter 25 Mitochondrial Toxicity
    1. UNIT 25.1 High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells
    2. UNIT 25.2 Measuring Mitochondrial Function in Permeabilized Cells Using the Seahorse XF Analyzer or a Clark-Type Oxygen Electrode
    3. UNIT 25.3 Assessment of Fatty Acid Beta Oxidation in Cells and Isolated Mitochondria
    4. UNIT 25.4 Identifying Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore in Isolated Rat Liver Mitochondria
    5. UNIT 25.5 Measuring Mitochondrial Membrane Potential with a Tetraphenylphosphonium-Selective Electrode
    6. UNIT 25.6 Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells
    7. UNIT 25.7 Seahorse Xfe24 Extracellular Flux Analyzer-Based Analysis of Cellular Respiration in Caenorhabditis elegans
  29. Appendix 1 Useful Information
    1. APPENDIX 1A Safe Use of Radioisotopes
    2. APPENDIX 1B Transgenic and Gene-Targeted Mouse Lines for Toxicology Studies
    3. APPENDIX 1C Radioactivity
  30. Appendix 2 Laboratory Stock Solutions and Equipment
    1. APPENDIX 2A Common Stock Solutions and Buffers
    2. APPENDIX 2B Standard Laboratory Equipment
  31. Appendix 3 Commonly Used Techniques
    1. APPENDIX 3A Molecular Biology Techniques
    2. APPENDIX 3B Techniques for Mammalian Cell Tissue Culture
    3. APPENDIX 3C Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
    4. APPENDIX 3D Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
    5. APPENDIX 3E Northern Blot Analysis of RNA
    6. APPENDIX 3F One-Dimensional SDS Gel Electrophoresis of Proteins
    7. APPENDIX 3G Spectrophotometric Determination of Protein Concentration
    8. APPENDIX 3H Dialysis and Concentration of Protein Solutions
    9. APPENDIX 3I The Colorimetric Detection and Quantitation of Total Protein