Note: The Comet Assay in Toxicology -- Contents -- Section I: Genesis of Comet Assay -- Chapter 1 The Comet Assay: A Versatile Tool for Assessing DNA Damage -- 1.1 Introduction -- 1.2 Bacteria -- 1.3 Plant Models -- 1.3.1 The Comet Assay in Lower Plants -- 1.3.2 The Comet Assay in Higher Plants -- 1.4 Animal Models -- 1.4.1 Lower Animals -- 1.5 Higher Animals -- 1.5.1 Vertebrates -- 1.6 The Specificity, Sensitivity and Limitations of the Comet Assay -- 1.7 Conclusions -- Acknowledgements -- References -- Section II: Various Procedures for the Comet Assay -- Chapter 2 Detection of Oxidised DNA Using DNA Repair Enzymes -- 2.1 Introduction -- 2.2 Methods for Measuring DNA Oxidation Damage -- 2.3 Enzyme Specificity -- 2.4 Applications -- 2.5 Protocol -- 2.5.1 Equipment -- 2.5.2 Supplies -- 2.5.3 Reagents, Buffers and Enzymes -- 2.5.4 Procedure -- Acknowledgment -- References -- Chapter 3 Microplate-Based Comet Assay -- 3.1 Introduction -- 3.2 Microplate Comet Assay -- 3.3 Drinking-Water Disinfection Byproducts -- 3.4 Chinese Hamster Ovary Cells -- 3.5 CHO Cell Microplate Comet Assay Protocol -- 3.5.1 CHO Cell Treatment -- 3.5.2 Preparation of Comet Microgels -- 3.5.3 Comet Microscopic Examination -- 3.5.4 Normalisation of CHO Cell Comet Data and Statistical Analysis -- 3.6 Utility of the Microplate Comet Assay in Comparing Classes of DBPs -- 3.6.1 Microplate Comet Analysis of the Haloacetonitriles -- 3.6.2 Microplate Comet Analysis of the Haloacetamides -- 3.6.3 Comparison of SCGE Genotoxic Potency Values of the Haloacetonitriles and Haloacetamides -- 3.7 Advantages of the Mammalian CellMicroplate Comet Assay -- Acknowledgements -- References -- Chapter 4 The Use of Higher Plants in the Comet Assay -- 4.1 Introduction -- 4.2 Differences between the Animal and Plant Comet Assay -- 4.3 Cultivation and Treatment of Plants for the Comet Assay. , 4.3.1 Onion (Allium cepa) -- 4.3.2 Tobacco (Nicotiana tabacum) -- 4.3.3 Broad Bean (Vicia faba) -- 4.3.4 Plants used for In-Situ Studies -- 4.4 Isolation of Nuclei from Plant Tissues -- 4.4.1 Isolation of Nuclei via Protoplast Formation -- 4.4.2 Isolation of Nuclei by Mechanical Destruction of the Cell Wall -- 4.5 Preparation of Comet Assay Slides -- 4.6 DNA Unwinding and Electrophoresis -- 4.7 DNA Staining -- 4.8 Reading the Slides, Expressing DNA Damage, Statistics -- 4.9 Comet Assay Procedure -- 4.10 Reagents, Media, Buffers -- 4.11 Equipment and Software -- 4.12 Determination of Toxicity -- 4.13 Correlation between the DNA Damage Evaluated by the Comet Assay and Other Genetic Endpoints in Plants -- 4.14 The Utility of the Comet Assay for Genotoxic Studies in the Laboratory -- 4.15 The Utility of the Comet Assay as an In Situ Marker -- 4.16 Comet Assay with Irradiated Food of Plant Origin -- 4.17 Recommendations for Plant Comet Assay Users -- Abbreviations -- References -- Chapter 5 Methods for Freezing Blood Samples at -80°C for DNA Damage Analysis in Human Leukocytes -- 5.1 Introduction -- 5.2 Materials and Methods -- 5.2.1 Protocol I -- 5.2.2 Protocol II -- 5.2.3 Fresh Blood -- 5.2.4 Fresh Blood Stored on Ice Prior to Freezing -- 5.2.5 Image and Data Analysis -- 5.3 Results and Discussion -- References -- Chapter 6 Development and Applications of the Comet-FISH Assay for the Study of DNA Damage and Repair -- 6.1 Introduction -- 6.2 The Comet-FISH Assay Procedure -- 6.3 Applications of the Comet-FISH Assay -- 6.3.1 Discovery of the Comet-FISH Assay -- 6.3.2 Using Comet-FISH to Measure DNA Damage -- 6.3.3 Using Comet-FISH to Quantify DNA Repair -- 6.3.4 Summary of Studies -- 6.4 Limitations of Comet-FISH Assay -- 6.4.1 Practical Difficulties -- 6.4.2 Imaging Difficulties -- 6.4.3 Interpretation of Results -- 6.5 Conclusion -- References. , Chapter 7 Detection of DNA Damage in Drosophila and Mouse -- 7.1 General Protocol for the Assessment of DNA Damage Using the Alkaline Comet Assay -- 7.1.1 Chemicals and Materials -- 7.1.2 Preparation of Reagents -- 7.1.3 Preparation of Agarose-Coated (Base) Slides for the Comet Assay -- 7.1.4 Preparation of Microgel Slides for the Comet Assay -- 7.1.5 Electrophoresis of Microgel Slides -- 7.1.6 Evaluation of DNA Damage -- 7.2 The Alkaline Comet Assay in Multiple Organs of Mouse -- 7.2.1 Chemicals and Materials -- 7.2.2 Methodology -- 7.3 The Alkaline Comet Assay in Drosophila melanogaster -- 7.3.1 Chemicals and Materials -- 7.3.2 Methodology -- 7.4 Conclusions -- Acknowledgements -- References -- Section III: Applications of Comet Assay -- Chapter 8 Clinical Applications of the Comet Assay -- 8.1 Introduction -- 8.2 The Comet Assay Methodology -- 8.3 Clinical Studies -- 8.4 Discussion and Conclusions -- References -- Chapter 9 Applications of the Comet Assay in Human Biomonitoring -- 9.1 Biomonitoring and Biomarkers - An Introduction -- 9.2 The (Modified) Comet Assay -- 9.3 Guidelines for Biomonitoring Studies -- 9.4 Biomonitoring with the Comet Assay: Special Considerations -- 9.4.1 Surrogate and Target Cells -- The Use of White Blood Cells -- 9.4.2 Sampling Time and Transport -- 9.4.3 Reference Standards -- 9.4.4 What Affects the Background Level of DNA Damage? -- 9.5 DNA Damage as a Marker of Environmental Exposure and Risk -- 9.6 DNA Repair as a Biomarker of Individual Susceptibility -- 9.7 Protocols -- 9.7.1 Protocol for Blood Sample Collection and Long-Term Storage of Lymphocytes for the Measurement of DNA Damage and Repair -- 9.7.2 Comet Assay - Determination of DNA Damage (Strand Breaks and Oxidised Bases) -- 9.7.3 In Vitro Assays for DNA Repair -- 9.8 Solutions, etc. -- 9.8.1. Lysis Solution. , 9.8.2. Buffer F (Enzyme Reaction Buffer for FPG, Endonuclease III, and In Vitro BER Assay) -- 9.8.3 Buffer F+Mg (Used for In Vitro NER Assay) -- 9.8.4 Buffer A (Used in In Vitro Repair Assays) -- 9.8.5 Triton Solution -- 9.8.6 Ro 19-8022 (Photosensitiser) -- 9.8.7 Electrophoresis Solution -- 9.8.8 Neutralising Buffer -- 9.8.9 Agarose -- 9.8.10 Enzymes -- 9.9 Analysis and Interpretation of Results -- 9.9.1 Quantitation -- 9.9.2 Calculation of Net Enzyme-Sensitive Sites -- 9.9.3 Calibration -- 9.9.4 How to Deal with Comet Assay Data Statistically -- 9.10 Conclusions -- Acknowledgements -- References -- Chapter 10 The Comet Assay in Human Biomonitoring -- 10.1 Introduction -- 10.2 Human Monitoring -- 10.3 Environmental Exposure -- 10.4 Lifestyle Exposure -- 10.5 Occupational Exposure -- 10.6 Reviews -- 10.7 Usefulness of the Comet Assay in Human Monitoring -- 10.8 Conclusions -- References -- Chapter 11 Comet Assays in Dietary Intervention Trials -- 11.1 Introduction -- 11.2 Experimental Design of Human Studies -- 11.3 Indicator Cells and Media -- 11.4 Conventional SCGE Trials with Complex Foods and Individual Components - The Current State of Knowledge -- 11.5 Use of SCGE Trials to Detect Protection Against DNA-Reactive Carcinogens -- 11.6 Use of SCGE Experiments to Monitor Alterations of the DNA-Repair Capacity -- 11.7 What Have We Learned from Intervention Studies so Far? -- 11.8 Future Perspectives -- References -- Chapter 12 The Comet Assay for the Evaluation of Genotoxic Exposure in Aquatic Species -- 12.1 Introduction -- 12.2 Protocols, Cell Types and Target Organs -- 12.3 Application of the Comet Assay to Invertebrate Species -- 12.3.1 Freshwater Invertebrates -- 12.3.2 Marine Invertebrates -- 12.4 Application of the Comet Assay to Vertebrate Species -- 12.4.1 Freshwater Vertebrates -- 12.4.2 Marine Vertebrates -- 12.5 Conclusions -- References. , Chapter 13 The Alkaline Comet Assay in Prognostic Tests for Male Infertility and Assisted Reproductive Technology Outcomes -- 13.1 Introduction -- 13.2 Sites of DNA Damage in Sperm -- 13.2.1 Oxidative Stress, a Major Cause of DNA Damage -- 13.2.2 Oxidative Stress, Antioxidant Therapies -- 13.2.3 Sperm DNA Damage Tests -- 13.2.4 Modifications to the Alkaline Comet Assay for Use with Sperm -- 13.2.5 Sperm DNA Adducts and their Relationship with DNA Fragmentation -- 13.3 Can Sperm DNA Integrity Predict Success? Relationships with Assisted Conception Outcomes -- 13.4 Clinically Induced DNA Damage -- 13.4.1 Cryopreservation -- 13.4.2 Vasectomy -- 13.5 A Major Barrier to Progress -- 13.6 Opportunities and Challenges - The Establishment of Clinical Thresholds and the Integration of DNA Testing into Clinical Practice -- Acknowledgements -- References -- Chapter 14 The Comet Assay in Sperm - Assessing Genotoxins in Male Germ Cells -- 14.1 Introduction -- 14.2 Single-Cell Gel Electrophoresis -- 14.3 The Use of Sperm with the Comet Assay -- 14.3.1 Human Sperm -- 14.3.2 Modifying Existing Comet Protocols for the Use of Sperm -- 14.3.3 Sperm DNA and the Comet Assay -- 14.3.4 The Sperm Comet Assay and the Use of Repair Enzymes -- 14.3.5 Assessing the Sperm Comet -- 14.3.6 Comet-FISH on Sperm -- 14.3.7 Cryopreserved versus Fresh Sperm -- 14.3.8 Viability Considerations -- 14.3.9 Statistical Analysis -- 14.4 Utilising Male Germ Cells with the Comet Assay -- 14.4.1 In Vivo Comet Assay -- 14.4.2 In Vitro Comet Assay -- 14.5 The Sperm Comet Assay versus Other Assays Used in Reproductive Toxicology -- 14.6 Conclusions -- Acknowledgements -- References -- Section IV: Regulatory, Imaging and Statistical Considerations -- Chapter 15 Comet Assay - Protocols and Testing Strategies -- 15.1 Introduction -- 15.2 Applications of the In Vivo Comet Assay for Regulatory Purposes. , 15.3 Recommendations for Test Performance.

Note: Cover -- Preface -- Contents -- Section I: Genesis of Comet Assay -- Chapter 1 The Comet Assay: A Versatile Tool for Assessing DNA Damage -- 1.1 Introduction -- 1.1.1 Bacteria -- 1.2 Plant Models -- 1.2.1 The Comet Assay in Lower Plants and Fungi -- 1.2.2 The Comet Assay in Higher Plants -- 1.3 Animal Models -- 1.3.1 Lower Animals -- 1.4 Higher Animals -- 1.4.1 Vertebrates -- 1.5 The Specificity, Sensitivity and Limitations of the Comet Assay -- 1.6 Conclusions -- References -- Section II: Various Procedures for the Comet Assay -- Chapter 2 High-throughput Measurement of DNA Breaks and Oxidised Bases with the Comet Assay -- 2.1 Introduction -- 2.2 Methods for Measuring DNA Oxidation Damage -- 2.3 Enzyme Specificity -- 2.4 Applications -- 2.5 Protocol -- 2.5.1 Equipment -- 2.5.2 Supplies -- 2.5.3 Reagents, Buffers and Enzymes -- 2.5.4 Procedure -- Acknowledgments -- References -- Chapter 3 Microplate-based Comet Assay -- 3.1 Introduction -- 3.2 Microplate Comet Assay -- 3.3 Drinking-water Disinfection Byproducts -- 3.4 Chinese Hamster Ovary Cells -- 3.5 CHO Cell Microplate Comet Assay Protocol -- 3.5.1 CHO Cell Treatment -- 3.5.2 Preparation of Comet Microgels -- 3.5.3 Comet Microscopic Examination -- 3.5.4 Normalisation of CHO Cell Comet Data and Statistical Analysis -- 3.6 Utility of the Microplate Comet Assay in Comparing Classes of DBPs -- 3.6.1 Microplate Comet Analysis of the Haloacetonitriles -- 3.6.2 Microplate Comet Analysis of the Haloacetamides -- 3.6.3 Comparison of SCGE Genotoxic Potency Values of the Haloacetonitriles and Haloacetamides -- 3.7 Advantages of the Mammalian Cell Microplate Comet Assay -- Acknowledgments -- References -- Chapter 4 The Use of Higher Plants in the Comet Assay -- 4.1 Introduction -- 4.2 Differences between the Animal and Plant Comet Assay -- 4.3 Cultivation and Treatment of Plants for the Comet Assay. , 4.3.1 Onion (Allium cepa) -- 4.3.2 Tobacco (Nicotiana tabacum) -- 4.3.3 Broad Bean (Vicia faba) -- 4.3.4 Plants Used for In situ Studies -- 4.4 Isolation of Nuclei from Plant Tissues -- 4.4.1 Isolation of Nuclei via Protoplast Formation -- 4.4.2 Isolation of Nuclei by Mechanical Destruction of the Cell Wall -- 4.5 Preparation of Comet Assay Slides -- 4.6 DNA Unwinding and Electrophoresis -- 4.7 DNA Staining -- 4.8 Reading the Slides, Expressing DNA Damage, Statistics -- 4.9 Comet Assay Procedure -- 4.10 Reagents, Media, Buffers -- 4.11 Equipment and Software -- 4.12 Determination of Toxicity -- 4.13 Correlation between the DNA Damage Evaluated by the Comet Assay and Other Genetic Endpoints in Plants -- 4.14 The Utility of the Comet Assay for Genotoxic Studies in the Laboratory -- 4.15 The Utility of the Comet Assay as an In situ Marker -- 4.16 Comet Assay with Irradiated Food of Plant Origin -- 4.17 Recommendations for Plant Comet Assay Users -- Abbreviations -- References -- Chapter 5 Methods for Freezing Blood Samples at -80°C for DNA Damage Analysis in Human Leukocytes -- 5.1 Introduction -- 5.2 Materials and Methods -- 5.2.1 Protocol I -- 5.2.2 Protocol II -- 5.2.3 Fresh Blood -- 5.2.4 Fresh Blood Stored on Ice Prior to Freezing -- 5.2.5 Image and Data Analysis -- 5.3 Results and Discussion -- References -- Chapter 6 Development and Applications of the Comet-FISH Assay for the Study of DNA Damage and Repair -- 6.1 Introduction -- 6.2 The Comet-FISH Assay Procedure -- 6.3 Applications of the Comet-FISH Assay -- 6.3.1 Discovery of the Comet-FISH Assay -- 6.3.2 Using Comet-FISH to Measure DNA Damage -- 6.3.3 Using Comet-FISH to Quantify DNA Repair -- 6.3.4 Summary of Studies -- 6.4 Limitations of Comet-FISH Assay -- 6.4.1 Practical Difficulties -- 6.4.2 Imaging Difficulties -- 6.4.3 Interpretation of Results -- 6.5 Conclusion -- References. , Chapter 7 Detection of DNA Damage in Different Organs of the Mouse -- 7.1 Introduction -- 7.2 The Alkaline Comet Assay in Multiple Organs of Mice -- 7.2.1 Chemicals and Materials -- 7.2.2 Methodology -- 7.3 Conclusions -- Acknowledgments -- References -- Chapter 8 Detection of DNA Damage in Drosophila -- 8.1 Introduction -- 8.2 General Protocol for the Assessment of DNA Damage Using the Alkaline Comet Assay -- 8.2.1 Chemicals and Materials -- 8.2.2 Preparation of Reagents -- 8.2.3 Preparation of Agarose Coated (Base) Slides for the Comet Assay -- 8.2.4 Preparation of Microgel Slides for the Comet Assay -- 8.2.5 Electrophoresis of Microgel Slides -- 8.2.6 Evaluation of DNA Damage -- 8.3 The Alkaline Comet Assay in Drosophila melanogaster -- 8.3.1 Chemicals and Materials -- 8.3.2 Methodology -- 8.4 Conclusion -- Acknowledgments -- References -- Section III: Applications of Comet Assay -- Chapter 9 The Comet Assay: Clinical Applications -- 9.1 Introduction -- 9.2 The Comet Assay Methodology -- 9.3 Clinical Studies -- 9.4 Discussion and Conclusions -- References -- Chapter 10 Applications of the Comet Assay in Human Biomonitoring -- 10.1 Biomonitoring and Biomarkers - An Introduction -- 10.2 The (Modified) Comet Assay -- 10.3 Guidelines for Biomonitoring Studies -- 10.4 Biomonitoring with the Comet Assay: Special Considerations -- 10.4.1 Surrogate and Target Cells -- The Use of White Blood Cells -- 10.4.2 Sampling Time and Transport -- 10.4.3 Reference Standards -- 10.4.4 What Affects the Background Level of DNA Damage? -- 10.5 DNA Damage as a Marker of Environmental Exposure and Risk -- 10.6 DNA Repair as a Biomarker of Individual Susceptibility -- 10.7 Protocols -- 10.7.1 Protocol for Blood Sample Collection and Long-term Storage of Lymphocytes for the Measurement of DNA Damage and Repair. , 10.7.2 Comet Assay - Determination of DNA Damage (Strand Breaks and Oxidised Bases) -- 10.7.3 In vitro Assays for DNA Repair -- 10.8 Solutions, etc. -- 10.8.1 Lysis Solution -- 10.8.2 Buffer F (Enzyme Reaction Buffer for FPG, End on uclease III, and In vitro BER Assay) -- 10.8.3 Buffer F+Mg (Used for In vitro NER Assay) -- 10.8.4 Buffer A (Used in In vitro Repair Assays) -- 10.8.5 Triton Solution -- 10.8.6 Ro 19-8022 (Photosensitiser) -- 10.8.7 Electrophoresis Solution -- 10.8.8 Neutralising Buffer -- 10.8.9 Agarose -- 10.8.10 Enzymes -- 10.9 Analysis and Interpretation of Results -- 10.9.1 Quantitation -- 10.9.2 Calculation of Net Enzyme-sensitive Sites -- 10.9.3 Calibration -- 10.9.4 How to Deal with Comet Assay Data Statistically -- 10.10 Conclusions -- Acknowledgments -- References -- Chapter 11 Comet Assay in Human Biomonitoring -- 11.1 Introduction -- 11.2 Human Monitoring -- 11.3 Environmental Exposure -- 11.4 Lifestyle Exposure -- 11.5 Occupational Exposure -- 11.6 Reviews -- 11.7 Usefulness of the Comet Assay in Human Monitoring -- 11.8 Conclusions -- References -- Chapter 12 Use of Single-cell Gel Electrophoresis Assays in Dietary Intervention Trials -- 12.1 Introduction -- 12.2 Different Endpoints -- 12.3 Experimental Design of Human Studies -- 12.4 Indicator Cells and Media -- 12.5 Conventional SCGE Trials With Complex Foods and Individual Components-The Current State of Knowledge -- 12.5.1 Definition of the Quality Score (QS) -- 12.6 Use of SCGE Trials to Detect Protection Against DNA-reactive Carcinogens -- 12.7 Use of SCGE-experiments to Monitor Alterations of the DNA-repair Capacity (Base- and Nucleotide-excision Repair) -- 12.8 What Have We Learned From Intervention Studies So Far? -- 12.9 Future Perspectives -- 12.9.1 Hot Topics -- 12.9.2 Detection of Antioxidants -- 12.9.3 Standardization. , 12.9.4 Search for Mechanistic Explanations -- 12.9.5 Interpretation Problems -- References -- Chapter 13 The Application of the Comet Assay in Aquatic Environments -- 13.1 Introduction -- 13.2 Protocols, Cell Types and Target Organs -- 13.3 Application of the Comet Assay to Invertebrate Species -- 13.3.1 Freshwater Invertebrates -- 13.3.2 Marine Invertebrates -- 13.4 Application of the Comet Assay to Vertebrate Species -- 13.4.1 Freshwater Vertebrates -- 13.4.2 Marine Vertebrates -- 13.5 Conclusions -- References -- Chapter 14 The Alkaline Comet Assay in Prognostic Tests for Male Infertility and Assisted Reproductive Technology Outcomes -- 14.1 Introduction -- 14.2 Sites of DNA Damage in Sperm -- 14.2.1 Oxidative Stress, a Major Cause of DNA Damage -- 14.2.2 Oxidative Stress, Antioxidant Therapies -- 14.2.3 Sperm DNA Damage Tests -- 14.2.4 Modifications to the Alkaline Comet Assay for Use with Sperm -- 14.2.5 Sperm DNA Adducts and their Relationship with DNA Fragmentation -- 14.3 Can Sperm DNA Integrity Predict Success? Relationships with Assisted Conception Outcomes -- 14.4 Clinically Induced DNA Damage -- 14.4.1 Cryopreservation -- 14.4.2 Vasectomy -- 14.5 A Major Barrier to Progress -- 14.6 Opportunities and Challenges - The Establishment of Clinical Thresholds and the Integration of DNA Testing into Clinical Practice -- Acknowledgments -- References -- Chapter 15 The Comet Assay in Sperm-Assessing Genotoxins in Male Germ Cells -- 15.1 Introduction -- 15.2 The Comet Assay (Single-cell Gel Electrophoresis) -- 15.3 The Use of Sperm with the Comet Assay -- 15.3.1 Human Sperm -- 15.3.2 Sperm DNA and the Comet Assay -- 15.3.3 Modifying Existing Comet Protocols for Somatic Cells for Use with Sperm -- 15.3.4 The Two-tailed Sperm Comet Assay -- 15.3.5 The Sperm Comet Assay and the Use of Repair Enzymes -- 15.3.6 Assessing the Sperm Comet. , 15.3.7 Comet-FISH on Sperm.