BOOK
The Comet Assay in Toxicology
(2016)
Additional Information
Book Details
Table of Contents
| Section Title | Page | Action | Price |
|---|---|---|---|
| Cover | Cover | ||
| Preface | vii | ||
| Contents | ix | ||
| Section I: Genesis of Comet Assay | 1 | ||
| Chapter 1 The Comet Assay: A Versatile Tool for Assessing DNA Damage | 3 | ||
| 1.1 Introduction | 3 | ||
| 1.1.1 Bacteria | 5 | ||
| 1.2 Plant Models | 5 | ||
| 1.2.1 The Comet Assay in Lower Plants and Fungi | 5 | ||
| 1.2.2 The Comet Assay in Higher Plants | 8 | ||
| 1.3 Animal Models | 10 | ||
| 1.3.1 Lower Animals | 10 | ||
| 1.4 Higher Animals | 18 | ||
| 1.4.1 Vertebrates | 18 | ||
| 1.5 The Specificity, Sensitivity and Limitations of the Comet Assay | 32 | ||
| 1.6 Conclusions | 34 | ||
| References | 35 | ||
| Section II: Various Procedures for the Comet Assay | 65 | ||
| Chapter 2 High-throughput Measurement of DNA Breaks and Oxidised Bases with the Comet Assay | 67 | ||
| 2.1 Introduction | 67 | ||
| 2.2 Methods for Measuring DNA Oxidation Damage | 69 | ||
| 2.3 Enzyme Specificity | 71 | ||
| 2.4 Applications | 73 | ||
| 2.5 Protocol | 76 | ||
| 2.5.1 Equipment | 76 | ||
| 2.5.2 Supplies | 76 | ||
| 2.5.3 Reagents, Buffers and Enzymes | 77 | ||
| 2.5.4 Procedure | 78 | ||
| Acknowledgments | 87 | ||
| References | 87 | ||
| Chapter 3 Microplate-based Comet Assay | 93 | ||
| 3.1 Introduction | 93 | ||
| 3.2 Microplate Comet Assay | 94 | ||
| 3.3 Drinking-water Disinfection Byproducts | 94 | ||
| 3.4 Chinese Hamster Ovary Cells | 96 | ||
| 3.5 CHO Cell Microplate Comet Assay Protocol | 96 | ||
| 3.5.1 CHO Cell Treatment | 96 | ||
| 3.5.2 Preparation of Comet Microgels | 97 | ||
| 3.5.3 Comet Microscopic Examination | 98 | ||
| 3.5.4 Normalisation of CHO Cell Comet Data and Statistical Analysis | 99 | ||
| 3.6 Utility of the Microplate Comet Assay in Comparing Classes of DBPs | 101 | ||
| 3.6.1 Microplate Comet Analysis of the Haloacetonitriles | 102 | ||
| 3.6.2 Microplate Comet Analysis of the Haloacetamides | 103 | ||
| 3.6.3 Comparison of SCGE Genotoxic Potency Values of the Haloacetonitriles and Haloacetamides | 105 | ||
| 3.7 Advantages of the Mammalian Cell Microplate Comet Assay | 105 | ||
| Acknowledgments | 106 | ||
| References | 106 | ||
| Chapter 4 The Use of Higher Plants in the Comet Assay | 112 | ||
| 4.1 Introduction | 112 | ||
| 4.2 Differences between the Animal and Plant Comet Assay | 113 | ||
| 4.3 Cultivation and Treatment of Plants for the Comet Assay | 113 | ||
| 4.3.1 Onion (Allium cepa) | 113 | ||
| 4.3.2 Tobacco (Nicotiana tabacum) | 114 | ||
| 4.3.3 Broad Bean (Vicia faba) | 114 | ||
| 4.3.4 Plants Used for In situ Studies | 114 | ||
| 4.4 Isolation of Nuclei from Plant Tissues | 115 | ||
| 4.4.1 Isolation of Nuclei via Protoplast Formation | 115 | ||
| 4.4.2 Isolation of Nuclei by Mechanical Destruction of the Cell Wall | 115 | ||
| 4.5 Preparation of Comet Assay Slides | 115 | ||
| 4.6 DNA Unwinding and Electrophoresis | 116 | ||
| 4.7 DNA Staining | 117 | ||
| 4.8 Reading the Slides, Expressing DNA Damage, Statistics | 117 | ||
| 4.9 Comet Assay Procedure | 118 | ||
| 4.10 Reagents, Media, Buffers | 119 | ||
| 4.11 Equipment and Software | 121 | ||
| 4.12 Determination of Toxicity | 121 | ||
| 4.13 Correlation between the DNA Damage Evaluated by the Comet Assay and Other Genetic Endpoints in Plants | 122 | ||
| 4.14 The Utility of the Comet Assay for Genotoxic Studies in the Laboratory | 122 | ||
| 4.15 The Utility of the Comet Assay as an In situ Marker | 126 | ||
| 4.16 Comet Assay with Irradiated Food of Plant Origin | 127 | ||
| 4.17 Recommendations for Plant Comet Assay Users | 127 | ||
| Abbreviations | 128 | ||
| References | 128 | ||
| Chapter 5 Methods for Freezing Blood Samples at -80°C for DNA Damage Analysis in Human Leukocytes | 134 | ||
| 5.1 Introduction | 134 | ||
| 5.2 Materials and Methods | 135 | ||
| 5.2.1 Protocol I | 135 | ||
| 5.2.2 Protocol II | 136 | ||
| 5.2.3 Fresh Blood | 136 | ||
| 5.2.4 Fresh Blood Stored on Ice Prior to Freezing | 136 | ||
| 5.2.5 Image and Data Analysis | 137 | ||
| 5.3 Results and Discussion | 137 | ||
| References | 141 | ||
| Chapter 6 Development and Applications of the Comet-FISH Assay for the Study of DNA Damage and Repair | 143 | ||
| 6.1 Introduction | 143 | ||
| 6.2 The Comet-FISH Assay Procedure | 144 | ||
| 6.3 Applications of the Comet-FISH Assay | 149 | ||
| 6.3.1 Discovery of the Comet-FISH Assay | 149 | ||
| 6.3.2 Using Comet-FISH to Measure DNA Damage | 149 | ||
| 6.3.3 Using Comet-FISH to Quantify DNA Repair | 155 | ||
| 6.3.4 Summary of Studies | 156 | ||
| 6.4 Limitations of Comet-FISH Assay | 157 | ||
| 6.4.1 Practical Difficulties | 157 | ||
| 6.4.2 Imaging Difficulties | 157 | ||
| 6.4.3 Interpretation of Results | 158 | ||
| 6.5 Conclusion | 159 | ||
| References | 159 | ||
| Chapter 7 Detection of DNA Damage in Different Organs of the Mouse | 164 | ||
| 7.1 Introduction | 164 | ||
| 7.2 The Alkaline Comet Assay in Multiple Organs of Mice | 165 | ||
| 7.2.1 Chemicals and Materials | 166 | ||
| 7.2.2 Methodology | 169 | ||
| 7.3 Conclusions | 172 | ||
| Acknowledgments | 173 | ||
| References | 173 | ||
| Chapter 8 Detection of DNA Damage in Drosophila | 177 | ||
| 8.1 Introduction | 177 | ||
| 8.2 General Protocol for the Assessment of DNA Damage Using the Alkaline Comet Assay | 178 | ||
| 8.2.1 Chemicals and Materials | 178 | ||
| 8.2.2 Preparation of Reagents | 180 | ||
| 8.2.3 Preparation of Agarose Coated (Base) Slides for the Comet Assay | 181 | ||
| 8.2.4 Preparation of Microgel Slides for the Comet Assay | 181 | ||
| 8.2.5 Electrophoresis of Microgel Slides | 182 | ||
| 8.2.6 Evaluation of DNA Damage | 183 | ||
| 8.3 The Alkaline Comet Assay in Drosophila melanogaster | 183 | ||
| 8.3.1 Chemicals and Materials | 184 | ||
| 8.3.2 Methodology | 184 | ||
| 8.4 Conclusion | 187 | ||
| Acknowledgments | 188 | ||
| References | 188 | ||
| Section III: Applications of Comet Assay | 193 | ||
| Chapter 9 The Comet Assay: Clinical Applications | 195 | ||
| 9.1 Introduction | 195 | ||
| 9.2 The Comet Assay Methodology | 196 | ||
| 9.3 Clinical Studies | 197 | ||
| 9.4 Discussion and Conclusions | 229 | ||
| References | 230 | ||
| Chapter 10 Applications of the Comet Assay in Human Biomonitoring | 238 | ||
| 10.1 Biomonitoring and Biomarkers - An Introduction | 238 | ||
| 10.2 The (Modified) Comet Assay | 239 | ||
| 10.3 Guidelines for Biomonitoring Studies | 240 | ||
| 10.4 Biomonitoring with the Comet Assay: Special Considerations | 241 | ||
| 10.4.1 Surrogate and Target Cells; The Use of White Blood Cells | 242 | ||
| 10.4.2 Sampling Time and Transport | 242 | ||
| 10.4.3 Reference Standards | 243 | ||
| 10.4.4 What Affects the Background Level of DNA Damage? | 244 | ||
| 10.5 DNA Damage as a Marker of Environmental Exposure and Risk | 244 | ||
| 10.6 DNA Repair as a Biomarker of Individual Susceptibility | 245 | ||
| 10.7 Protocols | 246 | ||
| 10.7.1 Protocol for Blood Sample Collection and Long-term Storage of Lymphocytes for the Measurement of DNA Damage and Repair | 246 | ||
| 10.7.2 Comet Assay - Determination of DNA Damage (Strand Breaks and Oxidised Bases) | 249 | ||
| 10.7.3 In vitro Assays for DNA Repair | 252 | ||
| 10.8 Solutions, etc. | 254 | ||
| 10.8.1 Lysis Solution | 254 | ||
| 10.8.2 Buffer F (Enzyme Reaction Buffer for FPG, End on uclease III, and In vitro BER Assay) | 254 | ||
| 10.8.3 Buffer F+Mg (Used for In vitro NER Assay) | 254 | ||
| 10.8.4 Buffer A (Used in In vitro Repair Assays) | 254 | ||
| 10.8.5 Triton Solution | 255 | ||
| 10.8.6 Ro 19-8022 (Photosensitiser) | 255 | ||
| 10.8.7 Electrophoresis Solution | 255 | ||
| 10.8.8 Neutralising Buffer | 255 | ||
| 10.8.9 Agarose | 255 | ||
| 10.8.10 Enzymes | 255 | ||
| 10.9 Analysis and Interpretation of Results | 256 | ||
| 10.9.1 Quantitation | 256 | ||
| 10.9.2 Calculation of Net Enzyme-sensitive Sites | 257 | ||
| 10.9.3 Calibration | 257 | ||
| 10.9.4 How to Deal with Comet Assay Data Statistically | 257 | ||
| 10.10 Conclusions | 258 | ||
| Acknowledgments | 258 | ||
| References | 259 | ||
| Chapter 11 Comet Assay in Human Biomonitoring | 264 | ||
| 11.1 Introduction | 264 | ||
| 11.2 Human Monitoring | 265 | ||
| 11.3 Environmental Exposure | 268 | ||
| 11.4 Lifestyle Exposure | 273 | ||
| 11.5 Occupational Exposure | 276 | ||
| 11.6 Reviews | 288 | ||
| 11.7 Usefulness of the Comet Assay in Human Monitoring | 291 | ||
| 11.8 Conclusions | 292 | ||
| References | 293 | ||
| Chapter 12 Use of Single-cell Gel Electrophoresis Assays in Dietary Intervention Trials | 314 | ||
| 12.1 Introduction | 314 | ||
| 12.2 Different Endpoints | 315 | ||
| 12.3 Experimental Design of Human Studies | 315 | ||
| 12.4 Indicator Cells and Media | 318 | ||
| 12.5 Conventional SCGE Trials With Complex Foods and Individual Components–The Current State of Knowledge | 319 | ||
| 12.5.1 Definition of the Quality Score (QS) | 319 | ||
| 12.6 Use of SCGE Trials to Detect Protection Against DNA-reactive Carcinogens | 319 | ||
| 12.7 Use of SCGE-experiments to Monitor Alterations of the DNA-repair Capacity (Base- and Nucleotide-excision Repair) | 333 | ||
| 12.8 What Have We Learned From Intervention Studies So Far? | 333 | ||
| 12.9 Future Perspectives | 337 | ||
| 12.9.1 Hot Topics | 337 | ||
| 12.9.2 Detection of Antioxidants | 338 | ||
| 12.9.3 Standardization | 338 | ||
| 12.9.4 Search for Mechanistic Explanations | 339 | ||
| 12.9.5 Interpretation Problems | 339 | ||
| References | 341 | ||
| Chapter 13 The Application of the Comet Assay in Aquatic Environments | 354 | ||
| 13.1 Introduction | 354 | ||
| 13.2 Protocols, Cell Types and Target Organs | 356 | ||
| 13.3 Application of the Comet Assay to Invertebrate Species | 357 | ||
| 13.3.1 Freshwater Invertebrates | 357 | ||
| 13.3.2 Marine Invertebrates | 358 | ||
| 13.4 Application of the Comet Assay to Vertebrate Species | 359 | ||
| 13.4.1 Freshwater Vertebrates | 359 | ||
| 13.4.2 Marine Vertebrates | 360 | ||
| 13.5 Conclusions | 361 | ||
| References | 361 | ||
| Chapter 14 The Alkaline Comet Assay in Prognostic Tests for Male Infertility and Assisted Reproductive Technology Outcomes | 369 | ||
| 14.1 Introduction | 369 | ||
| 14.2 Sites of DNA Damage in Sperm | 370 | ||
| 14.2.1 Oxidative Stress, a Major Cause of DNA Damage | 371 | ||
| 14.2.2 Oxidative Stress, Antioxidant Therapies | 372 | ||
| 14.2.3 Sperm DNA Damage Tests | 372 | ||
| 14.2.4 Modifications to the Alkaline Comet Assay for Use with Sperm | 373 | ||
| 14.2.5 Sperm DNA Adducts and their Relationship with DNA Fragmentation | 375 | ||
| 14.3 Can Sperm DNA Integrity Predict Success? Relationships with Assisted Conception Outcomes | 376 | ||
| 14.4 Clinically Induced DNA Damage | 377 | ||
| 14.4.1 Cryopreservation | 378 | ||
| 14.4.2 Vasectomy | 379 | ||
| 14.5 A Major Barrier to Progress | 379 | ||
| 14.6 Opportunities and Challenges – The Establishment of Clinical Thresholds and the Integration of DNA Testing into Clinical Practice | 379 | ||
| Acknowledgments | 380 | ||
| References | 380 | ||
| Chapter 15 The Comet Assay in Sperm–Assessing Genotoxins in Male Germ Cells | 390 | ||
| 15.1 Introduction | 390 | ||
| 15.2 The Comet Assay (Single-cell Gel Electrophoresis) | 391 | ||
| 15.3 The Use of Sperm with the Comet Assay | 392 | ||
| 15.3.1 Human Sperm | 392 | ||
| 15.3.2 Sperm DNA and the Comet Assay | 393 | ||
| 15.3.3 Modifying Existing Comet Protocols for Somatic Cells for Use with Sperm | 394 | ||
| 15.3.4 The Two-tailed Sperm Comet Assay | 396 | ||
| 15.3.5 The Sperm Comet Assay and the Use of Repair Enzymes | 397 | ||
| 15.3.6 Assessing the Sperm Comet | 398 | ||
| 15.3.7 Comet-FISH on Sperm | 398 | ||
| 15.3.8 Cryopreserved Versus Fresh Sperm | 399 | ||
| 15.3.9 Viability Considerations | 400 | ||
| 15.3.10 Statistical Analysis | 400 | ||
| 15.4 Utilizing Male Germ Cells with the Comet Assay | 401 | ||
| 15.4.1 In vivo Comet Assay | 401 | ||
| 15.4.2 In vitro Comet Assay | 417 | ||
| 15.5 The Sperm Comet Assay Versus Other Assays Used in Reproductive Toxicology | 418 | ||
| 15.6 Conclusion | 418 | ||
| References | 420 | ||
| Chapter 16 Genotoxic Effects in Peripheral Blood and Sperm in Humans in Healthy Individuals and Those with Disease States | 457 | ||
| 16.1 Introduction | 457 | ||
| 16.2 Assessing Ecotoxins and Novel Compounds | 460 | ||
| 16.2.1 Assessing Ecotoxins | 460 | ||
| 16.2.2 Novel Compounds | 460 | ||
| 16.3 Basic Research into DNA Damage and Repair | 465 | ||
| 16.4 Human Biomonitoring and Molecular Epidemiology | 466 | ||
| References | 473 | ||
| Section IV: Regulatory, Imaging and Statistical Considerations | 477 | ||
| Chapter 17 Application of the Comet Assay in Nanotoxicology | 479 | ||
| 17.1 Introduction | 479 | ||
| 17.1.1 Nanotechnology and Nanomaterials | 479 | ||
| 17.1.2 Nanomaterials and Genotoxicity | 480 | ||
| 17.1.3 Comet Assay for Genotoxicity Testing of Nanomaterials | 480 | ||
| 17.2 Experimental Design and Methodological Considerations | 482 | ||
| 17.2.1 Physico-chemical Characterization of Nanomaterial Dispersion | 482 | ||
| 17.2.2 In vitro Models for Detecting Genotoxicity of NMs with the Comet Assay | 483 | ||
| 17.2.3 Exposure Time | 483 | ||
| 17.2.4 Selection of Concentration Range | 483 | ||
| 17.2.5 Positive and Negative Controls and Reference Standards | 484 | ||
| 17.2.6 Possible Interference of the Comet Assay with the Nanomaterials | 485 | ||
| 17.2.7 Cytotoxicity Assessment as Part of Genotoxicity Testing | 486 | ||
| 17.3 Comet Assay Protocol | 486 | ||
| 17.3.1 Preparation of Reagents, Positive Controls, and Enzymes | 486 | ||
| 17.3.2 Procedure | 488 | ||
| 17.4 Statistical Analyses of the Data | 493 | ||
| 17.5 Final Remarks | 494 | ||
| Acknowledgments | 494 | ||
| References | 494 | ||
| Chapter 18 Comet Assay – Protocols and Testing Strategies | 498 | ||
| 18.1 Introduction | 498 | ||
| 18.2 Applications of the In vivo Comet Assay for Regulatory Purposes | 499 | ||
| 18.3 Recommendations for Test Performance | 500 | ||
| 18.3.1 Genetic Endpoint of the Comet Assay | 500 | ||
| 18.3.2 Basic Considerations for Test Protocol | 501 | ||
| 18.3.3 Selection of Tissues and Cell Preparation | 502 | ||
| 18.3.4 Image Analysis | 503 | ||
| 18.3.5 Assessment of Cytotoxicity - A Potential Confounding Factor | 504 | ||
| 18.3.6 Ongoing Validation Exercises | 504 | ||
| 18.4 Applications of the In vivo Comet Assay for Regulatory Purposes | 505 | ||
| 18.4.1 Follow-up Testing of Positive In vitro Cytogenetics Assays | 505 | ||
| 18.4.2 Follow-up Testing of Tumourigenic Compounds | 506 | ||
| 18.4.3 Assessment of Local Genotoxicity | 507 | ||
| 18.4.4 Assessment of Germ Cell Genotoxicity | 507 | ||
| 18.4.5 Assessment of Photogenotoxicity | 508 | ||
| 18.4.6 Genotoxicity Testing of Chemicals | 509 | ||
| 18.5 Conclusions | 510 | ||
| References | 511 | ||
| Chapter 19 Imaging and Image Analysis in the Comet Assay | 515 | ||
| 19.1 Introduction | 515 | ||
| 19.1.1 Experimental Design and Applications | 516 | ||
| 19.2 Comet Sample Preparation | 516 | ||
| 19.3 Comet Fluorescence Staining and Visualization | 517 | ||
| 19.4 Fluorescence Microscopy for Comet Imaging | 519 | ||
| 19.4.1 Light Sources | 519 | ||
| 19.4.2 Epifluorescence Light Path | 523 | ||
| 19.4.3 Fluorescence Filter Sets | 523 | ||
| 19.4.4 Microscope Objectives | 525 | ||
| 19.4.5 Beam-splitter and C-mount Adapter | 526 | ||
| 19.5 Image Detection—CCD, EMCCD and CMOS Cameras | 527 | ||
| 19.5.1 Practical Matters | 531 | ||
| 19.6 Image Processing and Comet Scoring | 533 | ||
| 19.6.1 Image Analysis | 534 | ||
| 19.6.2 Segmentation | 535 | ||
| 19.6.3 Identifying Comet Head and Tail | 538 | ||
| 19.6.4 Analysis of the Comet, Head and Tail Distributions | 539 | ||
| 19.6.5 Comet Analysis - Other Approaches | 542 | ||
| 19.7 How Many Cells, How Many Replicates? | 543 | ||
| 19.7.1 Data Storage and GLP Compliance | 543 | ||
| 19.7.2 Presentation and Preparation for Analysis | 544 | ||
| 19.7.3 Statistical Analyses | 547 | ||
| 19.7.4 Data Storage and Management | 547 | ||
| 19.8 Conclusions | 548 | ||
| References | 548 | ||
| Chapter 20 Statistical Analysis of Comet Assay Data | 551 | ||
| 20.1 Introduction | 551 | ||
| 20.2 Experimental Design and Statistical Analysis | 552 | ||
| 20.3 Study Design | 553 | ||
| 20.4 Endpoints | 554 | ||
| 20.5 The Experimental Unit and Experimental Design | 558 | ||
| 20.6 Statistical Methods | 560 | ||
| 20.7 Use of Control Groups | 564 | ||
| 20.8 Assessment of Results | 565 | ||
| 20.9 Multiple Comparison Issues | 566 | ||
| 20.10 Power and Sample Size | 569 | ||
| 20.11 OECD Guidelines | 570 | ||
| 20.12 JaCVAM Validation Study | 571 | ||
| 20.13 Human Studies | 572 | ||
| 20.14 Standardization and Inter-laboratory Comparisons | 574 | ||
| References | 575 | ||
| Subject Index | 581 |