Single-Cell Mutation Monitoring Systems, Softcover reprint of the original 1st ed. 1984
Methodologies and Applications
Topics in Chemical Mutagenesis Series

There is general agreement that increased environmental pollution poses a potential health hazard to humans and that effective control of such genetic injury requires monitoring the exposed individuals for genetic damage and identifying chemicals that may cause mutation or cancer. Tests available for identifying mutagens or carcinogens range from relatively simple, rapid assays in prokaryotes and test systems utilizing mammalian cells in tissue culture to highly elaborate tests in intact animals. No single test can provide data for an unequivocal assessment of the mutagenicity of a given chemical and the risk it might pose to human health. A tier approach, therefore, was suggested for mutagenicity testing in which the suspected agents would be initially evaluated with simple, inexpensive tests that would give qualitative results. Chemicals found to be positive in the first-tier testing would then be evaluated with more complex tests, including those based on mammalian cells in culture. Testing in the final tier requires whole-animal studies, and is expensive and time-consum­ ing, and even the results from these studies need to be extrapolated for human risk assessment. The mutation systems based on whole animals require scoring large num­ bers of animals, and therefore are not practical for the routine testing of muta­ gens. As an alternative to monitoring the pedigree, cells from exposed individ­ uals may be considered for screening for point mutations through the use of an appropriate marker protein.

1. Somatic-Cell Mutation Monitoring System Based on Human Hemoglobin Mutants.- 1. Introduction.- 1.1. The Approach.- 1.2. Previous Studies.- 1.3. Requirements of a Red Cell Screening System.- 2. The Hemoglobin Mutants.- 2.1. Hemoglobin Loci.- 2.2. Types of Mutations.- 3. Hemoglobin in Mutation Research: Gametal Mutation Rates.- 3.1. Indirect Estimates.- 3.2. Direct Estimates.- 4. A System for Detecting Somatic Mutations of Hemoglobin.- 4.1. Appropriate Mutants.- 4.2. Immunochemical Detection of Abnormal Hemoglobins in Single Cells.- 4.3. Detection of Rare Mutant Red Cells by Fluorescent Microscopy.- 4.4. Screening for “S Cells” or “C Cells” in Blood of Genetically A/A Subjects.- 4.5. Minimum Frequencies of Somatic Mutations at Globin-Chain Loci.- 4.6. Relationship between Somatic Mutation Frequencies and Gametal Mutation Rates.- 4.7. Relationship between Frequencies of Somatic-Cell Mutants and Compartments at Which Mutations Occur.- 5. Methodological Aspects: Monospecific Anti-Mutant-Hemoglobin Antibodies.- 5.1. Immunizations.- 5.2. Sepharose-Hb.- 5.3. Purification.- 5.4. Red Cell Labeling.- 6. Methodological Aspects: Monoclonal Anti-Globin-Chain Antibodies.- 6.1. Immunizations.- 6.2. Screening.- 6.3. Semiquantitative Assessment of Ab-Hb Binding.- 6.4. Mapping the Sites of Ab-Hb Binding.- 6.5. Possible Recognition of Mutant Hemoglobins in Animals.- References.- 2. Use of Fluorescence-Activated Cell Sorter for Screening Mutant Cells.- 1. Introduction.- 2. Immunologic Identification and Flow Detection of Erythrocytes Containing Amino Acid-Substituted Hemoglobin.- 2.1. Production of Antibodies.- 2.2. Suspension Labeling of Red Cells with Hemoglobin Antibodies.- 2.3. Flow Cytometric Processing.- 2.4. Results Using Hemoglobin S- and C-Specific Antibodies.- 3. Future of the Hemoglobin-Based Assay.- 4. Detection of Erythrocytes with Mutationally Altered Glycophorin A.- 4.1. Background.- 4.2. Gene Expression Loss Variants.- 4.3. Single-Amino Acid-Substitution Variants.- 5. Summary and Conclusions.- References.- 3. Development of a Plaque Assay for the Detection of Red Blood Cells Carrying Abnormal or Mutant Hemoglobins.- 1. Introduction.- 2. Principle of the Method.- 3. Reagents.- 3.1. Anti-Mouse RBC Ghost Sera.- 3.2. Anti-Mouse Hb Antibodies.- 3.3. Indicator Cells. Methods for Coupling Antibodies to Sheep RBC.- 3.4. Complement.- 4. Equipment.- 4.1. Plaque Chambers.- 4.2. Additional Materials.- 5. Procedure for the RBC-Antibody Plaque Assay.- 5.1. Factors Affecting the RBC Plaque Formation.- 5.2. Specificity of the RBC Plaque Assay.- 6. RBC-Protein A Plaque Assay.- 7. Conclusions.- References.- 4. Direct Assay by Autoradiography for 6-Thioguanine-Resistant Lymphocytes in Human Peripheral Blood.- 1. Introduction.- 1.1. Human Mutagenicity Monitoring.- 1.2. 6-Thioguanine-Resistant (TGr) Human Peripheral Blood T Lymphocytes (T-PBLs).- 1.3. Direct Enumeration of TGr T-PBLs by Autoradiography.- 1.4. Phenocopies.- 2. Autoradiographic TGr T-PBL Assay Method.- 2.1. Cell Preparation.- 2.2. Cryopreservation.- 2.3. Cell Culture.- 2.4. Termination, Coverslip Preparation, and Autoradiography.- 2.5. Enumeration of TGr T-PBLs and Calculation of TGr T-PBL Variant Frequency (Vf).- 3. Sample Results.- 3.1. TGr T-PBL VfAssay: Appearance of Slides.- 3.2. TGr T-PBL VfAssay: Sample Data.- 4. Statistical Analysis Methods.- 4.1. Notation and Basic Assumptions.- 4.2. Confidence Intervals for a Single Variant Frequency.- 4.3. Confidence Intervals for Ratios of Variant Frequencies.- 4.4. Sample Size Determinations.- 5. Discussion.- References.- 5. Application of Antibodies to 5-Bromodeoxyuridine for the Detection of Cells of Rare Genotype.- 1. Introduction.- 1.1. Flow Cytometry.- 1.2. The Use of 5-Bromodeoxyuridine for the Detection of Cell Proliferation.- 2. Materials and Methods.- 2.1. Cell Culture.- 2.2. Labeling with BrdUrd.- 2.3. Immunological Methods.- 2.4. Flow Cytometry.- 2.5. Hybridoma Production.- 3. Results.- 3.1. Determination of Antibody Specificity.- 3.2. Flow Cytometric Method for Immunofluorescent Detection of DNA Replication.- 3.3. Application of the Flow Immunofluorometric Anti-BrdUrd Technique to the Assessment of DNA Damage.- 3.4. Reconstruction Experiments for the Detection of Thioguanine-Resistant Variants by the Immunofluorescent Anti-BrdUrd Method.- 3.5. Preparation of Monoclonal Antibodies against 5-Bromodeoxyuridine or 5-Iododeoxyuridine.- 4. Discussion.- 5. Summary.- References.- 6. Cytogenetic Abnormalities as an Indicator of Mutagenic Exposure.- 1. Introduction.- 2. Lymphocyte Assay Methodology.- 2.1. In Vitro Cultures.- 2.2. Sampling Time.- 2.3. Fixation and Slide Preparation.- 2.4. Analysis of Cells.- 3. Chromosome Aberration Analysis Following Radiation or Chemical Exposure.- 3.1. The Sensitivity of the Lymphocyte Assay.- 3.2. Background Aberration Frequencies and “Matched” Control Groups.- 4. The Analysis of Bone Marrow Samples.- 5. The Plausibility of Estimating Genetic or Carcinogenic Risk from Aberration Frequencies in Lymphocytes.- 6. Concluding Remarks.- References.- 7. Sister Chromatid Exchange Analysis in Lymphocytes.- 1. Introduction.- 2. Methodology.- 2.1. Background.- 2.2. General Design Considerations.- 2.3. Technical.- 3. Selected Applications.- Appendix A. A Procedure for Growing and Preparing Human Lymphocytes for SCE Analysis.- Appendix B. A Procedure for Growing and Preparing Rat Lymphocytes for SCE Analysis.- References.- 8. Unscheduled DNA Synthesis as an Indication of Genotoxic Exposure.- 1. Introduction.- 2. Four Laboratory Approaches to Measuring Unscheduled DNA Synthesis.- 2.1. Liquid Scintillation Counting Measurements of UDS in Human Fibroblast DNA.- 2.2. Autoradiographic Measurements of UDS in Human Diploid Fibroblasts.- 2.3. Autoradiographic Measurements of UDS in Primary Cultures of Rat Hepatocytes.- 2.4. Measurements of UDS in Hepatocytes following in Vivo Treatment.- 3. Methods.- 3.1. Procedures for LSC UDS Assays.- 3.2. Establishment of Primary Cultures of Rat Hepatocytes from Treated or Untreated Animals.- 3.3. The in Vivo Rat Hepatocyte UDS Assay.- 3.4. Autoradiography.- 4. Reagents, Solutions, Stains, and Media.- 4.1. Reagents.- 4.2. Solutions and Stains.- 4.3. Media.- 5. Equipment and Supplies.- 5.1. Balances.- 5.2. Calculators.- 5.3. Centrifuges.- 5.4. Computer Equipment.- 5.5. Filters.- 5.6. Grain Counters.- 5.7. Incubators and Related Apparatus.- 5.8. Laminar-Flow Hoods.- 5.9. Microscopes.- 5.10. Mixer.- 5.11. Pipetting Apparatus.- 5.12. Pump Apparatus.- 5.13. Rocker Platform.- 5.14. Scintillation Counter.- 5.15. Spectrophotometer.- 5.16. Tissue Culture Supplies.- 5.17. Water Baths.- 5.18. Water System.- 5.19. Miscellaneous Equipment.- 5.20. Miscellaneous Supplies.- References.- 9. The Micronucleus Test as an Indicator of Mutagenic Exposure.- 1. Historical Background.- 2. Rationale of the Test System.- 3. Technical Procedure.- 3.1. Animals.- 3.2. Administration of the Test Substance.- 3.3. Determination of Dosage.- 3.4. Sampling Times.- 3.5. Preparation of the Bone Marrow Smears.- 3.6. Scoring of Micronuclei.- 3.7. Data Evaluation.- 3.8. Other Useful Information on the Micronucleus Test.- 3.9. Manpower and Costs.- 4. Results and Comparative Studies.- 5. Related Assay Systems.- 6. Advantages.- 7. Limitations.- 8. Application of the Micronucelus Test.- References.- 10. The Identification of Somatic Mutations in Immunoglobulin Expression and Structure.- 1. Introduction.- 2. Immunoglobulin Protein and Gene Structure.- 3. Methods for the Isolation of Mutants.- 3.1. Mutagenesis.- 3.2. Screening Techniques.- 3.3. Selective Techniques.- 4. Frequency and Phenotypes of Mutants.- 4.1. Frameshift Mutants.- 4.2. Point Mutants.- 4.3. Internal Deletion Mutants Associated with Changes in DNA or RNA.- 4.4. Class- and Subclass-Switch Mutants.- 5. Discussion.- References.- 11. Detection of Chemically Induced Y-Chromosomal Nondisjunction in Human Spermatozoa.- 1. Introduction.- 2. Background.- 3. Agents That Increase YFF Bodies in Human Sperm.- 3.1. Methodology.- 3.2. Results to Date.- 4. Discussion.- References.