Beschreibung
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.
Autorenporträt
Inhaltsangabe1. 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
Herstellerkennzeichnung:
Springer Verlag GmbH
Tiergartenstr. 17
69121 Heidelberg
DE
E-Mail: juergen.hartmann@springer.com
