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Quantitative mammalian cell mutagenesis and mutagen screening
437
Mutation Research, 64 ( 1 9 7 9 ) 4 3 7 - - 4 4 0 © Elsevier/North-Holland Biomedical Press
Meeting Report QUANTITATIVE MAMMALIAN CELL MUTAGENESIS AND MUTAGEN SCREENING A REPORT ON BANBURY CONFERENCE II *
A B R A H A M W. H S I E , J. P A T R I C K O ' N E I L L a n d V I C T O R K. M c E L H E N Y a
Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.) and a Banbury Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y 11724 (U.S.A.) ( R e c e i v e d 14 J u l y 1 9 7 9 ) (Accepted 6 August 1979)
The Banbury Center of the Cold Spring Harbor Laboratory, New York, is designed for small conferences on life sciences and public policy, with emphasis on assessing the potential environmental risks to humans. The first Banbury Conference, Assessing Chemical Mutagens: The Risk to Humans, was held in May 1978, and the proceedings were published as Banbury Report 1. The present conference, the second of the series, was held May 6--9, 1979, and included 31 invited U.S. mammalian somatic cell geneticists from various sectors of the scientific community, including universities, industries, research institutes, and regulatory agencies. Organized by A.W. Hsie and J.P. O'Neill of Oak Ridge National Laboratory and V.K. McElheny of Banbury Center, the conference was called to evaluate the current progress in gene mutation, quantitative mutagenesis, and mutagen screening with mammalian cells in culture, and to identify future research directions in these fields through informal presentations and round table discussions. The conference cor~sisted of seven sessions. Session 1, Gene Mutation, was chaired by E.A. Adelberg*(Yale University). T.T. Puck (University of Colorado) presented a historical perspective of mutation studies by discussing the induction, characterization, and use of auxotrophic mutants in Chinese hamster ovary (CHO) cells and the application of a CHO--human cell hybrid containing a single human chromosome in screening
By acceptance of this article, the publisher or r e c i p i e n t a c k n o w l e d g e s t h e right of the U.S. Government t o retain a nonexclusive, royalty-free license in and to any copyright covering the article. * The research presented by Hsie and O'Neill in this c o n f e r e n c e has b e e n s u p p o r t e d jointly by t h e National Cancer for Toxicological Research, Environmental P r o t e c t i o n A g e n c y , and t h e Office of Health and Environmental Research, U.S. Department of Energy, u n d e r c o n t r a c t W-7405-eng-26 w i t h t h e U n i o n Carbide Corporation.
438
for chromosomal deletions. L. Siminovitch (University of Toronto) gave an overview on the availability of both dominant and recessive mutation systems, with emphasis on diptheria toxin resistance, in CHO cells and human cells, and their potential application for mutagenesis studies. Based on variants of Chinese hamster V79 cells resistant to 8-azaguanine (AG) and the techniques of immunologic and peptide analysis, T.C. Caskey (Baylor College of Medicine) presented evidence for mutations affecting enzyme kinetic properties and thermal stability; he proposed the use of DNA cloning methodologies to probe mutagen-induced lesions at the nucleotide level. As the chairperson of the first round table session on Definition of Criteria to Define a Genetic Event, R.L. Davidson (Children's Hospital Medical Center), using 5-bromodeoxyuridine (BUdR) as an example, discussed both the need to fully characterize variant phenotypes and the possible differential mutagenic actions between bacteria and mammalian cells. Adelberg emphasized that in the absence of direct proof for gene mutation at the level of alteration of nucleotide sequence or amplification of a specific gene, one should vigorously search for positive evidence for gene mutation such as changes in the amino acid sequence or peptide mapping rather than simply ruling out epigenetic possibilities. Caskey pointed out the need to use genetically and biochemically wellcharacterized specific gene mutational systems to study quantitative mutagenesis. Puck proposed guideline criteria, including a reasonable frequency of spontaneous mutation and reversion, phenotype stability in mutants and revertants, phenotypic expression as a single gene mutation in hybrid cells, increased frequency of forward mutation and reversion by known mutagens, and the identification of altered gene product(s). Session 2, Quantitative Mutagenesis with Rodent Cells, was chaired by Siminovitch. The formal presentation included the detailed experimental protocols of 3 systems: the CHO cell/hypoxanthine--guanine phosphoribosyl transferase (CHO/HGPRT) system, the mouse lymphoma L5178 cell/thymidine kinase (L5178/TK) system, and the Chinese hamster V79 ceU/ouabain resistance (V79/Oua) system, described by O'Neill, M.M. Moore-Brown (Environmental Protection Agency), and C.-C. Chang (Michigan State University), respectively. While O'Neill and Moore-Brown emphasized the definition of the system, variant characterization, and factors affecting quantitation, especially phenotypic expression time, selection stringency, and other experimental conditions, Chang's talk centered on the advantage of the ouabain resistance and the use of the V79/ouabain system to detect tumor promoters and antipromoters. The round table discussions, Quantitative Mutational Systems: Evidence for Genetic Events, was chaired by L.A. Chasin (Columbia University). O'Neill, MooreBrown, and Chang presented genetic, physiological and biochemical evidence consistent with the notion that each assay has determined the frequency of variants which arose through genetic events. Session 3 was a round table discussion on Criteria for a Mutagen Screening System, chaired by D.A. Casciano (National Center for Toxicological Research). Short and informal presentations were made by J. Bradlaw (Food and Drug Administration), B.E. Butterworth (Chemical Industry Institute of Toxicology), L. Kier (Montesano Chemical Co.), D.F. Krahn (E.I. Du Pont de Nemours and Co.), M. Waters (Environmental Protection Agency), and E.
439
Zeiger (National Institute for Environmental Health Sciences). Diverse viewpoints and emphases were expressed by participants coming from different backgrounds. Despite this, all speakers and participants concurred that in addition to having the best scientific basis, a useful mammalian cell system should be broad enough to detect sensitively and quantitatively such varied multiple end points as gene mutation and chromosomal aberrations, including deletion. Routine feasibility and intralaboratory reproducibility have been emphasized. Additional features, such as the ability to detect crude or analytically separated environmental mixtures and to discriminate known carcinogenic vs. non-carciN nogenic agents, should be demonstrated. The lack of an "ideal" metabolic activation system caused a general concern over the quantitative nature of mutation induced by promutagens. Session 4 concerned Genetic, Biochemical and Molecular Analysis o f Mutation. In his opening remarks, chairperson P. O. P. Ts'o (Johns Hopkins University) discussed the relevance of genetic analysis to studies of neoplastic transformation. Adelberg discussed techniques of selecting for various membrane mutants affecting transport of amino acid and potassium ion in CHO and L cells. L. Thompson (Lawrence Livermore Laboratory) analyzed conditional aminoacyl-tRNA synthetase mutants genetically and biochemically, and described conditions for quantifying mutagenesis at HGPRT anci adenine phosphoribosyl transferase (APRT) loci in suspension cultures of CHO cells. Chasin discussed the isolation and characterization of dihydrofolate reductase mutants of CHO cells and their utility in studying reversion, fine-structural mapping, and definition of the mutational events. M. Taylor (Indiana University) discussed biochemical aspects of the APRT system and suggested the possibility of DNA sequencing for studying molecular events of mutation. Davidson presented evidence for the dissociation of BUdR incorporation into DNA and mutagenicity of BUdR, and described the complexicity of using BUdR selection for TK-negative mutants in Syrian hamster cells. R.M. Baker (Massachusetts Institute of Technology) detailed his view on the use of the ouabain resistance assay with CHO and human (HeLa) cells, analyzed the merits and disadvantages of the system, and cautioned its proper use in quantitative mutagenesis. Zeiger chaired Session 5, Use o f Mutagen Screening Systems, a discussion of the application of quantitative mammalian cell mutation systems to screening for environmental chemical mutagens. Krahn described the application of the CHO/HGPRT assay to the determination of the mutagenicity of gases and volatile liquids and outlined factors affecting metabolic activation of promutagens. J. Irr (E.I. Du Pont de Nemours and Co.) presented a statistical test designed for mutagenicity estimation in the CHO/HGPRT assay. D. Amacher (Pfizer Inc.) discussed the adaptation and modification of the standard protocol of L5178/TK assay and the reproducibility, detection spectrum, and limitations of the assay. J. Dent (Chemical Industry Institute of Toxicology) gave a general consideration with biochemical analysis of the use of rodent hepatocyte, $9, and bacterial extract (from Salmonella typhimurium) as as metabolic activation systems. Applying the considerations of Dent, D.B. Couch (Chemical Industry Institute of Toxicology) described the mutagenicity of dinitrotoluenes in the CHO/HGPRT assay as affected by the activation source under aerobic and anaerobic conditions. O'Neill discussed modifications of the experimental pro-
440
tocol of the CHO/HGPRT system for routine mutagen screening. In Session 6, Quantitative Human Cell Mutagenesis, chaired by O'Neill, the formal presentations dealt with the use of human cells in mutagenesis studies and their utility for mutagen screening. R. DeMars (University of Wisconsin} detailed the selection, characterization, and stability of resistance to AG(HGPRT locus) and 2,6-diaminopurine (APRT locus) in human fibroblasts, with emphasis on the utility of the APRT system for mutagen screening from human cells heterozygous for the APRT gene. W. Thilly (Massachusetts Institute of Technology) discussed different genetic loci studied and differences in responses among loci and chemicals; he also described the isolation of mutator/ antimutator variants of a human lymphoblast cell line. V. Maher (Michigan State University) reported on mutagenesis in diploid fibroblasts from normal human and xeroderma pigmentosum patients and emphasized the role of replication and repair of DNA in mutagenesis using the AG or 6-thioguanine (TG) resistance system. R.J. Albertini (University of Vermont) discussed TG-resisrant peripheral blood lymphocytes arising in vivo as indicators of somatic cell mutation and presented data showing that this technique could be used directly in industrial hygiene monitoring. The round table discussion on Genetic Basis of Mutation in Human Cell Systems was chaired by D. Clive (Burroughs-Wellcome Co.). Much of the discussion echoed the points discussed earlier for rodent cells, with emphasis on the advantage of using human cells as a model in estimating human risk from environmental mutagens. Session 7, Mutation, Cancer and Progress with Mutagen Testing was chaired by Bradlaw. Ts'o pointed out the role of somatic mutation in neoplastic transformation through characterization and quantitative comparison between gene mutation and transformation in Syrian hamster embryo cells. Hsie gave a progress report on the CHO/HGPRT system, emphasizing studies on apparent exposure dose, mutagenicity as related to DNA lesions, the structure--activity relationships among 132 agents, and the correlation between CHO mutagenicity and animal carcinogenicity. Clive detailed the characterization and analysis of TK- mutants, presented evidence on structural gene vs. chromosomal mutations, and discussed the relevance to risk estimates using L5178/TK assay. M. Hollstein (University of California) outlined data collation for validation of short-term screening systems, with special reference to differences in detection spectra among assays and the limitation of validation efforts. Waters discussed the EPA Gene-Tox Program, which intends to systematically evaluate the current state of the art and the utility of selected mutagenicity test systems from published literature; he also talked about the possible role of mammalian cell systems in environmental research and governmental regulation. Chasin gave the closing remarks and emphasized the need for "perfecting" metabolic activation systems as an urgent direction for research and development in the area of mammalian cell mutagenesis. The proceedings of this conference will be published as Banbury Report IL Other meetings of the Banbury series, scheduled for Autumn 1979 include: (a} Toward a Less Hazadous Cigarette, (b) Religious Groups as Low-Risk Populations, (c) Ethylene Dichloride: Economic Importance and Potential Health Risks.
Mutation Research, 64 ( 1 9 7 9 ) 4 3 7 - - 4 4 0 © Elsevier/North-Holland Biomedical Press
Meeting Report QUANTITATIVE MAMMALIAN CELL MUTAGENESIS AND MUTAGEN SCREENING A REPORT ON BANBURY CONFERENCE II *
A B R A H A M W. H S I E , J. P A T R I C K O ' N E I L L a n d V I C T O R K. M c E L H E N Y a
Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.) and a Banbury Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y 11724 (U.S.A.) ( R e c e i v e d 14 J u l y 1 9 7 9 ) (Accepted 6 August 1979)
The Banbury Center of the Cold Spring Harbor Laboratory, New York, is designed for small conferences on life sciences and public policy, with emphasis on assessing the potential environmental risks to humans. The first Banbury Conference, Assessing Chemical Mutagens: The Risk to Humans, was held in May 1978, and the proceedings were published as Banbury Report 1. The present conference, the second of the series, was held May 6--9, 1979, and included 31 invited U.S. mammalian somatic cell geneticists from various sectors of the scientific community, including universities, industries, research institutes, and regulatory agencies. Organized by A.W. Hsie and J.P. O'Neill of Oak Ridge National Laboratory and V.K. McElheny of Banbury Center, the conference was called to evaluate the current progress in gene mutation, quantitative mutagenesis, and mutagen screening with mammalian cells in culture, and to identify future research directions in these fields through informal presentations and round table discussions. The conference cor~sisted of seven sessions. Session 1, Gene Mutation, was chaired by E.A. Adelberg*(Yale University). T.T. Puck (University of Colorado) presented a historical perspective of mutation studies by discussing the induction, characterization, and use of auxotrophic mutants in Chinese hamster ovary (CHO) cells and the application of a CHO--human cell hybrid containing a single human chromosome in screening
By acceptance of this article, the publisher or r e c i p i e n t a c k n o w l e d g e s t h e right of the U.S. Government t o retain a nonexclusive, royalty-free license in and to any copyright covering the article. * The research presented by Hsie and O'Neill in this c o n f e r e n c e has b e e n s u p p o r t e d jointly by t h e National Cancer for Toxicological Research, Environmental P r o t e c t i o n A g e n c y , and t h e Office of Health and Environmental Research, U.S. Department of Energy, u n d e r c o n t r a c t W-7405-eng-26 w i t h t h e U n i o n Carbide Corporation.
438
for chromosomal deletions. L. Siminovitch (University of Toronto) gave an overview on the availability of both dominant and recessive mutation systems, with emphasis on diptheria toxin resistance, in CHO cells and human cells, and their potential application for mutagenesis studies. Based on variants of Chinese hamster V79 cells resistant to 8-azaguanine (AG) and the techniques of immunologic and peptide analysis, T.C. Caskey (Baylor College of Medicine) presented evidence for mutations affecting enzyme kinetic properties and thermal stability; he proposed the use of DNA cloning methodologies to probe mutagen-induced lesions at the nucleotide level. As the chairperson of the first round table session on Definition of Criteria to Define a Genetic Event, R.L. Davidson (Children's Hospital Medical Center), using 5-bromodeoxyuridine (BUdR) as an example, discussed both the need to fully characterize variant phenotypes and the possible differential mutagenic actions between bacteria and mammalian cells. Adelberg emphasized that in the absence of direct proof for gene mutation at the level of alteration of nucleotide sequence or amplification of a specific gene, one should vigorously search for positive evidence for gene mutation such as changes in the amino acid sequence or peptide mapping rather than simply ruling out epigenetic possibilities. Caskey pointed out the need to use genetically and biochemically wellcharacterized specific gene mutational systems to study quantitative mutagenesis. Puck proposed guideline criteria, including a reasonable frequency of spontaneous mutation and reversion, phenotype stability in mutants and revertants, phenotypic expression as a single gene mutation in hybrid cells, increased frequency of forward mutation and reversion by known mutagens, and the identification of altered gene product(s). Session 2, Quantitative Mutagenesis with Rodent Cells, was chaired by Siminovitch. The formal presentation included the detailed experimental protocols of 3 systems: the CHO cell/hypoxanthine--guanine phosphoribosyl transferase (CHO/HGPRT) system, the mouse lymphoma L5178 cell/thymidine kinase (L5178/TK) system, and the Chinese hamster V79 ceU/ouabain resistance (V79/Oua) system, described by O'Neill, M.M. Moore-Brown (Environmental Protection Agency), and C.-C. Chang (Michigan State University), respectively. While O'Neill and Moore-Brown emphasized the definition of the system, variant characterization, and factors affecting quantitation, especially phenotypic expression time, selection stringency, and other experimental conditions, Chang's talk centered on the advantage of the ouabain resistance and the use of the V79/ouabain system to detect tumor promoters and antipromoters. The round table discussions, Quantitative Mutational Systems: Evidence for Genetic Events, was chaired by L.A. Chasin (Columbia University). O'Neill, MooreBrown, and Chang presented genetic, physiological and biochemical evidence consistent with the notion that each assay has determined the frequency of variants which arose through genetic events. Session 3 was a round table discussion on Criteria for a Mutagen Screening System, chaired by D.A. Casciano (National Center for Toxicological Research). Short and informal presentations were made by J. Bradlaw (Food and Drug Administration), B.E. Butterworth (Chemical Industry Institute of Toxicology), L. Kier (Montesano Chemical Co.), D.F. Krahn (E.I. Du Pont de Nemours and Co.), M. Waters (Environmental Protection Agency), and E.
439
Zeiger (National Institute for Environmental Health Sciences). Diverse viewpoints and emphases were expressed by participants coming from different backgrounds. Despite this, all speakers and participants concurred that in addition to having the best scientific basis, a useful mammalian cell system should be broad enough to detect sensitively and quantitatively such varied multiple end points as gene mutation and chromosomal aberrations, including deletion. Routine feasibility and intralaboratory reproducibility have been emphasized. Additional features, such as the ability to detect crude or analytically separated environmental mixtures and to discriminate known carcinogenic vs. non-carciN nogenic agents, should be demonstrated. The lack of an "ideal" metabolic activation system caused a general concern over the quantitative nature of mutation induced by promutagens. Session 4 concerned Genetic, Biochemical and Molecular Analysis o f Mutation. In his opening remarks, chairperson P. O. P. Ts'o (Johns Hopkins University) discussed the relevance of genetic analysis to studies of neoplastic transformation. Adelberg discussed techniques of selecting for various membrane mutants affecting transport of amino acid and potassium ion in CHO and L cells. L. Thompson (Lawrence Livermore Laboratory) analyzed conditional aminoacyl-tRNA synthetase mutants genetically and biochemically, and described conditions for quantifying mutagenesis at HGPRT anci adenine phosphoribosyl transferase (APRT) loci in suspension cultures of CHO cells. Chasin discussed the isolation and characterization of dihydrofolate reductase mutants of CHO cells and their utility in studying reversion, fine-structural mapping, and definition of the mutational events. M. Taylor (Indiana University) discussed biochemical aspects of the APRT system and suggested the possibility of DNA sequencing for studying molecular events of mutation. Davidson presented evidence for the dissociation of BUdR incorporation into DNA and mutagenicity of BUdR, and described the complexicity of using BUdR selection for TK-negative mutants in Syrian hamster cells. R.M. Baker (Massachusetts Institute of Technology) detailed his view on the use of the ouabain resistance assay with CHO and human (HeLa) cells, analyzed the merits and disadvantages of the system, and cautioned its proper use in quantitative mutagenesis. Zeiger chaired Session 5, Use o f Mutagen Screening Systems, a discussion of the application of quantitative mammalian cell mutation systems to screening for environmental chemical mutagens. Krahn described the application of the CHO/HGPRT assay to the determination of the mutagenicity of gases and volatile liquids and outlined factors affecting metabolic activation of promutagens. J. Irr (E.I. Du Pont de Nemours and Co.) presented a statistical test designed for mutagenicity estimation in the CHO/HGPRT assay. D. Amacher (Pfizer Inc.) discussed the adaptation and modification of the standard protocol of L5178/TK assay and the reproducibility, detection spectrum, and limitations of the assay. J. Dent (Chemical Industry Institute of Toxicology) gave a general consideration with biochemical analysis of the use of rodent hepatocyte, $9, and bacterial extract (from Salmonella typhimurium) as as metabolic activation systems. Applying the considerations of Dent, D.B. Couch (Chemical Industry Institute of Toxicology) described the mutagenicity of dinitrotoluenes in the CHO/HGPRT assay as affected by the activation source under aerobic and anaerobic conditions. O'Neill discussed modifications of the experimental pro-
440
tocol of the CHO/HGPRT system for routine mutagen screening. In Session 6, Quantitative Human Cell Mutagenesis, chaired by O'Neill, the formal presentations dealt with the use of human cells in mutagenesis studies and their utility for mutagen screening. R. DeMars (University of Wisconsin} detailed the selection, characterization, and stability of resistance to AG(HGPRT locus) and 2,6-diaminopurine (APRT locus) in human fibroblasts, with emphasis on the utility of the APRT system for mutagen screening from human cells heterozygous for the APRT gene. W. Thilly (Massachusetts Institute of Technology) discussed different genetic loci studied and differences in responses among loci and chemicals; he also described the isolation of mutator/ antimutator variants of a human lymphoblast cell line. V. Maher (Michigan State University) reported on mutagenesis in diploid fibroblasts from normal human and xeroderma pigmentosum patients and emphasized the role of replication and repair of DNA in mutagenesis using the AG or 6-thioguanine (TG) resistance system. R.J. Albertini (University of Vermont) discussed TG-resisrant peripheral blood lymphocytes arising in vivo as indicators of somatic cell mutation and presented data showing that this technique could be used directly in industrial hygiene monitoring. The round table discussion on Genetic Basis of Mutation in Human Cell Systems was chaired by D. Clive (Burroughs-Wellcome Co.). Much of the discussion echoed the points discussed earlier for rodent cells, with emphasis on the advantage of using human cells as a model in estimating human risk from environmental mutagens. Session 7, Mutation, Cancer and Progress with Mutagen Testing was chaired by Bradlaw. Ts'o pointed out the role of somatic mutation in neoplastic transformation through characterization and quantitative comparison between gene mutation and transformation in Syrian hamster embryo cells. Hsie gave a progress report on the CHO/HGPRT system, emphasizing studies on apparent exposure dose, mutagenicity as related to DNA lesions, the structure--activity relationships among 132 agents, and the correlation between CHO mutagenicity and animal carcinogenicity. Clive detailed the characterization and analysis of TK- mutants, presented evidence on structural gene vs. chromosomal mutations, and discussed the relevance to risk estimates using L5178/TK assay. M. Hollstein (University of California) outlined data collation for validation of short-term screening systems, with special reference to differences in detection spectra among assays and the limitation of validation efforts. Waters discussed the EPA Gene-Tox Program, which intends to systematically evaluate the current state of the art and the utility of selected mutagenicity test systems from published literature; he also talked about the possible role of mammalian cell systems in environmental research and governmental regulation. Chasin gave the closing remarks and emphasized the need for "perfecting" metabolic activation systems as an urgent direction for research and development in the area of mammalian cell mutagenesis. The proceedings of this conference will be published as Banbury Report IL Other meetings of the Banbury series, scheduled for Autumn 1979 include: (a} Toward a Less Hazadous Cigarette, (b) Religious Groups as Low-Risk Populations, (c) Ethylene Dichloride: Economic Importance and Potential Health Risks.