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O XV A.3 Validation and strategic use of in vivo transgenic gene mutation assays
S-XV: Transgenic model for studying environmental mutagenesis
S145
SESSION XV: Transgenic model for studying environmental mutagenesis A. Use for mutagenesis studies ORALS
10 xv A.II
The use of transgenic anImals carrying the E. coli lacl gene In understanding mutagenesis and eardnogenesis
Barry W. Glickman, Paul Kotturi, Andrew Suri, Greg Stuart, David Walsh, Ken Sojonky, James Holcroft, Johan de Boer. Centre fo r Environmental Health and the Department ofBiology, University of Victoria, Victoria, BC, Canada The development of a number of transgenic rodents has provided new and powerful tools for the study of environmental mutagenesis and carcinogenesis. It has now become possible to efficiently examine mutation In vivo in living mammals. As a consequence it is now possible to study mutation in the specific target tissue, the effects of sex, aging and complex lifestyle issues such as diet. It is also now possible to examine mutation m neonates and look at transplacental and development effects. In addition, with the development of both mouse and rat Big Blu~ transgemc ammals carrying the E coli lad gene it has also become possible to make inter-species comparisons. Moreover, the ease of recovery and DNA sequencing, and the extensive existing lad data base, makes the Big BlueO!> an attractive system for the study mutational specificity In vivo. We examine some examples of how different carcinogens produce mutation specifically in those organs and tissues that are the target tissues for carcmogenesis. We show for example that B[a]p, DMBA, UV and ENU produce distinctly different mutational spectra. In terms of tissue specificity, we will demonstrate how a kidney carcinogen produces mutation tn the kidney, but not the liver. In the case of liver mutagenesis by AFB I we demonstrate that mutation occurs only in the rat and not the mouse which is precisely the situation observed for carcinogenesis. These, and other examples will be used demonstrate the potential use of transgenic systems to study mutagenesis. These systems provide powerful methods for both the detection of new mutagens and potential carcinogens, as well as mechanistic studies. Some of the limitations experienced with these systems and consideration of other target endpoints will also be discussed.
10 XV A.21
A proposal for the use of In vivo mutation assays In cancer assessments
Nancy J. Gorelick', Jon C. Mirsalisz. I Procter & Gamble Company, Cincinnati, OH, USA; 2 SRI International, Menlo Park, CA, USA Transgenic rodent models that carry a recoverable target gene provide the opportunity to evaluate the mutagenic potential of a chemical in an in vivo environment, which may be more relevant than that provided by bacteria or mammalian cells in vitro. These assays detect primarily basepair substitutions, frameshift mutations, and small deletions and insertions. Based on data developed to date. three uses in cancer assessments are proposed. First, when tumors are produced in a rodent carcinogenicity study, the transgenic mutation assays may be used to identifY whether a mutagenic mechanism is likely to be involved in the observed carcinogenic response. A transgenic mutation system could be used to evaluate mutations in the tissue, species, and sex under dosing conditions in which tumors were observed. A unique contribution of the transgenic systems is the measurement of gene mutation, compared to alternative in vivo measures currently employed [UDS or DNA adducts J, which only presume the potential induction of gene mutation. Furthermore, it is possible to develop mutational spectra in transgenic target tissues. which may provide insight into the molecular
mechanisms of mutagenesis and, hence, potentially affect dose response extrapolations and risk assessment. A negative transgenic result would be consistent with (but not prove) a nongenotoxic mechanism in vivo whereas a positive result would support a genotoxic mechanism. The second anticipated application of transgenic rodent models is when a compound is genotoxic in vitro, and targets in current standard assays (bone marrow and liver) are nonoptimal sites for evaluation of in vivo genotoxicity. Potential site-of-contact mutagens are in this category. Target sites include skin for dermally applied products with limited absorption or to evaluate potential photo-chemical mutagenicity, lung for inhaled products and, for orally administered products, the stomach. The third and most challenging application of the transgenic mutation assays is in cases where positive in vitro gene mutation results cannot be explained by mechanistic understanding. A negative result in a wellconducted in vivo mutation assay might obviate the need for a carcinogenicity bioassay, depending on the complete weight of evidence for safety. A positive result in vivo would confirm the observations in vitro. Keyword(s): Transgenic; mutagenesis; risk assessment
10 XV A.31
Validation and strategic use of in vivo transgenic gene mutation assays
Stephen Dean. Cooance, Otley Road, Harrogate, North Yorkshire, HG3 IPY, UK There is much interest in the polential uses of in vivo transgenic gene mutanon (TGM) models. The two best known, Big Blue and Mutamouse, are being used in many laboratories around the world and for many genetic toxicologists, the main interest is that the assays become sufficiently validated to be used to generate data for risk assessment within the regulatory process. Validationwill depend upon the application of the test system and there are two strategically distinct roles for TGM assays. Firstly, and this has already been acknowledged by ICH, is the investigation of chemicals which produce unexpected results in rodent cancer studies, to identify genotoxic and nongenotoxic carcinogens. Each study must be designed specifically to address the profile of the individual chemical. Confidence in the outcome could be increased by testing structurally or pharmacologically similar mutagenic and non-mutagenic chemicals at the same time. This provides additional, and very relevant, validation at the time of the test. Indeed, we are already using TGM assays for regulatory purposes in the same way as we would use data from any other non-standard test. TGM also offers gene mutation to complement unscheduled DNA synthesis (UDS) and cytogenetic endpoints which are already well established in vivo. Clearly, the potential to look at an endpoint more relevant to cancer, in any tissue, makes these systems unique. Presently, there are carcinogenic chemicals which are potent in vitro mutagens but which are ineffectively detected by UDS and micronucleus tests. Site-of-contact mutagens such as MNNG are excellent examples. Several clearly induce mutations in TGM models at the site of exposure and in the locations predicted from cancer studies. We must ensure thaI the target tissues (e.g.• skm, stomach, lungs) are validated individually and that the protocol optimises detection in these tissues. In this way, we can provide a more relevant endpoint and tissue alternative for chemicals that we know or suspect do not reach the bone marrow or liver. Keyword(s): Transgenic; Mutagenesis; in vivo
S145
SESSION XV: Transgenic model for studying environmental mutagenesis A. Use for mutagenesis studies ORALS
10 xv A.II
The use of transgenic anImals carrying the E. coli lacl gene In understanding mutagenesis and eardnogenesis
Barry W. Glickman, Paul Kotturi, Andrew Suri, Greg Stuart, David Walsh, Ken Sojonky, James Holcroft, Johan de Boer. Centre fo r Environmental Health and the Department ofBiology, University of Victoria, Victoria, BC, Canada The development of a number of transgenic rodents has provided new and powerful tools for the study of environmental mutagenesis and carcinogenesis. It has now become possible to efficiently examine mutation In vivo in living mammals. As a consequence it is now possible to study mutation in the specific target tissue, the effects of sex, aging and complex lifestyle issues such as diet. It is also now possible to examine mutation m neonates and look at transplacental and development effects. In addition, with the development of both mouse and rat Big Blu~ transgemc ammals carrying the E coli lad gene it has also become possible to make inter-species comparisons. Moreover, the ease of recovery and DNA sequencing, and the extensive existing lad data base, makes the Big BlueO!> an attractive system for the study mutational specificity In vivo. We examine some examples of how different carcinogens produce mutation specifically in those organs and tissues that are the target tissues for carcmogenesis. We show for example that B[a]p, DMBA, UV and ENU produce distinctly different mutational spectra. In terms of tissue specificity, we will demonstrate how a kidney carcinogen produces mutation tn the kidney, but not the liver. In the case of liver mutagenesis by AFB I we demonstrate that mutation occurs only in the rat and not the mouse which is precisely the situation observed for carcinogenesis. These, and other examples will be used demonstrate the potential use of transgenic systems to study mutagenesis. These systems provide powerful methods for both the detection of new mutagens and potential carcinogens, as well as mechanistic studies. Some of the limitations experienced with these systems and consideration of other target endpoints will also be discussed.
10 XV A.21
A proposal for the use of In vivo mutation assays In cancer assessments
Nancy J. Gorelick', Jon C. Mirsalisz. I Procter & Gamble Company, Cincinnati, OH, USA; 2 SRI International, Menlo Park, CA, USA Transgenic rodent models that carry a recoverable target gene provide the opportunity to evaluate the mutagenic potential of a chemical in an in vivo environment, which may be more relevant than that provided by bacteria or mammalian cells in vitro. These assays detect primarily basepair substitutions, frameshift mutations, and small deletions and insertions. Based on data developed to date. three uses in cancer assessments are proposed. First, when tumors are produced in a rodent carcinogenicity study, the transgenic mutation assays may be used to identifY whether a mutagenic mechanism is likely to be involved in the observed carcinogenic response. A transgenic mutation system could be used to evaluate mutations in the tissue, species, and sex under dosing conditions in which tumors were observed. A unique contribution of the transgenic systems is the measurement of gene mutation, compared to alternative in vivo measures currently employed [UDS or DNA adducts J, which only presume the potential induction of gene mutation. Furthermore, it is possible to develop mutational spectra in transgenic target tissues. which may provide insight into the molecular
mechanisms of mutagenesis and, hence, potentially affect dose response extrapolations and risk assessment. A negative transgenic result would be consistent with (but not prove) a nongenotoxic mechanism in vivo whereas a positive result would support a genotoxic mechanism. The second anticipated application of transgenic rodent models is when a compound is genotoxic in vitro, and targets in current standard assays (bone marrow and liver) are nonoptimal sites for evaluation of in vivo genotoxicity. Potential site-of-contact mutagens are in this category. Target sites include skin for dermally applied products with limited absorption or to evaluate potential photo-chemical mutagenicity, lung for inhaled products and, for orally administered products, the stomach. The third and most challenging application of the transgenic mutation assays is in cases where positive in vitro gene mutation results cannot be explained by mechanistic understanding. A negative result in a wellconducted in vivo mutation assay might obviate the need for a carcinogenicity bioassay, depending on the complete weight of evidence for safety. A positive result in vivo would confirm the observations in vitro. Keyword(s): Transgenic; mutagenesis; risk assessment
10 XV A.31
Validation and strategic use of in vivo transgenic gene mutation assays
Stephen Dean. Cooance, Otley Road, Harrogate, North Yorkshire, HG3 IPY, UK There is much interest in the polential uses of in vivo transgenic gene mutanon (TGM) models. The two best known, Big Blue and Mutamouse, are being used in many laboratories around the world and for many genetic toxicologists, the main interest is that the assays become sufficiently validated to be used to generate data for risk assessment within the regulatory process. Validationwill depend upon the application of the test system and there are two strategically distinct roles for TGM assays. Firstly, and this has already been acknowledged by ICH, is the investigation of chemicals which produce unexpected results in rodent cancer studies, to identify genotoxic and nongenotoxic carcinogens. Each study must be designed specifically to address the profile of the individual chemical. Confidence in the outcome could be increased by testing structurally or pharmacologically similar mutagenic and non-mutagenic chemicals at the same time. This provides additional, and very relevant, validation at the time of the test. Indeed, we are already using TGM assays for regulatory purposes in the same way as we would use data from any other non-standard test. TGM also offers gene mutation to complement unscheduled DNA synthesis (UDS) and cytogenetic endpoints which are already well established in vivo. Clearly, the potential to look at an endpoint more relevant to cancer, in any tissue, makes these systems unique. Presently, there are carcinogenic chemicals which are potent in vitro mutagens but which are ineffectively detected by UDS and micronucleus tests. Site-of-contact mutagens such as MNNG are excellent examples. Several clearly induce mutations in TGM models at the site of exposure and in the locations predicted from cancer studies. We must ensure thaI the target tissues (e.g.• skm, stomach, lungs) are validated individually and that the protocol optimises detection in these tissues. In this way, we can provide a more relevant endpoint and tissue alternative for chemicals that we know or suspect do not reach the bone marrow or liver. Keyword(s): Transgenic; Mutagenesis; in vivo