Chemicals are generally assumed to initiate carcinogenesis by binding to cellular DNA, and thereby turning a critical gene(s) on or off. The binding of nitrosamines is thought to be mediated by an unstable Alpha-hydroxynitrosamine metabolite, which rapidly decomposes to the final alkylating intermediate. We propose to study the metabolism of several nitrosamines whose mechanism of action is not understood and for which the alkylation pathway is not documented and perhaps not totally responsible for the genotoxicity of these carcinogenic compounds. We are suggesting that the cytochrome P-450 and NADPH-cytochrome-P-450-reductase dependent denitrosation process, which has been historically considered a detoxification pathway, may play a role in the genotoxicity of nitrosamines. This hypothesis is based on the formation of nitric oxide in the metabolism of nitrosamines. Nitric oxide itself, or by spontaneous in vivo oxidation, may initiate genotoxicity. Using dimethylnitrosamine, N-nitrosopyrrolidine, N-nitrosopiperidine and N-methyl-N-phenylnitrosamine we are proposing to determine the relative significance of denitrosation and Alpha-hydroxylation (DNA alkylation). This will be done by studying their metabolism under a variety of conditions and determining how their genotoxicity is altered as measured by five indicators of toxicity. The five indicators are: a) formation of revertant colonies (Ames assay); b) covalent binding of the nitrosamine to DNA; c) detection of promutagenic lesions; d) deamination of DNA bases; and e) single strand breaks, alkali labile lesions and DNA-cross-linking as measured by alkaline elution or DNA-unwinding/hydroxylapatite chromatography. The Alpha-acetoxy derivatives of the four nitrosamines will be studied using the same indicators in order to assess the contribution of Alpha-oxidation (Alpha-hydroxylation) to the genotoxicity exhibited by the parent nitrosamines. All of the above studies will be done using S. typhimurium TA1535 and/or TA102 as the DNA-containing substrate. Results of these studies will allow us to relate a specific route of metabolism and specific metabolites to particular indicators of genotoxicity. These relationships and correlations should provide insight into the roles of dealkylation and denitrosation in the carcinogenic activation of nitrosamines.
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