DNA DAMAGE BY BIOACTIVATED XENOBIOTICS

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DESCRIPTION (Adapted from the Investigator's Abstract): Mutagenesis by environmental agents is believed to be an initiating event in carcinogenesis. Many environmental chemicals are metabolically converted into mutagens. Mutagenesis in cultured cells treated with exogenously supplied preformed mutagens has been studied extensively but there is little information available on mutagenesis by mutagens produced within cells at physiologically relevant levels. The intrinsic differences between cells treated with exogenous reactive metabolites and metabolically competent cells treated with metabolic precursors include both the subcellular localization and the rate of exposure to reactive metabolites. Quantitative differences in the macromolecules which are covalently modified by the reactive metabolites cause alterations in cytotoxicity and genotoxicity. The few investigations comparing genotoxicity of reactive metabolites supplied exogenously and produced endogenously demonstrated dramatic differences. Clearly, more work using metabolically competent cells which more closely resemble the in vivo exposure condition is needed. The long term goal of the current proposal is to understand the mechanisms of mutagenesis by metabolically activated environmental agents considered prime candidates for initiation of carcinogenesis in exposed human populations. The working hypothesis that the gene specific distribution of DNA adducts is enhanced in cells metabolically producing mutagens and it is the distribution of adducts that determines the toxicological response. The specific aims are: 1. To determine the aspects of chromatin structure contributing to preferential DNA damage in expressed genes, and 2. to investigate the role of dose rate in mutagenesis and gene specific DNA damage. The proposed studies will use CYP1Al -expressing human cells and benzo[a]pyrene metabolites as a model system. Gene specific DNA damage levels determined by quantitative PCR will be compared between genes and correlated with transcription rates determined by quantitative reverse transcription PCR. Mutagenesis will be followed by HPRT mutant induction and the effect of altering dose rates will be determined. The distribution of DNA damage within the HPRT and p53 genes will be monitored by ligation mediated - PCR (LM-PCR). Mutation spectra will be determined by sequencing RT-PCR produced mutant HPRT cDNAs and correlated with DNA damage spectra. Adducted proteins will be analyzed to determine potential contributions of protein modification to the toxicological response. These studies will provide needed information elucidating the interplay between chromatin structure in active genes and dose rate in determining gene specific DNA damage by a model carcinogen. The data obtained will provide a model on which to base investigations of other bioactivated chemical carcinogens.