The goal of this project is to characterize the potential mutagenic consequences of DNA damage induced by NO, with particular attention to how specific types of damage may contribute to tumor initiation and development. The ability of macrophages and neutrophils to overproduce NO and reactive oxygen species is well established, as is the ability of these reactive species to induce lethal and mutagenic DNA damage. The chemistry of cellular damage induced by NO, although complex, may result from only two fundamental processes, reaction with oxygen to form nitrosating species and reaction with superoxide to form peroxynitrite (ONOO-). This project is designed to test this hypothesis through characterization of the mutagenic consequences of DNA damage produced by NO and ONOO- in chemically defined systems, as well as by activated phagocytes. Our first specific aim will be to determine the effects of CO2 and hydroxyl radical damage on mutagenicity and mutational spectrum induced by ONOO- in the supF gene of pSP189 replicated in human cells; it recently has been established by others that CO2 at mM concentration decreases the lifetime of peroxynitrite in a manner that may greatly alter its DNA damaging properties. Second, we shall characterize mutations in target genes within cells co-cultivated in vitro with mouse RAW264.7 macrophages activated to produce NO and reactive oxygen species. Our third line of investigation will probe mutagenesis in bacterial transgenes associated with NO overproduction in genetically altered SJL mice, a system we have developed for the study of nitric oxide toxicology in vivo. In our last specific aim, we propose to identify the DNA lesion(s) giving rise to the mutations observed in NO treated cells. Oligonucleotides will be synthesized containing modified bases formed by NO. These oligonucleotides will be inserted into the genome of an M13 derivative and replicated within E. coli. Finally, the type and amount of genetic change induced, if any, will be characterized. in addition, the genetic requirements for mutagenesis by specific lesions will be defined. These data will rank the possible involvement of specific lesions as the precursors to the mutations observed in the experiments described above.
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