TPA is widely used as a tumor promoting agent, which causes DNA strand breaks and chromosomal aberrations through indirect action mediated by reactive oxygen intermediates (ROI). Although it is considered non- or poorly mutagenic, TPA induced mutations in a cell line with which we have analyzed mutations induced by NiC12, Na2Cr04, CdC12, and NMU. This cell line, 6m2 cells, is a normal rat kidney fibroblast cell line infected with MuSVts110, a mutant of MuSV which confers conditional expression of V-mos and thus temperature-dependent transformation on the cells. CdC12, which is mutagenic in 6m2 cells, has also been reported to damage DNA through the mediation of ROIs. The viral proteins from cadmium-induced revertants (reversion to the MuSV phenotype) have been examined and in one of these revertant lines, evidence for a deletion of approximately 50 nucleotides has been found. Since both TPA and CdC12 believe to act through ROIs, and since these are mutagenic in 6m2 cells, it appears that 6m2 cells provide a system with which to study oxidative DNA damage. With this system, the molecular nature of mutations can be studied at the protein level or at the DNA sequence level. The proposed research is designed 1) to determine the nature of the mutations generated by CdC12 and TPA, compounds which are thought to act through ROIs, 2) to determine whether induction of these mutations involve ROIs, and 3) to ascertain whether there are detectable differences between mutations induced by gamma-irradiation, which acts primarily via hydroxyl radicals, and TPA, CdC12, or superoxide anion generated by the xanthine oxidase system
