Induction of activating mutations in the human c-Ha-ras-1 proto-oncogene by oxygen free radicals.

The mutagenicity of oxygen free radicals was studied in a forward mutation system. pEC plasmid containing the human c-Ha-ras-1 proto-oncogene was reacted with oxygen free radicals generated by Cu2+ and H2O2 and was then transfected into NIH/3T3 cells. Transformed foci were observed with oxygen free radical-modified DNA but not with unmodified DNA. The mutations responsible for the Ha-ras-1 gene activation in 11 transformed foci were characterized. G-->T mutations at the second base of codon 12 were found in two transformed foci, A-->T transversions at the second base of codon 61 in five foci, and G-->T mutations at the third position of codon 61 in four transformed foci. These observed mutations are identical to those commonly found in human skin carcinomas, suggesting that reactive oxygen species may play an important role in the carcinogenesis of these tumors. Interestingly, a significant proportion of mutations was found at the second and third base of codon 61 (CAG). In a previous study, the same oxygen free radical-generating system was found to cause an intrastrand cross-link between adjacent purine nucleotides at AG sites in DNA (Carmichael et al., Carcinogenesis 13:1127-1135, 1992). These data demonstrate that oxygen radicals can induce DNA damage that can result in a specific activation of a human proto-oncogene.