Carbon dioxide modulation of peroxynitrite-induced mutagenesis of the supF gene in pSP189

Peroxynitrite (ONOO(-)), a potent oxidant formed by the reaction of nitric oxide with superoxide anion, may play a significant role as an intermediate in NO(*)-related cytotoxicity and genotoxicity. In addition to causing other types of toxicity, peroxynitrite damages DNA and induces mutations in genetic targets following in vitro or in vivo exposure. It has recently been established that the reaction of CO(2) with ONOO(-) significantly changes its chemistry in biological media. The objective of this investigation was to characterize impacts of CO(2) on peroxynitrite-induced mutagenesis of the supF gene in the shuttle vector pSP189. The dose-response relationship between ONOO(-) concentration and mutation frequency was determined following bolus exposure of the plasmid suspended in 150 mM phosphate buffer at pH 7.4, with and without addition of 25 mM sodium bicarbonate. After treatment, plasmids were replicated in Escherichia coli MBL50 cells for mutant identification. CO(2) significantly reduced the mutagenic potency of peroxynitrite, in that increases in mutant fraction were reduced by 47-77% at ONOO(-) doses ranging from 0 to 4 mM. We also characterized the spectrum of mutations induced under these conditions and found mutational hotspots at positions 110, 113, 116, 141, 156, 168, and 172 in mutants induced in the presence of CO(2) and at positions 113, 124, 126, 141, and 156 in those induced in its absence. Among the 22 guanines present in the 84 nucleotide supF sequence, 18 were sites of mutation, including four mutational hotspots (G113, G116, G141, and G156), in plasmids exposed to ONOO(-) in the presence of bicarbonate. After exposure in the absence of bicarbonate, 14 of the 22 guanines were mutation sites, with hotspots located at five (G113, G124, G126, G141, and G156). Remaining mutations were located almost exclusively at cytosine residues. It is evident from these data that reactive intermediates formed through reaction of ONOO(-) with CO(2) play an important role in the mutagenicity of ONOO(-).