PEROXYNITRITE-INDUCED MUTATION SPECTRA OF PSP189 FOLLOWING REPLICATION IN BACTERIA AND IN HUMAN-CELLS

Peroxynitrite is a powerful oxidant formed through reaction of nitric oxide with superoxide. Because activated macrophages can produce both nitric oxide and superoxide, it has been proposed that peroxynitrite m ay contribute to cytotoxicity and increased cancer risks associated wi th the inflammatory response during chronic infections. We therefore i nvestigated mutagenicity of peroxynitrite in the supF gene of the pSP1 89 shuttle vector as a mutation target. The plasmid was exposed to 2.5 mM peroxynitrite in vitro, then replicated in Escherichia coli MBL50 and in human AD293 cells. Mutation frequency increased 21-fold in pSP1 89 replicated in E. coli and 9-fold in plasmid replicated in human cel ls. Mutations were clustered within the 5' region of the supF gene in plasmids replicated in bacteria. The hot spots were located at positio ns 108, 113, 116, 124, 126 and 141; more than 25% of all mutations occ urred at position 124. Following replication in human cells, mutations were more widely distributed over the gene, with hot spots at positio ns 113, 124, 133, 156 and 164; 15% occurred at position 124. In both s ystems, the majority of mutations occurred at G:C base pairs, predomin antly involving G:C --> T:A transversions (65% when replication was in bacteria and 63% when in human cells). G:C --> C:G transversions were observed at lower frequency (28% in MBL50 and 11% in AD293 cells), an d 11% of mutations found in vectors replicated in AD293 cells were G:C --> A:T transitions. A greater number of large deletions, insertions, tandem and multiple mutations occurred in plasmid replicated in AD293 cells. Differences in mutation spectra following replication in the t wo systems may be attributable to differences in recognition and repai r of the lesions and/or properties of the replication apparatus.