Mutations Induced in the supF Gene of pSP189 by Hydroxyl Radical and Singlet Oxygen:  Relevance to Peroxynitrite Mutagenesis

Abstract

We previously showed that the oxidant peroxynitrite (ONOO-) was strongly mutagenic in the supF shuttle vector pSP189 replicated in bacteria or human cells. Qualitative characteristics of the mutational spectra induced by ONOO- differed significantly from those reportedly caused by hydroxyl radical (OH.) in other experimental systems but showed similarities to spectra reportedly produced by singlet oxygen (1O2). The molecular mechanisms of ONOO--mediated DNA damage are unknown. The objective of the present set of experiments was to characterize mutational effects induced in the supF gene of pSP189 by OH* and 1O2 to permit direct comparison with mutational spectra induced by ONOO- in this system. Base substitutions were the major form of mutation induced in plasmids replicated in human (AD293) cells by ONOO- (84%) and 1O2 (71%), whereas OH* induced fewer of them (49%). In plasmids replicated in bacteria (Escherichia coli MBL50), frequencies of base substitutions induced by the three treatments were similar. G:C-to-T:A transversions were the most common form of base substitution induced by ONOO- (75% and 67%, respectively, in AD293- and MBL50-replicated plasmids) and 1O2 (68% and 71%); they were induced at lower frequencies by OH. (51% and 47%). G:C-to-C:G transversions or G:C-to-A:T transitions were induced at almost equal frequencies by both ONOO- and 1O2, whereas OH* induced these mutations at different frequencies in the AD293 system. Collectively, our results confirm that in several important respects mutational spectra induced by ONOO- have greater similarity to spectra induced by 1O2 than to those induced by OH* and suggest that genotoxic derivatives of ONOO- are likely to include species that have DNA-damaging properties resembling those of 1O2 in selectivity for guanine but not identical in sequence specificity.

Ion Currents as Initiators of Light-Induced Effects: Studies Using Photosensitizers

“…A variety of reactions can generate 1 O2 in vivo (13) including the reaction of ONOO -with H2O2 (14) , with O2- (15) or with model organic peroxides (16) . In fact, some effects attributed to ONOO -bility supported by the observation that mutations induced by ONOO -differ significantly from those induced by OH · , but show similarities to those reported for 1 O2 (17) .…”

Critical Roles for Polymerase ζ in Cellular Tolerance to Nitric Oxide–Induced DNA Damage

“…Pol~-dependent TLS past apurinic/apyrimidinic sites likely results in extensive accumulation of point mutations. Indeed, G:C to T:A transversion is the most common form of base substitution induced by peroxynitrite (50) and most frequently seen in p53 gene from liver and lung cancers (51). Future biochemical study on Polm ay allow for evaluation of the contribution of Pol~to each type of single base substitutions.…”

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution

“…It is not yet known whether the mechanism of decomposition of ONOO Ϫ to NO Ϫ and 1 O 2 is operative in physiological systems or leads to the nitration of tyrosine or to DNA damage (54). Wogan and coworkers (17) have shown that ONOO Ϫ is mutagenic in the supF shuttle vector pS189 in bacteria or human cells; the mutational spectra induced by peroxynitrite are similar to the mutational patterns induced by 1 O 2 . They suggested that genotoxic derivatives of peroxynitrite are likely to include species that have DNA-damaging properties resembling those of 1 O 2 in selectivity.…”

“…There is evidence that peroxynitrite is formed in significant concentrations in vivo (7)(8)(9) and may contribute to an increased risk for cancer (10), artherosclerosis (11), stroke (12), and other diseases (13). Peroxynitrite is a stable anion in alkaline solution (pKa of 6.8); however, once protonated, it decomposes rapidly with a half-life of less than 1 s at physiological pH at 37°C (14), generating reactive species that readily react with biomolecules such as lipids (15), amino acids (16), and DNA (17). Central to the question of the biochemistry of peroxynitrite is the mechanism of decomposition and the identity of the reactive species, a subject of intense research and controversy (18)(19)(20)(21).…”