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

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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.

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