MUTATIONS INDUCED IN THE SUPF GENE OF PSP189 BY HYDROXYL RADICAL AND SINGLET OXYGEN - RELEVANCE TO PEROXYNITRITE MUTAGENESIS

We previously showed that the oxidant peroxynitrite (ONOO-) was strong ly 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 b y hydroxyl radical (OH.) in other experimental systems but showed simi larities to spectra reportedly produced by singlet oxygen (O-1(2)). Th e molecular mechanisms of ONOO--mediated DNA damage are unknown. The o bjective of the present set of experiments was to characterize mutatio nal effects induced in the supF gene of pSP189 by OH. and O-1(2) to pe rmit direct comparison with mutational spectra induced by ONOO- in thi s system. Base substitutions were the major form of mutation induced i n plasmids replicated in human (AD293) cells by ONOO- (84%) and O-1(2) (71%), whereas OH. induced fewer of them (49%). In plasmids replicate d in bacteria (Escherichia coli MBL50), frequencies of base substituti ons induced by the three treatments were similar. G:C-to-T:A transvers ions were the most common form of base substitution induced by ONOO- ( 75% and 67%, respectively, in AD293- and MBL50-replicated plasmids) an d O-1(2) (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 O-1(2), whereas OH. induced these mutations at different frequencies in the AD293 sys tem. Collectively, our results confirm that in several important respe cts mutational spectra induced by ONOO- have greater similarity to spe ctra induced by O-1(2) than to those induced by OH. and suggest that g enotoxic derivatives of ONOO- are likely to include species that have DNA-damaging properties resembling those of O-1(2) in selectivity for guanine but not identical in sequence specificity.