Role of G→T Transversions in the Mutagenicity of Alkylperoxyl Radicals:  Induction of Alkali-Labile Sites in Bacteriophage M13mp19

The mutagenicity of peroxyl radicals, ubiquitous products of lipid peroxidation, was assessed using an in vitro M13 forward mutational assay. Single-stranded M13mp19 plasmids were incubated with a range of concentrations of the azo initiator 2,2‘-azobis(2-amidinopropane) hydrochloride, and then transfected into competent, SOS-induced Escherichia coli JM105 cells. Incubation with peroxyl radicals produced a concentration-dependent decrease in phage survival, with a 500 μM concentration of the azo initiator reducing the transfection efficiency by more than 90% while inducing a corresponding 6-fold increase in lacZ α mutation frequencies. Peroxyl radical-induced mutagenesis was completely prevented by the peroxyl radical scavenger Trolox. Automated DNA sequence analysis of the lacZ α gene of 100 peroxyl radical-induced mutants revealed that the most frequent sequence changes were base pair substitutions (92/95), with G→T transversions predominating (73/92). Alkaline treatment prior to transfection diminished the mutagenicity of damaged plasmids to a level resembling that of unmodified DNA. While abasic sites might account for the sensitivity to alkaline cleavage, the possibility that unidentified nonabasic alkaline-labile lesions also contribute to peroxyl radical mutagenesis cannot be excluded. Collectively, these findings raise the possibility that DNA damage caused by a major class of endogenous radicals contributes to one of the most common spontaneous mutational events, the G→T transversion.