Mutations at G : C base pairs predominate after replication of peroxyl radical-damaged pSP189 plasmids in human cells

The mutagenicity of peroxyl radicals, important participants in lipid perox idation cascades, was investigated using a plasmid-based mutational assay s ystem. Double-stranded pSP189 plasmids were incubated with a range of conce ntrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amid inopropane) hydrochloride (AAPH). Following replication in human Ad293 cell s, the plasmids were screened for supF mutations in indicator bacteria. Exp osure to peroxyl radicals caused strand nicking and a decrease in transfect ion efficiency, which was accompanied by a significant increase in supF mut ants. Each of these effects was abolished in the presence of the water-solu ble vitamin E analogue Trolox. Automated sequencing of 76 AAPH-induced muta nt plasmids revealed that substitutions at G:C base pairs were the most com mon changes, accounting for 85.5% of all identified mutations. Of these, mo st comprised G:C-->T:A transversions (53.5%), with lesser contributions by G:C-->A:T transitions (23.9%) and G:C-->C:G transversions (22.5%). Collecti vely, these data confirm our previous findings concerning the spectrum of m utations produced upon bacterial replication of peroxyl radical-damaged pha ge DNA and extend them by showing that such damage has mutagenic consequenc es during replication in more complex eukaryotic systems.