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 peroxidation cascades, was investigated using a plasmid-based mutational assay system. Double-stranded pSP189 plasmids were incubated with a range of concentrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Following replication in human Ad293 cells, the plasmids were screened for supF mutations in indicator bacteria. Exposure to peroxyl radicals caused strand nicking and a decrease in transfection efficiency, which was accompanied by a significant increase in supF mutants. Each of these effects was abolished in the presence of the water-soluble vitamin E analogue Trolox. Automated sequencing of 76 AAPH-induced mutant plasmids revealed that substitutions at G:C base pairs were the most common changes, accounting for 85.5% of all identified mutations. Of these, most 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%). Collectively, these data confirm our previous findings concerning the spectrum of mutations produced upon bacterial replication of peroxyl radical-damaged phage DNA and extend them by showing that such damage has mutagenic consequences during replication in more complex eukaryotic systems.
Burcham
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PC
Department of Clinical and Experimental Pharmacology, University of Adelaide, Australia. pburcham@medicine.adelaide.edu.au
Harkin
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LA
D000578:Amidines / Q000494:pharmacology; D020029:Base Pairing / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D002461:Cell Line, Transformed; D003597:Cytosine Nucleotides / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000187:drug effects; D004622:Embryo, Mammalian; D005609:Free Radicals / Q000494:pharmacology; D016153:Genes, Suppressor / Q000187:drug effects; D006150:Guanine Nucleotides / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D009154:Mutation / Q000187:drug effects* / Q000235:genetics; D016877:Oxidants / Q000494:pharmacology; D010545:Peroxides / Q000494:pharmacology*; D010957:Plasmids / Q000187:drug effects* / Q000235:genetics; D012343:RNA, Transfer / Q000187:drug effects; D014162:Transfection
D000578:Amidines; D003597:Cytosine Nucleotides; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D005609:Free Radicals; D006150:Guanine Nucleotides; D016877:Oxidants; D010545:Peroxides; C096275:supF tRNA; C049375:perhydroxyl radical; C046728:2,2'-azobis(2-amidinopropane); D012343:RNA, Transfer