title: Copper-dependent site-specific mutagenesis by benzoyl peroxide in the supF gene of the mutation reporter plasmid pS189.
abstract: Benzoyl peroxide (BzPO) enhances tumor promotion and malignant conversion in mouse epidermis. DNA damage may contribute to these processes. BzPO reacts with Cu(I) to produce the benzoyloxyl radical, which in turn causes strand breaks in plasmid DNA. In this study we investigated whether BzPO with or without Cu(I) caused promutagenic DNA damage in the supF gene of the mutation reporter plasmid pS189 replicating in human Ad293 cells. Exposure of pS189 in vitro to BzPO (0.1-1 mM) inhibited plasmid replication; however, addition of Cu(I) (0.1 mM) did not augment BzPO-induced plasmid toxicity. Exposure to BzPO with or without 0.1 mM Cu(I) was also associated with a concentration-dependent increase in mutation frequency, up to > 100-fold above the spontaneous mutation frequency. Supplemental Cu(I) was not required for mutagenesis; however, it both raised the maximal mutation frequency observed and lowered the threshold concentration of BzPO necessary to discern mutagenesis above the spontaneous background. Neither the hydroxyl radical scavengers mannitol or DMSO, the spin trap N-tert-butyl-alpha-phenylnitrone, nor reduced glutathione altered BzPO/Cu(I)-induced mutagenesis; however, mutagenesis was suppressed by the chelator EDTA. Twenty-four of 32 individual BzPO/Cu(I)-induced mutants characterized by sequencing contained point mutations; 22/25 point mutations occurred at G-C base pairs. There were five large deletions and four small deletions. Three additional BzPO-induced mutants contained four point mutations, all occurring at G-C base pairs. Two BzPO/Cu(I)-induced mutational clusters at d(pGGG)-d(pCCC) sites were observed. These data suggest that BzPO may interact with Cu(I) bound to G-C base pairs in DNA to produce site-specific promutagenic DNA damage.
authors.affiliation: Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA 91010.
authors.forename: S A
authors.initials: SA
authors.lastname: Akman
authors.forename: J H
authors.initials: JH
authors.lastname: Doroshow
authors.forename: T W
authors.initials: TW
authors.lastname: Kensler
mesh_terms: D001483:Base Sequence; D001585:Benzoyl Peroxide / Q000633:toxicity*; D002478:Cells, Cultured; D002874:Chromosome Mapping; D003300:Copper / Q000378:metabolism / Q000633:toxicity*; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D016153:Genes, Suppressor / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008353:Mannitol / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000187:drug effects* / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000187:drug effects / Q000235:genetics*
chemical_list: D008353:Mannitol; D003300:Copper; D004247:DNA; D001585:Benzoyl Peroxide; D004121:Dimethyl Sulfoxide