Benzoyl peroxide (BzPO) is both a tumor promoter and progressor in mouse skin; however, BzPO is neither an initiator nor a complete carcinogen in this tissue. Although not mutagenic, BzPO has been observed to produce strand breaks in DNA of exposed cells. These actions are presumed to be mediated by freeradical derivatives of BzPO. Previous studies suggested that the metabolism of BzPO in keratinocytes proceeds via the initial cleavage of the peroxide bond, yielding benzoyloxy radicals which, in turn, can either fragment to form phenyl radicals and carbon dioxide or abstract H atoms from biomolecules to yield benzoic acid. Benzoic acid is the major stable metabolite of BzPO produced by keratinocytes. In the present study we have investigated the role of BzPO and its metabolites in the generation of strand scissions in a cell-free system using ΦX-174 plasmid DNA. In this system BzPO produced DNA damage that was dose-dependent over a concentration range of 0.1–1 mM and required the presence of copper but not other transition metals. By contrast, benzoic acid did not produce DNA damage in this system, either in the presence or in the absence of copper. The inclusion of spin trapping agents, such as N-tert-butyl-α-phenylnitrone (PBN), 3,5-dibromo-4-nitrosobenzenesulfonate, and nitrosobenzene, in incubations was found to significantly reduce the extent of DNA damage generated via the copper-mediated activation of BzPO. Electron paramagnetic resonance spectroscopy studies suggested that the primary radical trapped by PBN following copper mediated decomposition of BzPO was the benzoyloxy radical. By contrast, formation of either phenyl radicals or carbon dioxide was not detected in this system. Compounds that serve as facile H donors, such as glutathione and ergothioneine, were also effective inhibitors of BzPO-mediated DNA strand breakage. BzPO does not appear to readily undergo addition reactions with DNA in that no covalent binding of BzPO to DNA was produced in incubations of radiolabeled BzPO, calf thymus DNA, and Cu+. Collectively, these observations suggest BzPO may be activated to DNA-damaging intermediates via copper-catalyzed cleavage of the peroxide bond, resulting in the formation of the benzoyloxyl radical which may then produce labile sites in DNA through H-abstraction reactions.
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