Hydroxyl radical formation and DNA damage mediated by human NADPH-cytochrome P450 reductase

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It is well recognized that reactive oxygen intermediates (ROI), in particular, hydroxyl radicals, can damage DNA and contribute to the carcinogenic process. Cytochrome P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4) is a microsomal flavoprotein present in almost all epithelial cells. Its major function is to transfer electrons to cytochrome P450. In the presence of oxygen and utilizing NADPH as a source of reducing equivalents, an unusual property of the enzyme is its ability to generate ROI in the absence of cytochrome P450 substrates. In the present studies we characterized this process using human recombinant cytochrome P450 reductase. We found that the enzyme readily synthesizes superoxide anion and hydrogen peroxide, as measured by superoxide-mediated oxidation of hydroethidine, and hydrogen peroxide and peroxidase-mediated oxidation of 10-acetyl-3,7-dihydroxyphenoxazine, respectively. These reactions were NADPH-dependent and not altered specifically by various recombinant P450’s and their substrates/inhibitors including zoxazolamine (10-200 μM) and α-naphthoflavone (1 μM), specific for CYP1A2, buprenorphine (3-100 μM) and ketoconazole (1 μM), specific CYP3A4, and chlorzoxazone (10-200 μM), a specific CYP2E1 substrate. Despite formation of superoxide anion and hydrogen peroxide by the enzyme, no hydroxyl radical formation was detected, as measured by oxidation of terephthalate, either in the absence or presence of cytochrome P450 substrates, unless redox active iron (Fe/EDTA complexes) was added to the reaction mix. In the presence of redox active iron, a dose-dependent formation of hydroxyl radical was detected with respect to NADPH and enzyme concentrations. Using plasmid DNA unwinding assays, NADPH-dependent cytochrome P450 reductase mediated formation of hydroxyl radicals, but not superoxide anion or hydrogen peroxide, was found to damage DNA as determined by alterations in migration of DNA in agarose gels. Taken together, our data indicate that cytochrome P450 reductase is able to continually generate ROI in the presence of NADPH. We speculate that regulation of production of DNA damaging hydroxyl radicals will depend on the composition of microsomal constituents including cytochrome P450 reductase, the cellular content of antioxidants, and the accessibility of redox active iron in the microsomal electron transport chain. Supported by NIH grants CA093798, CA100994 and ES05022.