Reduction of hexavalent chromium by human cytochrome b5:: Generation of hydroxyl radical and superoxide

The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b(5) and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure 53 Cr(VI). When 5-diethoxyphosphoryl-5-methyl-l-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO center dot) adduct was observed as well as a smaller signal for the superoxide (O-2(center dot-)) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 mu M). NADPH, Cr(VI), O-2, and the PLs were all required for significant HO center dot generation. Superoxide dismutase eliminated the O-2(center dot-) adduct and resulted in a 30% increase in the HO center dot adduct. Catalase largely diminished the HO center dot adduct signal, indicating its dependence on H2O2. Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H2O2 was not needed, indicating that it was generated by the PLs. Adding exogenous H2O2, however, did increase the amount of DEPMPO/HO center dot adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethyl sulfoxide (DMSO) plus the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO center dot. Quantification of the various species over time was consistent with a stoichiometric excess of HO center dot relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b5 in the generation of HO center dot. Overall, the simultaneous generation of Cr(V) and H2O2 by the PLs and the resulting generation of HO center dot at low Cr(VI) concentrations could have important implications for Cr(VI) toxicity. (c) 2006 Elsevier Inc. All rights reserved.