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

Abstract

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-Diethoxyphos-phoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO • ) adduct was observed as well as a smaller signal for the superoxide (O 2 •− ) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 μM). NADPH, Cr(VI), O 2 and the PLs were all required for significant HO • generation. Superoxide dismutase eliminated the O 2 • − adduct and resulted in a 30% increase in the HO • adduct. Catalase largely diminished the HO • adduct signal indicating its dependence on H 2 O 2 . 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 H 2 O 2 was not needed, indicating that it was generated by the PLs. Adding exogenous H 2 O 2 , however, did increase the amount of DEPMPO/ HO • adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethylsulfoxide (DMSO) plus the spin trap α-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO • . Quantification of the various species over time was consistent with a stoichiometric excess of HO • relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b 5 in the generation of HO • . Overall, the simultaneous generation of Cr(V) and H 2 O 2 by the PLs and the resulting generation of HO • at low Cr(VI) concentrations could have important implications for Cr(VI) toxicity.

Reductive activation of hexavalent chromium by human lung epithelial cells: Generation of Cr(V) and Cr(V)-thiol species

“…2). The g = 1.979 signal is similar to that for Cr(V) generated in vitro by human microsomes [30] and by proteoliposomes containing purified human P450 reductase and cytochrome b 5 [31,46]. The signal at g = 1.985 suggests a Cr(V) species that is dependent on thiols.…”

“…Consistent with this concept, co-treatment of clonal human bronchial fibroblasts with ascorbate as an extracellular Cr(VI) reductant prevents Cr uptake and clastogenic effects [55]. By decreasing Cr(VI) available for uptake, extracellular Cr(VI) reduction by the medium might similarly diminish Cr-associated ROS generation associated with intracellular reductants such as cytochrome b 5 and P450 reductase [32]. The potential impact of medium-generated extracellular Cr(V) on cells has not been explored, but the transient nature of Cr(V) suggests that it would not accummulate in the medium.…”

“…Human microsomal enzymes have been implicated in Cr(VI) reduction including the generation of Cr(V) [32]. While the expression of several microsomal enzymes typically declines markedly in cell culture, immunofluorescence demonstrated that BEAS-2B cells continue to express the microsomal proteins cytochrome b 5 , b 5 reductase, and P450 reductase (Fig.…”

“…Unlike many other cells, BEAS-2B cells maintain expression of key microsomal enzymes when grown in culture [59,63] including cytochrome b 5 and P450 reductase (Fig. 12) which can generate Cr(V) and hydroxyl radical by Cr-mediated redox cycling [32]. In human lungs, P450 reductase is localized primarily in the bronchial and bronchiolar epithelial cells [64].…”

“…In human systems, lung and liver microsomes generate Cr(V) during Cr(VI) reduction [30]. Purified human microsomal enzymes (P450 reductase plus cytochrome b 5 ) similarly generate Cr(V) [31,32].…”