Cr(III)-mediated hydroxyl radical generation via Haber-Weiss cycle

Abstract

ESR spin trapping and HPLC were utilized to investigate Cr(III)-mediated hydroxyl radical ( • OH) generation via the following Haber-Weiss reactions in vitro: Cr(III)+ O 2 •−→ Cr(II)+ O 2 Cr(II)+ H 2 O 2→ Cr(III)+ • OH+ OH − Xanthine and xanthine oxidase were used as a source of superoxide (O 2 •−) and H 2O 2. A mixture of xanthine and xanthine oxidase in the presence of the spin trapping agent, 5,5-dimethyl-pyrroline N-oxide (DMPO), generated DMPO/O 2 •−. Addition of Cr(III) to this mixture generated DMPO/ • OH . Catalase partially inhibited DMPO/ • OH formation, while the combination of catalase and superoxide dismutase (SOD) completely blocked the generation of DMPO/ • OH . The reaction of Cr(III) with H 2O 2, itself, also generated DMPO/ • OH . This H 2O 2 enhanced DMPO/ • OH formation was significantly increased in the presence of xanthine, and xanthine oxidase. Metal chelators, deferoxamine, 1,10-deferoxamine and EDTA, decreased Cr(III)-dependent • OH generation. Parallel ESR spin trapping measurements were carried out using Cr(VI). Although Cr(III) generated • OH via a Haber-Weiss cycle, the relative yield of the • OH formation was comparable to that of a Fe(II)-mediated one but lower than that generated by a Cr(VI)-mediated Haber-Weiss cycle. HPLC measurements also show that the • OH radical generated via a Cr(III)-mediated Haber-Weiss reaction was capable of causing 2 ′-deoxyguanosine (dG) hydroxylation to generate 8-hydroxyl-2 ′-deoxyguanosine (8-OHdG). The relative yield of 8-OHdG formation correlated with the generation of • OH as measured by ESR spin trapping. The results suggest that Cr(III)-mediated • OH radical generation may contribute to the mechanism of Cr(III)- and Cr(VI)-induced carcinogenesis.