Role of molecular oxygen in the generation of hydroxyl and superoxide anion radicals during enzymatic Cr(VI) reduction and its implication to Cr(VI)-induced carcinogenesis

Electron spin resonance (ESR) spin trapping measurements provide evidence for the generation of hydroxyl radicals (^·OH) in the reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH as a cofactor. Catalase inhibited the ^·OH generation, while the addition of H₂O₂ enhanced it, indicating that the ^·OH radical generation involves a Fenton-like reaction. The metal chelator, deferoxamine, inhibited the ^·OH generation with a concomitant generation of a deferoxamine nitroxide radical. EDTA and 1,10-phenanthroline also inhibited the ^·OH generation. Experiments performed under argon atmosphere decreased the yield of the ^·OH formation, showing that molecular oxygen plays a critical role. ESR spin trapping and measurements of fluorescence change of scopoletin in the presence of horseradish peroxidase show that reduction of Cr(VI) by GSSG-R/NADPH generates superoxide anion radicals (O₂^·-) as well as H₂O₂. It can be concluded that ^·OH radical is generated by the reaction of H₂O₂ with Cr(V), which is produced by enzymatic one-electron reduction of Cr(VI). H₂O₂ is produced by the reduction of molecular oxygen via O₂^·- as an intermediate. The ^·OH radicals generated by these reactions are capable of causing DNA strand breaks, which can be inhibited by catalase, formate, and experiments performed under argon.