Induction of 8-hydroxydeoxyguanosine in DNA by chromium(III) plus hydrogen peroxide and its prevention by scavengers

Abstract

The capability of Cr(III) to induce DNA lesions generated by oxidative damage was investigated in this study by examining the formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymus DNA by CrCl₃ and/or H₂O₂ in 10 mM phosphate buffer. In the presence of 0.5 mM H₂O₂, the formation of 8-OHdG markedly increased with increasing CrCl₃ concentration. In contrast, H₂O₂ or CrCl₃ alone did not cause any increase in 8-OHdG level above background. The amount of 8-OHdG induced by CrCl₃ plus H₂O₂ was time dependent; its generation increased linearly over an incubation period of 90 min. The formation of 8-OHdG was unfavorable in an acidic solution (pH < 6); the highest level of 8-OHdG was observed at pH 7–8. Scavengers of reactive oxygen species markedly inhibited the formation of 8-OHdG by CrCl₃ plus H₂O₂; the inhibition effect was sodium azide > D-mannitol > Tris-HCI at an equal concentration. The induction of 8-OHdG by CrCl₃ plus H₂O₂ remained unchanged in D₂O. Moreover, an addition of catalase (2.2 U/ml) to the reaction mixture completely inhibited the formation of 8-OHdG by CrCl₃/H₂O₂, whereas only 22% of that formation was inhibited by superoxide dismutase (11 U/ml). A large amount of bovine serum albumin (1.1 mg/ml) could reduce the formation of 8-OHdG by CrCl₃ plus H₂O₂, thereby implying that Cr(III)-mediated DNA-protein crosslinks are unfavorable for 8-OHdG formation. Furthermore, ascorbate could prevent the formation of 8-OHdG by CrCl₃ plus H₂O₂; the extent of prevention increased with increasing ascorbate concentration (10 μM-3 mM). Thus, ascorbate acts as a free radical scavenger in the CrCl₃/H₂O₂ system. The above findings suggest that Cr(III)/H₂O₂ could generate oxidative damage to DNA, possibly through a Fenton-like reaction, i.e. Cr(III) + H₂O₂ ↑ Cr(IV) + OH + OH⁻. This study also indicates that Cr(III), previously considered as the ultimate kinetically stable species of Cr(VI) metabolites, is capable of inducing carcinogenic lesions through interaction with a cellular oxygen species.