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 CrCl 3 and/or H 2O 2 in 10 mM phosphate buffer. In the presence of 0.5 mM H 2O 2, the formation of 8-OHdG markedly increased with increasing CrCl 3 concentration. In contrast, H 2O 2 or CrCl 3 alone did not cause any increase in 8-OHdG level above background. The amount of 8-OHdG induced by CrCl 3 plus H 2O 2 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 CrCl 3 plus H 2O 2; the inhibition effect was sodium azide > D-mannitol > Tris-HCl at an equal concentration. The induction of 8-0HdG by CrCl 3 plus H 2O 2 remained unchanged in D 2O. Moreover, an addition of catalase (2.2 U/ml) to the reaction mixture completely inhibited the formation of 8-OHdG by CrCl 3/ H 2O 2, 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 CrCl 3 plus H 2O 2, thereby implying that Cr(III)-mediated DNA-protein crosslinks are unfavorable for 8-0HdG formation. Furthermore, ascorbate could prevent the formation of 8-OHdG by CrCl 3 plus H 2O 2; the extent of prevention increased with increasing ascorbate concentration (10 μM-3 mM). Thus, ascorbate acts as a free radical scavenger in the CrCl 3/H 2O 2 system. The above findings suggest that Cr(III)/H 2O 2 could generate oxidative damage to DNA, possibly through a Fenton-like reaction, i.e. Cr(III) + H 2O 2 → 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.