Role of chromium(IV) in the chromium(VI)-related free radical formation, dG hydroxylation, and DNA damage

A reactive Cr(IV) ester was synthesized from a reaction of CrO3 with 2,4-dimethyl-2,4-pentanediol as verified by electron spin resonance (ESR) and magnetic susceptibility measurements. ESR spin trapping studies demonstrate that the Cr(IV) ester is capable of generating hydroxyl free radical ((OH)-O-.) through a Fenton-like mechanism (Cr(IV) + H2O2 --> Cr(V)+ (OH)-O-. + OH-) with a concomitant generation of Cr(V) species (g = 1.9787). Cr(IV) caused DNA strand breaks as measured by electrophoretic assays. H2O2 enhanced the DNA strand breaks via (OH)-O-. formation by a Cr(IV)-mediated Fenton-like reaction. In the Cr(IV)/H2O2 system, formate did not block Cr(V) formation, but prevented DNA damage, indicating that (OH)-O-. radicals, and not Cr(V), caused the DNA damage. Reaction of Cr(VI) with ascorbate was also used as a source of Cr(IV). Incubation of Cr(VI), ascorbate, and DNA caused DNA strand breaks. A free radical trap, 5,5-dimethyl-1-pyrroline (DMPO), only slightly inhibited the DNA damage. Addition of Mn(II), which inhibited Cr(IV), caused significant protection. H2O2 enhanced the DNA damage via Cr(IV)-mediated (OH)-O-. radical generation and Mn(II) inhibited the damage, again showing that Cr(IV) and its related (OH)-O-. generation caused DNA strand breaks. HPLC measurements showed that (OH)-O-. radicals generated by a Cr(IV)-mediated Fenton-like reaction generated 8-hydroxy-2'-deoxyguanosine from 2'-deoxyguanosine. The results demonstrate that Cr(IV) and its generated (OH)-O-. radicals are capable of damaging DNA. Moreover, in comparison with Cr(V), Cr(IV) is a more potent DNA damaging agent.