Effects of mannitol or catalase on the generation of reactive oxygen species leading to DNA damage by Chromium(VI) reduction with ascorbate.

ABSTRACT

Interaction of Cr(VI) and ascorbate in vitro generates Cr(V), Cr(IV), Cr(III), carbon-based alkyl radicals, COO(*)(-), (*)OH, and ascorbate radicals and induces DNA interstrand cross-links at guanines. To determine which specific Cr species and free radicals cause DNA damage, we investigated the effects of mannitol and catalase on the formation of reactive intermediates, Cr-DNA associations, DNA polymerase-stop sites, and 8-hydroxydeoxyguanosine (8-OHdG) adducts induced by Cr(VI)/ascorbate in a Hepes buffer. EPR spectra showed that mannitol trapped reactive Cr(V), forming a stable Cr(V)-diol complex, and inhibited the radicals induced by Cr(VI)/ascorbate, whereas catalase or heat-denatured catalase enhanced the levels of Cr(V) without altering the radical signals. Mannitol markedly inhibited the retarded gel electrophoretic mobility of supercoiled plasmids and the formation of DNA polymerase-stop sites induced by Cr(VI)/ascorbate, but catalase did not. On the other hand, mannitol reduced only 32% of the Cr-DNA adducts induced by Cr(VI)/ascorbate, suggesting that Cr monoadducts (possibly DNA-Cr-mannitol adducts) are the major lesions generated in the Cr(VI)/ascorbate/mannitol/DNA solution. Native catalase but not heat-denatured catalase protected approximately 25% of the Cr-DNA adducts induced by Cr(VI)/ascorbate, suggesting that hydrogen peroxide may be involved. Mannitol could not completely inhibit the formation of 8-OHdG adducts induced by Cr(VI)/ascorbate, indicating that this DNA damage may be generated before the action of mannitol to trap Cr(V) and reactive oxygen species. Alternatively, Cr-peroxide intermediates may also lead to 8-OHdG formation to account for the incomplete prevention by mannitol. Catalase or heat-denatured catalase partially protected the formation of 8-OHdG adducts induced by Cr(VI)/ascorbate, suggesting an effect of proteins. Together, the results from this study suggest that the primary species generated during the reduction of Cr(VI) by ascorbate are hydroxyl radicals and Cr(V) species, responsible for the formation of 8-OHdG and DNA cross-links, respectively.