Cr(IV) causes activation of nuclear transcription factor-κB, DNA strand breaks and dG hydroxylation via free radical reactions

Abstract

Electrophoretic mobility shift, DNA strand breakage assays and electron spin resonance (ESR) spin trapping were used to investigate the activation of nuclear transcription factor (NF)- κB, DNA strand breakage and 2′-deoxyguanosine hydroxylation induced by Cr(IV), as well the role of free radical reactions in these processes. Incubation of synthesized Cr(IV)-glutathione complex with cultured Jurkat cells resulted in activation of DNA binding activity of NF- κB. Cr(VI) is also able to induce NF- κB activation through Cr(V) and Cr(IV) intermediates generated during the reduction of Cr(VI) by the cells. Cr(III) did not cause observable NF- κB activation due to its inability to cross cell membranes. Cr(IV)-induced NF- κB activation is dose-dependent. Catalase inhibited the activation while superoxide dismutase enhanced it. The metal chelator, deferoxamine, and hydroxyl (OH) radical scavengers, sodium formate and aspirin, also inhibited the NF- κB activation. Electrophoretic assays using λ Hind III linear DNA showed that, in the presence of H 2O 2, Cr(IV) is capable of causing DNA strand breaks. Deferoxamine, sodium formate and aspirin inhibited the DNA strand breaks. HPLC measurements also show that OH radical generated by the Cr(IV)-mediated reaction with H 2O 2 was capable of causing 2′-deoxyguanosine (dG) hydroxylation to generate 8-hydroxyguanosine (8-OHdG). The relative magnitude of 8-OHdG formation correlated with the generation of OH radicals. ESR spin trapping measurements showed that reaction of Cr(IV) with H 2O 2 generated OH radicals, which were inhibited by deferoxamine, sodium formate and aspirin. The results show that Cr(IV) can cause NF- κB activation, DNA strand breaks and dG hydroxylation through OH radical-initiated reactions. This reactive chromium intermediate may play an important role in the mechanism of Cr(VI)-induced carcinogenesis. The results also suggest that the Cr(IV)-glutathione complex may be used as a model compound to study the role of Cr(IV) in Cr(VI) carcinogenicity.