Mechanism of DNA-protein cross-linking by chromium

Hexavalent chromium is a known inducer of DNA-protein cross-links (DPCs) that contribute to repression of inducible genes and genotoxicity of this metal. Lymphocytic DPCs have also shown potential utility as biomarkers of human exposure to Cr(VI). Here, we examined the mechanism of DPC formation by Cr(VI) and the impact of its main cellular reducers. In vitro reactions of Cr(VI) with one-electron reducing thiols (glutathione and cysteine) or two-electron donating ascorbate were all efficient at DPC production, indicating a dispensable role of Cr(V). No Cr(VI) reducer was able to generate DPC in the presence of Cr(III)-chelating EDTA or phosphate. A critical role of Cr(III) in DNA-protein linkages was further confirmed by dissociation of Cr(VI)-induced DPC by phosphate. EDTA was very inefficient in DPC dissociation, indicating its poor suitability for testing of Cr(III)-mediated bridging and reversal of complex DPC. Reactions containing only one Cr-modified component (protein or DNA) showed that Cr(III)-DNA adduction was the initial step in DPC formation. Cross-linking proceeded slowly after the rapid formation of Cr-DNA adducts, indicating that protein conjugation was the rate-limiting step in DPC generation. Experiments with depletion of glutathione and restoration of ascorbate levels in human lung A549 cells showed that high cellular reducing capacity promotes DPC yield. Overall, our data provide evidence for a three-step cross-linking mechanism involving (i) reduction of Cr(VI) to Cr(III), (ii) Cr(III)-DNA binding, and (iii) protein capture by DNA-bound Cr(III) generating protein-Cr(III)-DNA cross-links.