Mechanism of DNA-protein cross-linking by chromium

Abstract

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.

Figure 1. DPC formation by Cr(III) under different reaction conditions

Standard reaction mixtures contained 5 μg DNA, 60 μg BSA, 25 mM MES (pH 6.0) and indicated Cr(III) concentrations. Data are means ±SD (n=3). Where not seen, error bars were smaller than symbols. (A) Time-course of DNA-BSA crosslinking by Cr(III). (B) Yield of DPC as a function of BSA concentration (reaction time =3 hr). (C) Effect of pH on the formation of DPC (pH 6.0 – 25 mM MES, pH 7.0 – 25 mM MOPS).

Figure 2. Influence of reaction conditions on DPC formation by Cr(VI)

Standard reaction mixtures contained 5 μg DNA, 60 μg BSA, 25 mM MES (pH 6.0) or 25 mM MOPS (pH 7.0), 1 mM Asc and 0 or 100 μM Cr(VI). Data are means ±SD (n=3). Where not seen, error bars were smaller than symbols. (A) Time-course of DNA-BSA crosslinking. (B) DPC formation as a function of BSA concentration (reaction time =3 hr, MES buffer). (C) Importance of the initial reaction period in the DPC production (0 – 180 min, DNA was added before the start of Cr(VI) reduction; 5 – 180 min, DNA was added 5 min after the start of Cr(VI) reduction).

Figure 3. DPC formation by Cr(VI) in the presence of the different reducers and Cr(III) chelators

Standard reaction mixtures in panels A-C contained 5 μg DNA, 60 μg BSA, 25 mM MOPS (pH 7.0) or 25 mM Na-phosphate buffer (pH 7.0), Cr(VI) and indicated reducers. Data are means ±SD (n=3). Where not seen, error bars were smaller than symbols. (A) BSA-DNA crosslinking in reactions containing 1 mM Asc (Pi - phosphate buffer, EDTA – MOPS buffer containing 5 mM EDTA). (B) BSA-DNA crosslinking in the presence of 10 mM GSH or (C) 2 mM Cys. (D) DPC formation in purified A549 nuclei by Cr(VI) activated by a mixture of its main reducers (1 mM Asc, 2 mM GSH and 0.5 mM Cys).

Figure 4. Reversibility of Cr(VI)-induced DPC by Cr(III) chelators

Reaction conditions and definitions were as in Fig. 3. Stability of DPC was assessed during 24 hr incubations with 5 mM EDTA or 50 mM phosphate (pH 7.0) at 37°C. (A) Resistance of DNA-BSA crosslinks formed in Cr(VI)/1mM Asc reactions to dissociation by EDTA. EDTA was added 0.5 (left panel) or 3 hr (right panel) after the start of Cr(VI) reduction. (B) As panel A except that phosphate buffer (Pi) was tested for the DPC reversibility. (C) Reversibility of Cr(VI)-induced DPC in A549 nuclei. DPC were formed by incubating purified nuclei in 25 mM MOPS (pH 7.0) in the presence of 100 μM Cr(VI) and a mixture of its main biological reducers (1 mM Asc, 2 mM GSH and 0.5 mM Cys). Nuclei were processed for DPC measurements immediately after 3 hr initial reactions (−) and following additional 24 hr incubations at 37°C in 50 mM MOPS, pH 7.0 (MOPS) or 50 mM phosphate, pH 7.0 (Pi).

Figure 5. DPC formation using separately produced Cr-DNA and Cr-BSA adducts

DNA and BSA were separately modified in 30-min long reactions with Cr(III) or mixtures of Cr(VI)-1 mM Asc, purified using BioGel P-30 columns and then incubated for 3 hr with the unmodified component to produce DPC. Data are means ± SD (n=3). If not seen, error bars were smaller than symbols. (A) DPC formation using BSA or DNA modified with Cr(III) or (B) Cr(VI). (C) The presence of small ligands has no effect on DPC formation in reactions of BSA with pre-formed Cr-DNA adducts. DNA was modified with 100 μM Cr(VI)/1 mM Asc for 30 min, purified using BioGel P-30 columns and incubated for 3 hr with BSA in the presence of 2 mM Asc, Cys or GSH. (D) Influence of small ligands on DPC production during Cr(VI) reduction with 1 mM Asc. Ctrl – standard reaction, +Asc – additional 2 mM Asc was added, +Cys – 2 mM Cys was added, +GSH – 2 mM GSH was added.

Figure 6. Impact of Asc and GSH on DPC formation in A549 cells

All Cr(VI) exposures were for 3 hr in serum-free medium. Data are means±SD. (A) DPC levels in cells with and without pretreatment with 0.1 mM BSO for 24 hr. DPC were measured either immediately (left panel) or 18 hr after Cr(VI) treatments (right panel). (B) Formation of DPC in control and 1 mM Asc-preloaded cells. (C) Cr accumulation by A549 cells with and without Asc-preloading.