Chromium(VI) causes interstrand DNA cross-linking in vitro but shows no hypersensitivity in cross-link repair-deficient human cells

Abstract

Hexavalent chromium is a human carcinogen activated primarily by direct reduction with cellular ascorbate and to a lesser extent, by glutathione. Cr(III), the final product of Cr(VI) reduction, forms six bonds allowing intermolecular cross-linking. In this work, we investigated the ability of Cr(VI) to cause interstrand DNA cross-links (ICLs) whose formation mechanisms and presence in human cells are currently uncertain. We found that in vitro reduction of Cr(VI) with glutathione showed a sublinear production of ICLs, the yield of which was less than 1% of total Cr-DNA adducts at the optimal conditions. Formation of ICLs in fast ascorbate-Cr(VI) reactions occurred during a short reduction interval and displayed a linear dose dependence with the average yield of 1.3% of total adducts. In vitro production of ICLs was strongly suppressed by increasing buffer molarity, indicating inhibitory effects of ligand-Cr(III) binding on the formation of cross-linking species. The presence of ICLs in human cells was assessed from the impact of ICL repair deficiencies on Cr(VI) responses. We found that ascorbate-restored FANCD2-null and isogenic FANCD2-complemented cells showed similar cell cycle inhibition and toxicity by Cr(VI). XPA-null cells are defective in the repair of Cr-DNA monoadducts, but stable knockdowns of ERCC1 or XPF in these cells with extended time for the completion of cross-linking reactions did not produce any sensitization to Cr(VI). Our results together with chemical and steric considerations of Cr(III) reactivity suggest that ICL generation by chromate is probably an in vitro phenomenon occurring at conditions permitting the formation of Cr(III) oligomers.

Figure 1. Formation of ICLs during reduction of Cr(VI) with GSH

Reaction mixtures contained 10 mM GSH, 25 mM MOPS (pH 7.0), 2 μg DNA and indicated concentrations of potassium chromate. All reduction reactions were incubated for 3 hr at 37°C. Data are means+SD (n=3-4). Where not seen, error bars are smaller than data symbols. (A) Reduction of 100 μM Cr(VI) by GSH. (B) Formation of Cr-DNA adducts in Cr(VI)-GSH reactions. (C) Representative radiogram demonstrating the presence of dsDNA in Cr(VI)-treated samples after denaturation with 200 mM NaOH. (D) Dose-dependence of ICL formation by Cr(VI). (E) Yield of ICLs as a percentage of total Cr-DNA adducts.

Figure 2. Formation of ICLs during reduction of Cr(VI) with Asc

Reaction mixtures contained 1 mM Asc, 25 mM MOPS (pH 7.0), 2 μg DNA and indicated concentrations of Cr(VI). All reduction reactions except for data in panel F were incubated for 30 min at 37°C. Data are means±SD (n=3-5). Where not seen, error bars are smaller than data symbols. (A) Reduction of 100 μM Cr(VI) by Asc. (B) Formation of Cr-DNA adducts in Cr(VI)-Asc reactions. (C) Representative radiogram demonstrating the presence of dsDNA in Cr(VI)-treated samples after denaturation with 200 mM NaOH. (D) Dose-dependence of ICL formation by Cr(VI). (E) Yield of ICLs as a percentage of total Cr-DNA adducts. (F) Formation of ICLs during Cr(VI) reduction (0-30 min samples) and after the completion of Cr(VI) reduction (5-30 min samples). All reactions contained 50 μM Cr(VI).

Figure 3. Hypersensitivity of FANCD2-null human fibroblasts to cisplatin and mitomycin C

Isogenic FANCD2-null (FANCD2-/-) and FANCD2-complemented (FANCD2+) cells were treated with mitomycin C and cisplatin for 6 hr in complete media and cytotoxicity measurements were taken 72 hr later. Data are means±SD, n=4, *-p<0.05, **- p<0.01, ***-p<0.001. (A) Cell growth and (B) Viability of mitomycin C (MMC)-treated FANCD2-/- and FANCD2+ fibroblasts. (C) Cell growth and (D) Viability of cisplatin (cisPt)-treated FANCD2-/-and FANCD2+ fibroblasts.

Figure 4. Uptake and cytotoxicity of Cr(VI) in FANCD2-null and FANCD2-complemented human fibroblasts

Data are means±SD (n=3-6). Cells were preincubated with the indicated concentrations of DHA for 90 min prior to the addition of Cr(VI). All treatments with Cr(VI) were 6-hr long. (A) Cellular concentrations of Asc after preincubation with DHA. (B) Cr(VI) uptake by cells preincubated with 0.5 and 1 mM DHA. (C) Cell viability at 72 hr after exposure to Cr(VI). (D) Growth inhibition by Cr(VI). (E) Clonogenic survival of Cr(VI)-treated cells.

Figure 5. Importance of FANCD2 for recovery of cell cycle after exposure to mitomycin C but not Cr(VI)

(A) A flow chart describing an experimental approach for testing the ability of cells to recover from replication arrest and progress into mitosis. (B) A severe defect in the ability of FANCD2-null cells to recover from mitomycin C (MMC)-induced replication arrest. Cells were trapped in mitosis by the addition of 0.1 μg/mL nocodazole for 18 hr after removal of MMC. (C) A normal progression of FANCD2-null cells into mitosis after Cr(VI) exposure. Cells were trapped in mitosis by the addition of 0.1 μg/mL nocodazole for 18 hr after Cr(VI) exposure.

Figure 6. Cytotoxicity of Cr(VI) in cells with shRNA-depleted XPF-ERCC1 endonuclease

(A) Western blots demonstrating XPF and ERCC1 knockdowns in XPA-null human fibroblasts. (B) Cytotoxicity of Cr(VI) in control (shSCR) and XPF/ERCC1-depleted (shXPF/shERCC1) cells. Cells were preloaded with 2.5 mM Asc prior to 6-hr long treatment with Cr(VI) and cell viability was measured at 72 hr post-exposure. Data are means±SD (n=6).

Figure 7. Suppression of ICL formation by increasing buffer molarity

Reaction mixtures contained 10 mM GSH, MOPS buffer (pH 7.0), 2 μg DNA, and 0-200 μM Cr(VI) and were incubated for 3 hr at 37°C. Data are means±SD (n=3, ***-p<0.001). Where not seen, error bars are smaller than data symbols. (A) ICL formation in Cr(VI)-GSH reactions containing 25 or 50 mM MOPS. (B) Reduction of Cr(VI) by 10 mM GSH in the presence of 25 and 50 mM MOPS (pH 7.0).

Figure 8. A flow chart depicting Cr(VI) reactions leading to the formation of DNA monoadducts and DNA interstrand crosslinks

L - any buffer or other ligand capable of moderate or strong binding to Cr(III), Cr(III)_n – polymeric Cr(III) products.