Cultured human cells are invaluable
biological models for mechanistic studies of genotoxic chemicals and drugs. Continuing replacement of animals in toxicity testing will further increase the importance of in vitro cell systems, which should accurately reproduce key in vivo characteristics of toxicants such as their profiles of metabolites and DNA lesions. In this work, we examined how a common severe deficiency of cultured cells in ascorbate (Asc) impacts the formation of oxidative DNA damage by
hexavalent chromium (
chromate).
Cr(VI) is reductively activated inside the cells by both Asc and small
thiols but with different rates and spectra of intermediates and
DNA adducts. We found that
Cr(VI) exposure of H460 human lung epithelial cells in standard culture (<0.01 mM cellular Asc) induced biologically significant amounts of oxidative DNA damage. Inhibition of oxidative damage repair in these cells by stable XRCC1 knockdown strongly enhanced cytotoxic effects of
Cr(VI) and led to depletion of cells from G(1) and accumulation in S and G(2) phases. However, restoration of physiological levels of Asc (≈ 1 mM) completely eliminated
Cr(VI)
hypersensitivity of XRCC1 knockdown. The induction of
chromosomal breaks assayed by the micronucleus test in Asc-restored H460, primary human lung fibroblasts, and CHO cells was also unaffected by the XRCC1 status. Centromere-negative (clastogenic) micronuclei accounted for 80-90% of all
Cr(VI)-induced micronuclei. Consistent with the micronuclei results, Asc-restored cells also showed no increase in the levels of
poly(ADP-ribose), which is a
biochemical marker of single-stranded breaks. Asc had no effect on cytotoxicity of
O(6)-methylguanine, a lesion produced by direct
DNA alkylation. Overall, our results indicate that the presence of physiological levels of Asc strongly suppresses
pro-oxidant pathways in
Cr(VI) metabolism and that the use of standard cell cultures creates a distorted profile of its genotoxic properties.