title: Undetectable role of oxidative DNA damage in cell cycle, cytotoxic and clastogenic effects of Cr(VI) in human lung cells with restored ascorbate levels.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
authors.forename: Mindy
authors.initials: M
authors.lastname: Reynolds
authors.forename: Susan
authors.initials: S
authors.lastname: Armknecht
authors.forename: Tatiana
authors.initials: T
authors.lastname: Johnston
authors.forename: Anatoly
authors.initials: A
authors.lastname: Zhitkovich
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology*; D015153:Blotting, Western; D002274:Carcinogens, Environmental / Q000009:adverse effects*; D002453:Cell Cycle / Q000187:drug effects*; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002857:Chromium / Q000009:adverse effects*; D006224:Cricetinae; D003412:Cricetulus; D018736:DNA Adducts / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects*; D015162:Micronucleus Tests; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D000975:Antioxidants; D002274:Carcinogens, Environmental; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D002857:Chromium; C074702:chromium hexavalent ion; D006147:Guanine; C008449:O-(6)-methylguanine; D005978:Glutathione; D001205:Ascorbic Acid