Metabolism of Cr(VI) by ascorbate but not glutathione is a low oxidant-generating process

Genotoxic activity of hexavalent chromium (chromate) results from its reductive activation inside the cell. Cr(VI) metabolism in vivo is primarily driven by ascorbate (Asc) but in cultured cells by glutathione (GSH). Given the common use of cultured cells for mechanistic studies, it is important to establish whether Cr(VI) activated by Asc and GSH displays the same genotoxic properties. Using 2',7' dichlorofluorescin (DCFH) as a redox sensitive probe, we found that Asc-dependent reduction of Cr(VI) in vitro under physiological conditions generated 25-80 times lower yields of oxidants compared to GSH. When both reducers were present, Asc dominated Cr(VI) metabolism and inhibited DCFH oxidation. Consistent with the findings in defined chemical reactions, restoration of physiological levels of Asc in human lung H460 cells led to the loss of their hypersensitivity to clonogenic killing by Cr(VI) in the presence of methoxyamine, which inhibits base excision repair of oxidative DNA damage. Despite suppressed oxidative damage, Asc-containing cells formed a large number of DNA double-strand breaks after exposure to a dose of Cr(VI) corresponding to the drinking water standard of 100 ppb. Our results indicate that Asc-driven metabolism of Cr(VI) shifts its genotoxicity toward nonoxidative mechanisms.