Reduction with glutathione is a weakly mutagenic pathway in chromium(VI) metabolism

Although reductive metabolism of Cr(VI) always results in the production of Cr(III) and extensive Cr-DNA binding, cellular studies have indicated that different reduction processes are not equivalent in the induction of mutagenic events. Here, we examined mutagenicity and formation of Cr-DNA damage by Cr(VI) activated in vitro by one of its important reducers, glutathione (GSH). Our main focus was on reactions containing 2 mM GSH, corresponding to its average concentration in CHO (1.8 mM) and V79 (2.6 mM) mutagenicity models. We found that Cr(VI) reduction by 2 mM GSH produced only weak mutagenic responses in pSP189 plasmids replicated in human fibroblasts. Reductive activation of Cr(VI) with 5 mM GSH resulted in approximately 4-times greater DNA adduct-normalized yield of mutations. Mutagenic DNA damage formed in GSH-chromate reactions was caused by nonoxidative mechanisms, as blocking of Cr-DNA adduction led to a complete loss of mutagenesis. All GSH-mediated reactions also lacked significant DNA single-strand breakage. We developed a sensitive HPLC procedure for the detection of GSH-Cr-DNA cross-links based on the dissociation of DNA-conjugated GSH by Cr(III) chelation and its derivatization with monobromobimane. Weak mutagenicity of 2 mM GSH reactions was associated with a low production of mutagenic GSH-Cr-DNA cross-links (5.0% of total Cr-DNA adducts). In agreement with their greater mutation-inducing ability, 5 mM GSH reactions generated 4-5 times higher levels of GSH-DNA cross-linking. Overall, our results indicate that chromate reduction by physiological concentrations of GSH is a weakly mutagenic process, which is consistent with low mutagenicity of Cr(VI) in ascorbate-deficient cells.