Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.

Exposure of cells to carcinogenic Cr(VI) compounds results in the formation of several types of DNA lesions such as strand breaks, DNA-protein crosslinks and uncharacterized DNA-Cr adducts. Hexavalent chromium compounds are positive in most bacterial and eukaryotic mutagenic systems, although the nature of DNA modifications underlying the chromium-induced mutagenesis is not known. Hexavalent chromate(VI) is very active in cellular systems because it is actively transported into cells, but intracellularly it is ultimately reduced to Cr(III). Here we show that exposure of Chinese hamster ovary (CHO) cells to potassium chromate(VI) leads to the formation of stable complexes between DNA and amino acids or glutathione. Cysteine, glutamic acid and histidine were the major amino acids crosslinked to DNA in chromate-treated cells. Incubation of purified DNA in the presence of EDTA dissociated SDS stable amino acid-DNA complexes, which indicates that these DNA adducts are most likely to represent ternary coordination complexes mediated by Cr(III) rather than covalent linkage between amino acids/glutathione and DNA. The amino acids that were found complexed with DNA purified from chromate-exposed cells did not orginate from previously crosslinked proteins during DNA isolation, but represented authentic reactions of free amino acids and glutathione with chromium and DNA in cells. Ternary complexes of glutathione or amino acids with Cr(III) and DNA were estimated to account for as much as 50% of DNA-bound chromium following exposure to < or = 25 microM chromate.

No abstract