Activation of chromium(VI) by thiols results in chromium(V) formation, chromium binding to DNA and altered DNA conformation

Abstract

The ability of the thiols glutathione, cysteine, beta-mercaptoethanol and dithiothreitol to effect chromium(VI)-induced DNA damage in vitro has been investigated. Reaction of pBR322 DNA with chromium(VI) in the presence of the thiols led to formation of chromium(V) and chromium--DNA adducts. The extent of chromium binding to DNA differed by several orders of magnitude among the thiols tested, in the order dithiothreitol greater than beta-mercaptoethanol much greater than cysteine greater than or equal to glutathione. The maximal level of chromium(V) formed also differed among the thiols tested, in the order beta-mercaptoethanol greater than dithiothreitol much greater than glutathione greater than or equal to cysteine. Electronic spectral studies of these reactions indicated that the rate of reduction of chromium(VI) is dependent on the thiol tested, in the order cysteine greater than dithiothreitol greater than glutathione greater than beta-mercaptoethanol. Electron paramagnetic resonance studies of these reactions indicate that a significant level of chromium(III) is detected only with cysteine. Chromium--DNA adducts formed by reaction of chromium(VI) in the presence of glutathione or cysteine did not lead to DNA conformational changes detectable upon agarose gel electrophoretic analysis. Changes in DNA conformation were detected as altered electrophoretic mobility of pBR322 DNA on agarose gels after reaction with chromium(VI) in the presence of dithiothreitol or beta-mercaptoethanol. Effects on DNA electrophoretic mobility, which depended on whether the initial conformation of the plasmid was linear or supercoiled, included altered and heterogeneous mobility, as well as complete inhibition of migration of the plasmid. Transmission electron microscopy of chromium--DNA complexes revealed aggregates of several plasmids, as well as condensation of individual plasmids into compact kinked forms. These effects may be due to cross-linking of DNA induced by chromium metabolites. These studies indicate that the levels of chromium bound to DNA are related to the levels and stabilities of the chromium(V) species formed upon reaction of chromium(VI) with the various thiols. Chromium--thiol interactions may play an important role in chromium(VI) genotoxicity.