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, β-mercaptoethanol and dithiothreitol to effect chromluin(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 chromlum(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 > β-mercaptoethanol ≫ cysteine ≥ glutathione. The maximal level of chromlum(V) formed also differed among the thiols tested, in the order β-mercaptoethanol > dithlothreitol ≫ glutathione ≥ cysteine. Electronic spectral studies of these reactions indicated that the rate of reduction of chromiuni(VI) is dependent on the thiol tested, in the order cysteine > dithiothreitol > glutat hione > β-mercaptoethanol. Electron paramagnetic resonance studies of these reactions indicate that a significant level of chromium(III) is detected only with cystelne. 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 mobifity of pBR322 DNA on agarose gels after reaction with chromiuin(VI) in the presence of dithiothreitol or β mercaptoethanol. Effects on DNA electrophoretic mobility, which depended on whether the initial conformation of the plasmid was linear or supercoiled, induded 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 fonns. These effects may be due to crosslinking 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 chromiwn(VI) with the various thiols. Chromiuin-thiol interactions may play an important role In ch omium(VI) genotoxicity.