OXIDATIVE MECHANISMS IN CHROMIUM CARCINOGENESIS

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Chromium(VI) compounds pose a serious health risk to occupationally and environmentally exposed human populations. Exposure to Cr(VI) produces lung carcinomas in humans and laboratory animals. The overall objective of this research project is to elucidate the mechanism by which chromium(VI) compounds act as carcinogens. The hypotheses to be tested in this research project are: (1) that high valent +5 and +4 oxidation states of chromium are the primary intermediates that lead to oxidative DNA damage via direct DNA-metal interactions; (2) that reduction of Cr(VI) by intracellularly important reductants such as glutathione, ascorbate and cysteine form ligand-based radicals leading to oxidative DNA lesions but are of a lesser significance than oxidation by high valent chromium; (3) that these oxidative lesions are manifested in repair-deficient prokaryotic cell systems which are selectively sensitive to the DNA lesions detected in the in vitro studies. The specific aims of the proposed research are: (1) The mechanism of direct- or metal-centered oxidation of DNA by high valent chromium will be measured using model high valent Cr(V) compounds. Oxidation products arising from H-atom abstraction at the C1', C3', C4' and C5' of deoxyribose will be determined by HPLC and GC/MS using the model dinucleotide sugar oxidation substrate, 5',3'-di-O-Acetyl- d(TpT). Formation of guanine and cytosine base oxidation products will be determined using model dinucleotide substrates of d(GpG) and d(CpC). Base- and sequence-specificity of reactions with oligonucleotides will be determined by gel electrophoresis for formation of frank strand breaks and alkali-labile sites. The effect of aerobic vs anaerobic atmospheres will be determined on the above reactions. (2) The role of ligand-based radicals of glutathione, ascorbate and cysteine in the formation of DNA oxidation products will be probed by the specific (non-chromium) generation of these radical species and through their in situ formation by reduction with Cr(VI). The formation and fate of the radicals will be monitored by EPR. Measurement of sugar and base oxidation products as well as the formation of frank strand breaks and alkali-labile sites will be carried out as described in specific aim 1. (3) Selective lethality of Cr(VI) in DNA repair-deficient strains of E. coli will be determined. The synergistic effects of added ascorbate or modulation of intracellular glutathione levels will be determined. Transformation of a plasmid into the sensitive E. coli strains will be carried out for later extraction and measurement of base and sugar oxidation products and mutations. The proposed studies should give insight into the mechanisms of chromium(Vl)-induced DNA damage critical to the formation of cancer. Understanding these mechanisms may allow reduction of risk to exposed human populations.