Hexavalent chromium is a well established human carcinogen and is a contaminant at numerous Superfund toxic waste dump sites. Carcinogenesis by hexavalent chromium is thought to involve the formation of Cr-DNA adducts. About 50% of the chromium is bound to DNA as ternary complexes of Cr (III) with either amino acids or glutathione. Additionally, hexavalent chromium has been shown to interact with DNA in a non-random fashion targeting itself to promoters and coding regions of highly inducible genes. Previous studies in humans have suggested that there is a large intra-individual variability in the uptake of chromate. Using he bacterial repair enzymes UvrABC and the technique of ligation-mediated PCR, we will study whether the UvrABC bacterial enzymes can detect and differentiate Cr-DNA complexes including Cr(III)-DNA binary, as well as (Cr(III)-DNA ternary complexes involving either cysteine, histidine, or glutathione within exon 7 of the p53 gene. We will also investigate the effect of cytosine methylation on the formation of adducts in human lymphocytes following exposure to hexavalent chromium within other DNA regions that we can analyze for hotspots of Cr-DNA adducts. Bacterial repair enzymes will cut at Cr adducts, and Southern blotting will measure the hybridization signal in the cut region compared to the uncut DNA using probes, such as the ornithine decarboxylase (ODC) promoter and the p53 coding regions. We will examine hexavalent chromium uptake in 60 human volunteers and identify those with the highest uptake capacity and highest intracellular levels of chromium and high chromate uptake will be utilized to map the Cr-DNA adducts at a single nucleotide level in p53 exon 5 and 7, as well as other DNA regions. These studies attempt to develop biomarkers of exposure and effect of carcinogenic hexavalent chromium. We consider important parameters of genetic diversity including epigenetic differences in DNA methylation and the interindividual diversity of chromatic uptake into cells. These studies should lead to new methodologies for assessing the impact of environmental exposure of hexavalent chromium to the human population and identify factors of susceptibility that enhance Cr toxicity.
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