The overall objective of this research project is to understand the mechanism by which chromium(VI) compounds act as carcinogens. The three approaches we plan to use in attacking this problem are: (1) mitochondria will be used as a model system for studying the uptake-reduction mechanism proposed for chromium(VI) carcinogenicity since this organelle contains chromium(VI) reductase activity as well as DNA (mt DNA) of defined sequence; (2) the binding of chromium to repetitive DNA sequences of nuclear origin will be determined since nuclear DNA may differ as a target for chromium compounds from mitochondrial DNA; and (3) carcinogenic chromium(VI) compounds will be compared with noncarcinogenic chromium(III) compounds with respect to chromium binding to mt DNA and repetitive DNA in order to distinguish interactions related to the biological activity of the chromium compounds. We plan to test the hypothesis that reductive activation of chromium(VI) results in specific chromium-DNA adducts at defined DNA sequences which are preferentially attacked because of their DNA structure.
The specific aims of the proposed research are: (1) Compare chromium(VI) and chromium(III) for their ability to form chromium complexes with rat mitochondrial DNA and repetitive DNA in vivo and in tro. The formation and repair of chromium-DNA adducts in mt DNA and repetitive DNA fragments will be determined after treatment with 51Cr-labeled sodium dichromate and chromium(III) chloride. (2) Determine the sequence specificity of chromium interactions with mitochondrial and repetitive DNA. The sequence specificity and alkaline lability of the chromium-DNA adducts on mt DNA and repetitive DNA fragments will be determined using DNA sequencing techniques. (3) Determine the mechanism of chromium(VI) reduction by mitochondrial NADH-ubiquinone oxidoreductase. Metabolism of chromium(VI) by Complex I and isolated components will be determined. The generation of reactive chromium(V) and free radical species upon reduction of chromium(VI) by these systems will be monitored by EPR spectroscopy. (4) Determine the ability of cis-platinum(II) to bind to rat mitochondrial and repetitive DNA in vivo and in vitro; and (5) Determine the effect of chromium on the replication of mt DNA and on the expression of mt genes.