The overall objective of this research project is to understand the mechanism by which chromium(VI) compounds act as carcinogens. We plan to test the following hypotheses: (1) that metabolic activation of chromium(VI) results in """"""""reactive intermediates"""""""" which give rise to specific DNA lesions; (2) that the specific DNA lesions occur at defined DNA sequences which are preferentially attacked because of their DNA structure; and (3) that the specific chromium(VI)-induced DNA lesions affect the normal template activity of DNA by altering DNA-protein interactions. The following approaches which include both in vitro and in vivo experimental systems will be used in attacking this problem: (1) The chromium(V) and radical intermediates generated during the metabolic activation of chromium(VI) by redox-active cellular components will be determined. EPR spectroscopy will be used to detect chromium(V) species and spin traps will be used to detect singlet oxygen and hydroxyl and thiyl radical species formed upon reaction of chromium(VI) be pretreated with xenobiotics which specifically affect the levels of the various cellular redox components and change in the production of the chromium(V) and radical species in various tissues will be determined. (2) Chromium (VI)-induced DNA lesions, in the form of DNA strand breaks, DNA cross-links, chromium-DNA adducts and radical-DNA adducts, which result from the attack f """"""""active intermediates"""""""" formed during the metabolism of chromium(VI) will be determined in plasmid DNA and restriction fragments in vitro and in mitochondrial and nuclear DNA in vivo. These DNAs differ in size, conformation and associated proteins, and therefore, may differ as targets for chromium(VI)-induced DNA lesions. The sequence/conformation specificity of the chromium-DNA adducts will be examined, and the chromium-DNA adducts will be isolated, characterized and compared with synthetic chromium-nucleotide complexes. Antibodies will be raised to chromium-DNA adducts isolated from the in vitro reactions, and will be used to analyze DNA isolated from tissues of rats and chick embryos treated with chromium(VI) in vivo. (3) The effect of chromium-induced DNA lesions on protein-DNA interactions will be examined. The ability of DNA polymerase to synthesize daughter DNA strands in the presence of chromium-DNA adducts on the DNA template will be determined. The ability of gene regulatory proteins to bind to their specific DNA recognition elements after formation of chromium-DNA adducts within these sequences will also be determined. The interaction of lac repressor CAP and RNA polymerase with a DNA restriction fragment containing the promoter and operator sequences of the E. coli lactose operon, and interaction of glucocorticoid receptor and metal regulatory proteins to a restriction fragment containing the matallothionein promoter will be examined. These studies should elucidate critical cellular pathways which lead to the carcinogenic activity of chromium(VI) compounds.
