Certain chromium compounds are environmentally and occupationally important human respiratory carcinogens that are mutagenic and carcinogenic at exposure levels which induce some growth arrest and apoptotic cell death. A growing body of evidence indicates that interference with DNA replication is not only a proximal stimulus for carcinogen-induced terminal growth arrest and apoptotic cell death, but may also instigate genomic changes which contribute to the early stages of neoplasia in the progeny of cells which have escaped cell death, We made the initial discovery that chromium-induced DNA-DNA interstrand crosslinks (Cr-DDC) are present in chromate-exposed cells and cause guanine-specific polymerase arrest (PAL) in in vitro replication systems. In the last grant period we made significant progress in studying the formation of this lesion and some of its biological consequences. Based on those studies we have formulated the following over-riding hypothesis: Cr-DDC are polymerase-stalling lesions that activate DNA repair pathways, which generate double-strand break (DSB) intermediates and induce key cellular survival pathways including recombination. Thus, the major objective of this research is to investigate the formation, repair, and biological and molecular consequences of chromium-induced polymerase arresting lesions, in yeast and human cellular/genetic paradigms that allow us to understand the contribution of these lesions to cellular stress and survival responses, The aims for the next period are (i) to conduct further biochemical analysis of Cr-induced polymerase arresting lesions, and to test the hypothesis that direct Cr-DNA binding and/or base oxidation is critical or sufficient for the formation of this base-specific lesion, (ii) to test the hypothesis that Cr-DDC (polymerase arresting lesions) and/or repair intermediates have a critical role in cellular toxicity and/or triggering survival responses. (iii) to mechanistically apply Differential Expression Profiling (DNA MicroArray Gene Chip technology) to decipher survival and repair pathways that are activated when key DNA repair genes are missing, and (iv) to test the hypothesis that recombination is activated by Cr-DDC/PAL (in both cellular and in vitro recombination systems), and is critical to the survival of genetically damaged cells. This research will help elucidate the molecular mechanisms of chromium toxicity and carcinogenesis and will have practical value in contributing to the evaluation of risk to humans in contact with chromates. ? ? ?
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