Chromium has been identified as a """"""""primary hazardous substance"""""""" at a large proportion of the Superfund Hazardous Waste Sites. Epidemiological studies have linked the exposure to chromium compounds in the work place to increased risk for development of lung cancer. Furthermore, certain forms of chromium have been shown to be carcinogenic in animals and mutagenic in bacteria and mammalian cell systems. Although the process of carcinogenesis is complex, it is clear that mutagenic activation of proto-oncogenes plays an important role. The purpose of the research proposed here is to understand the mechanisms by which chromium causes mutations in mammalian cells and to provide a link between the mutagenic and carcinogenic activities of chromium. We are using shuttle vectors in cultured mammalian cells and yeast to identify premutagenic DNA damage induced by chromium compounds and to determine the mutagenic specificity of this damage. We are also using a mammalian in vitro DNA replication system to investigate how the cellular DNA replication apparatus responds to chromium-induced template damage and whether DNA replication fidelity is affected by direct effects of chromium on DNA replication factors. In addition, we are seeking a link between chromium mutagenesis and carcinogenesis by using transgenic mice to examine the mutagenic effects of inhaled calcium chromate dust in the lung - the target organ for chromium carcinogenesis. The focus of this work will be to understand the role that biotransformation of chromium within the cell plays in the mutagenic activation of the compound. Apparently, chromium enters the cells as Cr(VI) where it can be reduced to reactive species Cr(V), Cr(VI) and Cr(III). Although a number of cellular macromolecules have been implicated in these transformations, it appears that intracellular glutathione may play a key role in chromium reduction and mutagenesis. We propose to test the following hypothesis: The intracellular reduction of chromium by glutathione is a major pathway for the generation of reactive intermediates that are responsible for chromium mutagenesis in the intact cell and for chromium carcinogenesis in animals. By comparing the spectra of mutations induced in a mutagenesis target gene by chromium compounds when plasmid DNA is treated in vitro and replicated in yeast or mammalian cells, when yeast or mammalian cells that contain plasmid are treated with chromium, and in the transgene in the lungs of mice following chromium inhalation, we hope to identify the types of premutagenic DNA damage that are important in chromium mutagenesis and determine how the mutagenic specificity is determined by the specific pathway for chromium reduction in the cell. An increased understanding of the mechanisms of chromium mutagenesis, should enhance our ability to evaluate the risks associated with human exposure to the environmental hazard, and perhaps suggest methods for intervention and prevention of adverse health effects of exposure.
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