Intact cultured human lymphocytes will be treated with chromate and DNA- protein complexes will be isolated. Chromate crosslinked proteins will be released from the DNA by treatment with EDTA, which chelates the trivalent form of chromium and should cause the release of proteins and peptides that are complexed to the DNA. We will measure the quantity of released proteins and peptides as an indicator of DNA-protein crosslinking. Antibodies to specific proteins will assess the quantity of protein released by EDTA. These antibodies will include those directed to p97 protein, to actin, as well as to other proteins that we have identified as complexed to DNA. In conjunction with the in vitro study in human cells, a parallel approach will be employed using mice exposed to chromate by chronic i.p. injection or by drinking water exposure. The chronic i.p. injection or oral exposure will examine whether the procedures that are being utilized in vitro in cultured human lymphocytes (i.e., studying the proteins and amino acids/peptides released by EDTA) can be used to detect chromate exposure in animals using their peripheral blood lymphocytes as a sentinel of exposure. A similar approach will also be utilized to study DNA-protein crosslink formation in humans occupationally-exposed to chromium or humans exposed from chromate ground contamination in Hudson County, New Jersey. Traditional methods of assessing chromium exposure will be utilized to test the sensitivity of the DNA-protein complex detection method as a biomarker. These studies should yield a very sensitive method to measure DNA-protein crosslinks, lesion that are known to be formed by chromate as well as several other agents. At present there is not an adequate method to detect human chromate exposure and the studies proposed here should fill this void.
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