Although arsenic is a well-established human carcinogen and inducers cancers of the skin, liver, bladder, and lung the underlying mechanism(s) is unknown. Using the human-hamster hybrid (AL) cells that are sensitive in detecting multi-locus deletions, we show recently that arsenic is indeed mutagenic to endogenous arsenite-treated cells. The first objective of this proposal is to determine if reactive oxygen species, particularly hydroxyl radicals, generated by arsenite result in oxidative DNA damage and mutagenesis in AL will be determined from arsenite-treated cultures using immunoperoxidase staining and the slicylate assay, respectively. The localization of the ROS formed by arsenite will be determined using confocal microscopy with the oxidative sensitive dye CM-H2DCFDA. To show that oxyradical induced by arsenite actually mediate the mutagenic events, the incidence and types of S1 mutants induced by equitoxic doses of either arsenite or hydrogen peroxide will be determined. The second objective is to determine the role of mitochondria in mediating the genotoxic response of arsenite. Mitochondria in AL cells will be functionally inactivated using rhodamine 6G. Cultures will be treated with graded doses of arsenite and rescued by fusion with cytoplasts to determine the mutagenic response. The AL cells contain only one copy of human chromosome 11 and mutations on marker genes located on this chromosome can be readily scored using an antibody complement lysis technique since the AL cells also contain the HPRT gene located on the hamster X chromosome, mutations induced by arsenic on an essential (- X) versus a non-essential chromosome (human chromosome 11) will provide useful information on the types and sizes of the induced molecular alterations.
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