The Applicant proposes experiments to study the effects of oxidant stress on the renal epithelial cell line LLC-PK1. The cells will be treated with hydrogen peroxide (a representative oxidant) or diamide (to deplete sulfhydryl groups) and then examined by northern blotting, western blotting and if necessary RT-PCR for the induction of a number of genes, including genes encoding antioxidant enzymes, the early response genes fos, jun, c-myc, and certain genes associated with apoptosis (e.g., p53) that are known to be related to oxidant stress. Changes in transcription factors will be studied by mobility shift assays, using antibody-induced supershifts for identification. Signal transduction pathways (Ca++, protein kinases, phospholipase A2) will be investigated, chiefly by inhibitor studies. Cell proliferation (3H)thymidine incorporation) and apoptosis (ethidium homodimer-1) will be measured, and antisense methods will be used to determine whether one or more of these genes affect the growth or survival of the challenged cells. After these studies have been carried out, the Applicant will look at the effects of selected antioxidants, including ascorbate, alpha- tocopherol, N-acetylcysteine and PQQ on the same parameters, as well as on cell proliferation and apoptosis. Finally, the cells will be transfected with vectors expressing Bcl-2, the adenovirus E1b gene product, and various antioxidant enzymes, and the peroxide-induced responses of cells so transfected will be studied as described above.
