INHIBITION OF CHEMICAL TRANSFORMATION BY ASPIRIN

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We will pursue our earlier findings that aspirin inhibits late stages of chemical transformation of C3H/10T1/2 cells. We will specifically in determine mechanisms by which aspirin inhibits chemical transformation of C3H/10T1/2 cells and whether prostaglandins or other eicosanoids play a role in late stages of chemical transformation of C3H/10T1/2 cells. Firstly, we will rigorously by HPLC and RIA methodologies determine whether transforming concentrations of MCA cause arachidonic acid release and biosynthesis of prostaglandins and other eicosanoids in C3H/10T1/2 cells. Then, we will determine whether levels of prostaglandin or leukotriene metabolites of arachidonic acid metabolism are reduced in cells undergoing transformation by 1Mu/ml 3-methylchlanthrene following addition of 20 Mug/ml of aspirin to cells, which we previously found markedly inhibits chemical transformation. We will treat C3H/10T1/2 cells with inhibitors of arachidoinic acid release, such as dexamethasone, with inhibitors of cyclooxygenase, such as aspirin, indomethacin, and flurbiprofen, with inhibitors of lipoxygenase acitivity (some also inhibit cyclooxygenase activity) such as 5,6 dehydro-arachidonic acid, nordihydroguariuretic acid, and BW 755C, and determine where in the eicosanoid biosynthetic pathway the greatest inhibitory effect is exerted on transformation. This will help us focus on the fewest number of eicosanoid metabolites to be examined as putative enhancers of chemcial transformation. We will then study aspirin-mediated inhibiton of partially purified cyclooxygenase in cell fractions of C3H/10T1/2 cells. We will also determine whether inhibition of cyclooxygenase activity in MCA-treated C3H/10T1/2 cells subsequently treated with aspirin correlates with inhibition of chemical transformation by the cyclooxygenase inhibitors aspirin and Indomethacin. Secondly, we will add arachidonic acid metabolites, both those formed via cyclooxygenase and also via the lipoxygenase pathways, to C3H/10T1/2 cells treated with an initiating concentration of MCA, 0.1 Mug/ml, and determine whether these metabolites enhance the transformation of C3H/10T1/2 cells or reverse inhibition of transformation incells treated with 1 Mug/ml of aspirin. In all transformation assays, we will also run in parallel our previously developed assay measuring induction of mutation to ouabain resistance to determine whether effects on enhancement of or inhibition of transformation are paralleled by similar enhancements or inhibitions of base substitution mutations.