ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

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Dioxin (TCDD) and PCBs are major environmental toxins, but neither the precise mechanism of their toxicity or biochemical mechanisms of defense are known. Furthermore there are large differences in species sensitivity to TCDD and PCBs which have yet to be explained. In this research we will extend our previous original observations that TCDD causes major increases in NADPH-dependent arachidonic acid (AA) metabolism. The factors regulating TCDD's effects and the role of AA metabolites in enhancing or limiting TCDD toxicity will be investigated, principally using a chick embryo model. TCDD's effects on AA metabolism in intact cells will be studied using cultured hepatocytes, Kupffer cells and endothelial cells to identify the types of liver cells in which AA metabolism is increased and in cultured cardiac myocytes. Its effects on AA metabolites formation in ovo will also be examined. TCDD's effects on AA release from membranes and on activation of phospholipases A2 and C and on diacylglycerol will be investigated using cultured hepatocytes and cardiac myocytes. Dose-response curves for TCDD's effects on AA metabolism and release and on deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin by cultured cells will be compared to asses relationships between TCDD's effects on eicosanoids and mixed function oxidases. Immunoinhibition of TCDD and PB induced AA metabolism in microsomes and cell homogenates by antibodies against purified P-450 from TCDD-and PB-microsomes and cell homogenates by antibodies against purified P-450s from TCDD- and PB-microsomes and to NADPH- cytochrome P-450 reductase will be used to prove that P-450 mediates TCDD's increase of NADPH-dependent eicosanoid metabolism, to identify the isozymes involved and determine if the same or different isozymes mediate TCDD's and PB's effects. TCDD's specificity will be probed by comparing its effects to those of representative toxic and nontoxic PCBs and to PB. A role for eicosanoids in TCDD's effects will be studied by investigating if HPLC-purified eicosanoids generated by TCDD-treated livers mimic or modify TCDD's effects on protein kinase C in cultured hepatocytes and myocytes or cardiac contractile responses to beta- adrenergic agonists and if inhibitors of AA metabolism diminish the effects. Structure of bio-active eicosanoids will be characterized by GC/MS. In vivo modifications of TCDD toxicity by pharmacologic agents which alter AA metabolism will be examined to learn if inhibiting P-450 mediated eicosanoid metabolism decreases TCDD toxicity. AA metabolism and MFO activity will be assyed concomitantly so that valid conclusions can be drawn.