ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

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TCDD and chemically related PCBs are of continuing public concern because of their high toxicity for some species. Mechanisms for their toxicity and the role of the large increases in specific cytochrome P450 isozymes are not understood. This proposal is based on the premise that elucidating mechanisms for TCDD toxicity and protective responses in a sensitive model species will strengthen the scientific basis for evaluating human risks. This laboratory is investigating the novel hypothesis that TCDD-induced P450 participates in TCDD toxicity by metabolizing endogenous compounds, such as the membrane fatty acid, arachidonic acid (AA) to biologically active metabolites that can affect cell signals and thereby modulate toxicity. We will continue to use principally a well established chick embryo model. TCDD treatment was found to increase metabolism of AA by P450 to specific AA epoxides and monohydroxylated products with biologic activities resembling changes in TCDD toxicity. TCDD increases AA epoxides by a specific TCDD-induced P450, TCDD/AA, distinct from the TCDD induced P-450 catalyzing AHH and 7- EROD, TCDD/AHH. TCDD treatment also increases AA release from liver cells, making AA available to TCDD/AA and depresses formation of constitutive omega-OH AA. In the next period we will investigate the mechanisms behind these changes and the increase by TCDD treatment in [Ca2+]/i and their consequences for cell function. SA1 will establish whether AA release is increased by TCDD in cells where P450 is not increased, whether it is preferentially increased by TCDD in response to hormonal stimuli and whether it occurs after TCDD exposure in cells in situ as well as in ovo. The role in AA release of phospholipases, protein kinase C, P450 and transcriptional events will be determined using freshly prepared hepatocyte and cardiac myocyte cell suspensions or cell cultures. SA2 will examine cellular consequences in liver and heart cells of changes by TCDD in AA release and P450 AA metabolites on AA metabolism, [Ca2+]/i, lipid composition, and protein kinase C, all major determinants of cell function. The mechanism for the inhibition of omega-OH AA by TCDD will also be examined. SA3 will investigate involvement of the Ah receptor in the increase by TCDD in AA metabolism, AA release and [Ca2+]/i by examining if known Ah receptor ligands and non ligands including PCB congeners elicit these effects and whether the increase by TCDD in P450 AA metabolism occurs in congenic mice differing in Ah receptor sensitivity. In SA4, it is proposed to clone and sequence TCDD/AA and TCDD/AHH cDNAs. The probes obtained will be used to examine effects of TCDD on mRNA expression and transcription for these P450s and to use SA5 to examine the cellular distribution of the genes for these P450s by in situ hybridization and of the P450 proteins by immunohistochemistry using monospecific antibodies.