ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

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The role of the Ah receptor in the induction of cytochrome P450 (CYP)1A enzymes by the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)is well understood, but the role of CYP1A in dioxin toxicity is not. In this competing renewal we will focus on the role of CYP1A induction in the biologic effects of dioxin. We will continue to use the well characterized chick embryo model whose many advantages include ease of introducing modified genes in vivo and of conducting molecular and biochemical studies in primary cell cultures and organs from the same species. We recently cloned the dioxin-induced CYP1A enzymes (CYP1A4 and 1A5 vs CYP1A1 and 1A2 in mammals). CYP1A4 is an aryl hydrocarbon hydroxylase. CYP1A5, like human CYP1A2, is an arachidonic acid epoxygenase. The ability of CYP1A to metabolize an important endogenous substrate to biologically active products supports a functional role for CYP1A. We found also that dioxin induces 1A4 and 1A5 in liver and kidney but CYPIA4 alone in the heart and-vascular endothelium, that dioxin depresses induction by endotoxin of the cardiovascular modulator iNOS in liver, and that dioxin is cardiotoxic, These findings point to the cardiovascular system as a majortargetof dioxin action. We will testthe hypothesis that CYP1A products, eicosanoids and reactive oxygen species, modulate effects of dioxin on expression or activity of biologically significant proteins in cardiovascular function. Unequivocal evidence for CYP1A involvement will be obtained by exploiting exciting new retroviralmolecular techniques specifically tailored to the chick and newly developed cDNA probes for CYP1A4 and 1A5. CYP1A4 and CYP1A5 will be suppressed or superexpressed by organ specific targetting of antisense or sense constructs in retroviral vectors to chick embryo heart, liver or kidney in ova in early development and the effects examined close to hatching. The effects of CYP1A modification on CYP1A4 and 1A5 and two potential cardiovascular targets of dioxin, SERCA and Na+/K+ATPase will be investigated in SpecificAim 1. The mechanisms by which dioxin depresses iNOS and impairs cardiac contractile responses and the role of CYP1A in those effects will be investigated in Specific Aims 2 and 3, respectively. All three SA will apply the same molecular techniques to modify CYP1A expression. This research will apply a powerful now technology for organ-specific gene manipulation to a significant toxicologic issue (CYP1A function) and will provide insights into important cardiovascular aspects of dioxin toxicity.