ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

Ah受体在细胞色素P450（CYP）1A诱导中的作用 酶的环境毒素2，3，7，8-四氯二苯并-p-二恶英（二恶英）是 CYP1A在二恶英毒性中的作用尚不清楚。在这场竞争中， 更新，我们将集中在CYP1A诱导的作用，在生物学效应， 二恶英我们将继续使用良好表征的鸡胚模型， 许多优点包括易于在体内引入修饰的基因， 在原代细胞培养和器官中进行分子和生物化学研究 来自同一物种。我们最近克隆了二恶英诱导的CYP1A酶 （哺乳动物中的CYP1A4和1A5与CYP1A1和1A2）。CYP1A4是芳基 烃羟化酶CYP1A5与人CYP1A2一样，是一种花生四烯酸， 环氧酶CYP1A代谢一种重要的内源性 生物活性产物的底物支持CYP1A的功能作用。我们 还发现二恶英在肝脏和肾脏中诱导1A4和1A5，而CYPIA4单独 在心脏和血管内皮中，二恶英抑制内毒素诱导 心血管调节剂iNOS在肝脏中，二恶英是心脏毒性，这些 研究结果指出，心血管系统是二恶英作用的主要目标。我们 将检验CYP1A产物、类花生酸和活性氧 物种，调节二恶英对生物学表达或活性的影响， 心血管功能的重要蛋白质。CYP1A的明确证据 通过开发令人兴奋的新逆转录病毒分子， 专门针对鸡和新开发的cDNA探针的技术， CYP1A4和1A5。CYP1A4和CYP1A5将被抑制或超表达， 逆转录病毒载体中反义或有义构建体的器官特异性靶向， 鸡胚心脏、肝脏或肾脏在卵内的早期发育及其影响 检查接近孵化。CYP1A修饰对CYP1A4和 1A5和二恶英的两个潜在心血管靶点SERCA和 将在SpecificAim 1中研究Na +/K + ATP酶。的机制 二恶英抑制iNOS并损害心脏收缩反应， 将分别在特定目的2和3中研究CYP1A在这些效应中的作用。 所有三种SA将应用相同的分子技术来修饰CYP1A 表情这项研究将应用一种强大的器官特异性 基因操作对重大毒理学问题（CYP1A功能）的影响， 提供深入了解二恶英毒性的重要心血管方面。

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.