ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

Ah受体在细胞色素P450(CYP)1A诱导中的作用 酶由环境毒素2，3，7，8-四氯二苯并对二恶英(Dioxin)所致 众所周知，但CyP1A在二恶英毒性中的作用尚不清楚。在这场竞争中 更新，我们将集中在细胞色素P1a的诱导在生物效应中的作用。 二恶英。我们将继续使用具有良好特性的鸡胚胎模型，其 许多优点包括易于在体内引入修饰基因，以及 在原代细胞培养和器官中进行分子和生化研究 来自同一物种。我们最近克隆了二恶英诱导的细胞色素P1A酶 (哺乳动物中的CYP1A4和1A5与CYP1A1和1A2相比)。CyP1A4是一种芳基 碳氢化合物羟基酶。和人类的CYP1A2一样，CyP1A5也是一种花生四烯酸 环氧合酶。细胞色素P1a代谢一种重要内源物质的能力 底物对生物活性产物支持细胞色素P450的功能作用。我们 二恶英还可诱导肝脏和肾脏中的1A4和1A5，但仅诱导细胞色素PIA4 在心脏和血管内皮细胞中，二恶英抑制内毒素诱导 肝脏中心血管调节剂iNOS的含量，以及二恶英对心脏的毒性，这些 研究结果表明，心血管系统是二恶英作用的主要目标。我们 将检验一种假设，即细胞色素P1a的产物、二十烷类化合物和活性氧 物种，调节二恶英对生物表达或活性的影响 心血管功能中的重要蛋白质。关于CYP1A的确凿证据 将通过开发令人兴奋的新逆转录病毒分子来获得参与 专为雏鸡量身定做的技术和新开发的用于 CYP1A4和1A5。CYP1A4和CYP1A5将被抑制或过度表达 逆转录病毒载体中反义或正义结构的器官特异性靶向 鸡胚心、肝或肾在卵子早期发育中的作用 在接近孵化的时候被检查。细胞色素P1A1a基因修饰对细胞色素P1A4和细胞色素P4的影响 1A5和二恶英的两个潜在心血管靶点SERCA和 Na+/K+ATPase将在专业目标1中进行研究。 二恶英抑制诱导型一氧化氮合酶损伤心脏收缩反应及其作用 这些影响将分别在特定目标2和3中进行研究。 这三种SA都将应用相同的分子技术来修饰CyP1A 表情。这项研究将把一项强大的NOW技术应用于器官特异性 针对重大毒理学问题的基因操作(CYP1A功能)和将 提供对二恶英毒性的重要心血管方面的见解。

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.