ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

TCDD和与化学有关的多氯联苯继续引起公众的关注，因为 它们对某些物种的高毒性。它们的毒性机制 以及特异性细胞色素P450同工酶大幅增加的作用 是不被理解的。这项提议是基于这样一个前提： TCDD的毒性和保护反应机制的阐明 敏感的模式物种将加强对 评估人类风险。这个实验室正在调查这部小说 假设TCDD诱导的P450通过以下途径参与TCDD毒性 代谢内源化合物，如膜脂肪酸， 花生四烯酸(AA)可影响生物活性代谢物 细胞发出信号，从而调节毒性。我们将继续使用 主要是建立了一个成熟的鸡胚模型。TCDD治疗为 发现通过P450将AA代谢增加到特定的AA环氧化物和 单羟基化产物，其生物活性类似于 TCDD毒性。TCDD通过特定的TCDD诱导增加AA环氧化物 P450，TCDD/AA，不同于TCDD诱导的P-450催化AHH和7- EROD、TCDD/AHH。TCDD治疗也增加肝脏中AA的释放 细胞，使AA对TCDD/AA可用，并抑制 结构型欧米伽-OHAA。在接下来的时间里，我们将调查 这些变化背后的机制以及TCDD治疗后的增加 [CA2]/i及其对细胞功能的影响。SA1将建立 TCDD是否增加P450不存在的细胞中AA的释放 增加，是否由TCDD优先增加以响应 激素刺激以及TCDD暴露后是否会发生这种情况 SITE和OVO都是如此。磷脂酶、蛋白质在AA释放中的作用 激酶C、P450和转录事件将用Fresh 制备肝细胞和心肌细胞悬液或细胞培养。 SA2将检测肝脏和心脏细胞变化的细胞后果 通过TCDD释放AA和P450 AA代谢产物对AA代谢的影响，[Ca]/i， 脂质成分和蛋白激酶C，所有细胞的主要决定因素 功能。TCDD抑制omega-OHAA的机制将 也要接受检查。SA3将研究AH受体在脑内的作用 TCDD增加AA代谢、AA释放和[Ca~(2+)]/i 检查是否已知AH受体配体和非配体，包括印刷电路板 同系物诱导了这些效应，以及TCDD在P450 AA中的增加 代谢发生在不同的AH受体敏感性不同的同源基因小鼠中。 在SA4中，建议克隆和测序TCDD/AA和TCDD/AHH cDNAs。 所获得的探针将用于检测TCDD对mRNA的影响 这些P450的表达和转录，并使用SA5来检测 P450基因在细胞内的原位分布 杂交法和免疫组化法检测P450蛋白 单特异性抗体。

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.