ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

四氯二苯并对二恶英和化学上与多氯联苯有关的物质继续受到公众的关注， 它们对某些物种的高毒性 其毒性机制 细胞色素P450同工酶的大量增加 不被理解。 这一提议的前提是， 阐明TCDD毒性和保护反应的机制， 敏感的模式物种将加强科学基础， 评估人类风险。 这个实验室正在研究 TCDD诱导P450参与TCDD毒性作用的假说 代谢内源性化合物，如膜脂肪酸， 花生四烯酸（AA）的生物活性代谢物，可以影响 细胞信号，从而调节毒性。 我们将继续使用 主要是建立良好的鸡胚模型。 TCDD治疗是 发现通过P450增加AA代谢为特定AA环氧化物， 生物活性类似于 TCDD毒性。 TCDD通过特异性TCDD诱导的AA环氧化物增加 P450，TCDD/AA，不同于TCDD诱导的P-450催化AHH和7- EROD、TCDD/AHH。 TCDD治疗也增加了AA从肝脏的释放 细胞，使AA可用于TCDD/AA和抑制形成 组成型ω-OH AA。 在下一个阶段，我们将研究 这些变化背后的机制和TCDD治疗增加， [Ca2+]/i及其对细胞功能的影响。 SA 1将建立 在P450不存在的细胞中，TCDD是否增加AA释放 增加，是否优先增加TCDD响应 激素刺激和是否发生后，TCDD暴露在细胞中， 既在原地也在卵内。 磷脂酶、蛋白质在AA释放中的作用 激酶C、P450和转录事件将使用新鲜的 制备肝细胞和心肌细胞的细胞悬浮液或细胞培养物。 SA 2将检查肝脏和心脏细胞变化的细胞后果 通过TCDD在AA释放和P450 AA代谢物对AA代谢的影响，[Ca 2 +]/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-OH AA的机制将 也要检查。 SA 3将研究Ah受体参与 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PCB 同源物引起这些影响，以及TCDD是否增加P450 AA 代谢发生在Ah受体敏感性不同的同类小鼠中。 在SA 4中，建议克隆和测序TCDD/AA和TCDD/AHH cDNA。 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SA 5来检查 这些P450基因的细胞分布通过原位 杂交和P450蛋白的免疫组化， 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.