ARACHIDONATE PRODUCTS IN DIOXIN AND PCB TOXICITY

TCDD 和化学相关的 PCB 一直受到公众关注，因为 它们对某些物种的高毒性。 其毒性机制 以及特定细胞色素 P450 同工酶大幅增加的作用 不被理解。 这个提议的前提是 阐明 TCDD 毒性和保护反应的机制 敏感模式物种将加强科学基础 评估人类风险。 该实验室正在研究这部小说 假设 TCDD 诱导的 P450 通过以下方式参与 TCDD 毒性： 代谢内源性化合物，例如膜脂肪酸， 花生四烯酸 (AA) 转化为生物活性代谢物，可影响 细胞信号从而调节毒性。 我们将继续使用 主要是一个完善的鸡胚模型。 TCDD治疗是 发现通过 P450 增加 AA 代谢为特定的 AA 环氧化物， 单羟基化产品的生物活性类似于 TCDD 毒性。 TCDD 通过特定的 TCDD 诱导增加 AA 环氧化物 P450、TCDD/AA，与 TCDD 诱导的 P-450 催化 AHH 和 7- 不同 EROD，TCDD/AHH。 TCDD 治疗还可增加肝脏 AA 的释放 细胞，使 AA 可用于 TCDD/AA 并抑制 组成型 omega-OH AA。 在接下来的一段时间内，我们将调查 这些变化背后的机制以及 TCDD 治疗的增加 [Ca2]/i 及其对细胞功能的影响。 SA1将建立 TCDD 是否会增加 P450 不增加的细胞中 AA 的释放 增加，是否优先增加TCDD以应对 激素刺激以及是否在细胞暴露于 TCDD 后发生 原位以及卵内。 AA 在磷脂酶、蛋白质释放中的作用 激酶 C、P450 和转录事件将使用新鲜测定 制备肝细胞和心肌细胞细胞悬液或细胞培养物。 SA2 将检查肝脏和心脏细胞变化的细胞后果 AA 释放中的 TCDD 和 AA 代谢中的 P450 AA 代谢物，[Ca2]/i， 脂质成分和蛋白激酶 C，细胞的所有主要决定因素 功能。 TCDD 抑制 omega-OH AA 的机制将 也予以检查。 SA3 将研究 Ah 受体参与 TCDD 增加 AA 代谢、AA 释放和 [Ca2]/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 P450 AA 中 TCDD 的增加是否 代谢发生在 Ah 受体敏感性不同的同源小鼠中。 在SA4中，建议对TCDD/AA和TCDD/AHH cDNA进行克隆和测序。 获得的探针将用于检查TCDD对mRNA的影响 这些 P450 的表达和转录，并使用 SA5 来检查 这些 P450 基因的细胞原位分布 使用免疫组织化学法对 P450 蛋白进行杂交和分析 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.