Arachidonate Products and CYP1A in Dioxin Toxicity

说明(申请人提供)：环境毒素2，3，7，8-四氯二苯并-对二恶英(TCDD，Dioxin)需要结合和激活ah受体(AhR)才能产生毒性作用。TCDD的毒性包括致命性消耗综合征和心血管功能障碍，但TCDD激活AhR如何产生这些影响尚不清楚。本研究旨在了解AhR的TCDD激活如何导致糖异生抑制、能量衰竭和死亡，并确定AhR激活的主要转录产物细胞色素P450(CYP)1A酶对毒性的贡献。将检验以下假设：(1)TCDD在转录和翻译后对聚集在PGC1上的信号通路，即Akt、AMPK、PKA和Sirt1的作用，有助于TCDD抑制糖异生。前列环素？通过糖异生途径调节通量的PEPCK和葡萄糖6-磷酸酶的转录；(2)抑制糖异生迫使能量依赖脂类获得能量，但也限制了脂类作为燃料来源的可用性；(3)TCDD对细胞膜脂质花生四烯酸(AA)依赖细胞色素P的代谢的影响导致肝脏和心脏的能量衰竭。这项研究将跟进之前拨款期间的发现，即TCDD导致心脏收缩功能障碍，增加肝脏中依赖CYP的AA环氧化物(EETs)的形成，减少20-HETE(具有主要生理心血管调节作用的AA产物)的形成，以研究它们在营养代谢中的作用。TCDD将被用作一个工具，以了解AhR的激活和CYP依赖的AA代谢的变化如何导致与消耗综合征(特定目标(SA)1)相关的糖和脂代谢以及离子通道和心肌功能(SA2)的生理性紊乱。对SA2的研究还将确定肝脏中EETs的增加是否会影响心脏，是否具有心脏保护作用或心脏毒性。接近孵化的鸡胚胎将继续被用作主要模型，基于它在研究TCDD毒性方面的记录，它在AA代谢方面与人类的相似性，以及它在这项研究中的特殊用途，允许独立于食物摄入的混杂影响来研究肝脏代谢影响。这些发现将在哺乳动物细胞中得到证实。通过在鸡胚胎肝细胞中沉默或过表达细胞色素P1A4和细胞色素P1A5，以及一种新的分子方法，即逆转录病毒载体中的正义和反义细胞色素P1A1a基因构建将在发育早期针对鸡胚的肝脏或心脏，并在后期阶段检测其影响，将寻找支持或反对细胞色素P1A1a在AhR效应中作用的确凿证据。这项研究有望表明，CYP1A酶有助于代谢和心血管调节途径，提高对糖和脂代谢之间的关系的理解，以及AhR在调节营养物质可获得性变化的生理和病理反应中的作用，并对常见相关疾病、心血管疾病和糖尿病具有意义。相关性：对营养供应和新陈代谢变化的反应会导致衰老和常见的人类疾病，包括糖尿病、心血管疾病和癌症。环境毒素TCDD通过与芳香烃受体(AhR)结合的单一作用，引发一种致命的耗竭综合征，其特征是无法合成葡萄糖，导致能量衰竭和死亡。通过了解TCDD激活AhR如何产生营养反应的大规模失调，我们预计将更多地了解身体如何正常地协调对不断变化的营养水平的反应，以及正常的调节过程如何失控，这对常见的人类疾病以及正常的生理都有影响。

DESCRIPTION (provided by applicant): Binding and activation of the Ah receptor (AhR) is required for the environmental toxin 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD, dioxin) to produce its toxic effects. TCDD toxicity includes a lethal wasting syndrome and cardiovascular dysfunction, but it is not known how TCDD activation of the AhR produces those effects. This research seeks to understand how TCDD activation of the AhR leads to suppression of gluconeogenesis, energy failure and death and to identify contributions of cytochrome P450 (CYP) 1A enzymes, the major transcriptional products of AhR activation, to the toxicity. The following hypothesis will be tested: (1) Transcriptional and posttranslational effects of TCDD on signaling pathways converging on PGC1?, i.e. Akt, AMPK, PKA and Sirt1, contribute to suppression of gluconeogensis by TCDD. PGC1? governs transcription of PEPCK and glucose 6-phosphatase, regulators of flux through the gluconeogenic pathway; (2) Suppression of gluconeogenesis forces reliance on lipids for energy but also limits availability of lipids as a fuel source; (3) Effects of TCDD on CYP-dependent metabolism of the membrane lipid arachidonic acid (aa) contribute to energy failure in liver and heart. The research will follow up on discoveries under prior funding periods of the grant that TCDD causes cardiac contractile dysfunction and increases hepatic formation of CYP-dependent aa epoxides, EETs, and decreases formation of 20-HETE, aa products with major physiologic cardiovascular regulatory effects, to examine their involvement in nutrient metabolism. TCDD will be used as a tool to learn how AhR activation and changes in CYP-dependent aa metabolism can lead to physiologic disturbances in glucose and lipid metabolism involved in the wasting syndrome (specific aim (SA) 1) and in ion channel and cardiac muscle function (SA2). Studies in SA2 will also determine whether increased production of EETs in liver affect the heart and are cardioprotective or cardiotoxic. The chick embryo close to hatching will continue to be used as the major model based on its track record in studying TCDD toxicity, its similarity to humans with respect to aa metabolism and its special utility for this research in permitting hepatic metabolic effects to be studied independently of confounding effects of food intake. Findings will be confirmed in mammalian cells. Definitive evidence for or against a role of CYP1A in AhR effects will be sought by silencing or overexpressing CYP1A4 and CYP1A5 in chick embryo hepatocytes and by a novel molecular approach in which sense and antisense CYP1A gene constructs in retroviral vectors will be targeted to liver or heart in chick embryos at early stages of development and the effects examined at a later stages. This research is expected to show that CYP1A enzymes contribute to metabolic and cardiovascular regulatory pathways, to improve understanding about relationships between glucose and lipid metabolism and the role of the AhR in regulating physiologic and pathologic responses to changes in nutrient availability, and to have implications for common related diseases, cardiovascular disease and diabetes. Relevance: Responses to changes in nutrient supply and metabolism contribute to aging and common human diseases including diabetes, cardiovascular disease and cancer. The environmental toxin, TCDD, by the single action of binding to the aryl hydrocarbon receptor (AhR), which is present in all our cells, initiates a lethal wasting syndrome characterized by a failure to synthesize glucose and leading to energy failure and death. By learning how TCDD activation of the AhR produces massive dysregulation of nutrient responses we expect to learn more about how the body normally orchestrates responses to changing levels of nutrients and how normal regulatory processes can spin out of control, with implications for common human diseases as well as normal physiology.