Arachidonate Products and CYP1A in Dioxin Toxicity

描述（由申请人提供）：环境毒素2,3,7,8-四氯二苯并-对二恶英（TCDD，二恶英）产生毒性作用需要Ah受体（AhR）的结合和激活。TCDD毒性包括致死性消耗综合征和心血管功能障碍，但目前尚不清楚TCDD如何激活AhR产生这些影响。本研究旨在了解TCDD激活AhR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。本文将检验以下假设：(1)TCDD对PGC1信号通路的转录和翻译后影响？Akt、AMPK、PKA和Sirt1参与了TCDD对糖异生的抑制。PGC1吗?调控PEPCK和葡萄糖6-磷酸酶的转录，调控糖异生途径的通量；(2)抑制糖异生迫使人们依赖于脂质作为能量来源，但也限制了脂质作为燃料来源的可用性；(3) TCDD对cypp依赖性膜脂花生四烯酸（aa）代谢的影响导致肝脏和心脏能量衰竭。这项研究将继续追踪在先前资助期内的发现，即TCDD会导致心脏收缩功能障碍，增加肝脏中cypp依赖性aa环氧化物（EETs）的形成，并减少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将被用作了解AhR激活和cypp依赖性aa代谢变化如何导致消耗综合征（specific aim (SA) 1）和离子通道和心肌功能（SA2）中涉及的糖和脂质代谢的生理紊乱的工具。SA2的研究还将确定肝脏中eet的增加是否会影响心脏，是保护心脏还是毒害心脏。鉴于鸡胚在TCDD毒性研究中的记录、其在aa代谢方面与人类的相似性以及其在允许独立于食物摄入的混杂效应研究肝脏代谢效应方面的特殊效用，接近孵化的鸡胚将继续被用作主要模型。研究结果将在哺乳动物细胞中得到证实。通过沉默或过表达鸡胚肝细胞中的CYP1A4和CYP1A5，以及通过一种新的分子方法，在逆转录病毒载体中构建的正义和反义CYP1A基因将靶向鸡胚胎早期发育阶段的肝脏或心脏，并在后期检查其影响，将寻求支持或反对CYP1A在AhR效应中作用的明确证据。本研究预计将表明CYP1A酶参与代谢和心血管调节途径，提高对糖脂代谢之间关系的理解，以及AhR在调节营养可利用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有启示。相关性：对营养供应和代谢变化的反应有助于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通过与存在于我们所有细胞中的芳烃受体（AhR）结合的单一作用，引发了一种致命的消耗综合征，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如何产生大量营养反应失调，我们希望更多地了解身体通常如何协调对营养水平变化的反应，以及正常调节过程如何失控，这对人类常见疾病和正常生理都有影响。

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.