Molecular Mechanisms of Dioxin Action

描述（由申请人提供）：先天性心血管畸形是新生儿和婴儿死亡的主要原因，也是成人心脏功能不全的主要原因。瓣膜狭窄和左心发育不全综合征是最常见的畸形，每1000个活产婴儿中就有8个发生，占人类心血管畸形病例的25-30%。迫切需要了解导致这些疾病的机制。我们的长期目标是确定这些心脏疾病的机制和原因，并开发工具来预防它们的发生。这些研究的目的是确定遗传和环境因素之间的关联是否是导致疾病发病率的最终原因。我们的中心假设是，小鼠产前暴露于2,3,7,8-四氯二苯并-对二恶英（TCDD；二恶英），一种有机氯环境污染物，介导芳烃受体（AHR）依赖的同源盒转录因子NKX2.5的抑制，NKX2.5是心脏形态发生的关键决定因素。我们的理由是，NKX2.5基因突变和母亲在怀孕期间暴露于卤代烃、二恶英和多氯联苯是已知的这些人类发育性心脏畸形的主要危险因素。在此背景下的关键是我们发现AHR抑制NKX2.5的表达。完成我们的具体目标将实现以下短期目标：(1)表征AHR在心血管发展和NKX2.5调节中的作用；(2)鉴定TCDD暴露导致心血管发育过程中调控基因表达的变化；(3)确定胚胎发育期间接触TCDD是否会导致长期的心血管畸形。这些目的验证了一种假说，即有机氯环境因子在心脏胚胎发育过程中破坏ahr调节的功能，并导致长期的心血管损伤。我们将使用启动子介导的选择方法来模拟NKX2.5、AHR和二恶英暴露之间的基因-环境相互作用，并使用全局基因表达分析和染色质免疫沉淀来鉴定分化过程中TCDD效应的遗传和表观遗传特征。我们将在子宫内暴露于TCDD的新生小鼠和成年小鼠的组织中检测靶基因簇的表达、表观遗传变化和心脏畸形的发生率。本工作的意义在于确定了AHR和TCDD发挥心脏作用的分子机制，为AHR、二恶英与心血管疾病之间的因果关系提供了检验。我们提出的新方法将为其他环境因素的许多类似研究确定一条适用的路径。我们的工作是创新的，因为它提出将分子工具和胚胎干细胞研究相结合，这在环境因子的作用机制研究中从未使用过。我们期望在TCDD暴露、AHR激活的基因和心脏畸形之间建立强有力的机制联系。

DESCRIPTION (provided by applicant): Congenital cardiovascular malformations are the leading cause of neonatal and infant death and a major cause of adult cardiac insufficiency. Valvular stenosis and hypoplastic left heart syndrome are the most common malformations occurring in eight out of 1000 live births and constituting 25-30% of all cases of human cardiovascular malformations. There is a critical need to understand the mechanisms that cause these diseases. Our long-term goal is to identify the mechanisms and causes of these cardiac diseases and to develop tools to prevent their incidence. The objective of these studies is to determine whether an association between genetic and environmental factors is the ultimate cause responsible for disease incidence. Our central hypothesis is that prenatal exposure of mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), an organochlorinated environmental pollutant, mediates the aryl hydrocarbon receptor (AHR)-dependent repression of the homeobox transcription factor NKX2.5, a key determinant of cardiac morphogenesis. Our rationale is that mutations in the NKX2.5 gene and maternal exposure to halogenated hydrocarbons, dioxins and polychlorinated biphenyls during pregnancy are the main known risk factors for these human developmental cardiac malformations. Key in this context is our finding that AHR represses NKX2.5 expression. Completion of our specific aims will achieve the following short-term goals: (1), to characterize the AHR role in cardiovascular development and NKX2.5 regulation; (2), to identify regulatory gene expression changes during cardiovascular development that result from TCDD exposure; (3) to determine if TCDD exposure during embryonic development causes long-lasting cardiovascular malformations. These aims test the working hypothesis that organochlorinated environmental agents disrupt AHR-regulated functions during cardiac embryonic development and cause long-lasting cardiovascular injury. We will use promoter-mediated selection methods to model the gene-environment interactions established between NKX2.5, AHR and dioxin exposure and use global gene expression analyses and chromatin immunoprecipitation to identify genetic and epigenetic signatures characteristic of TCDD effects during differentiation. We will probe tissues of newborn and adult mice exposed to TCDD in utero for the expression of target gene clusters, epigenetic changes and incidence of cardiac malformations. The significance of this work lies on the identification of the molecular mechanisms by which AHR and TCDD exert their cardiac effects, providing a test of the causal connections between AHR, dioxin and cardiovascular disease. The novel approach that we propose will define a path applicable to many similar studies of other environmental agents. Our work is innovative because it proposes to use a combination of molecular tools and ES cell research never before employed in the study of the mechanisms of action of environmental agents. We expect to establish a strong mechanistic link between TCDD exposure, the genes that the AHR activates and cardiac malformations. PUBLIC HEALTH RELEVANCE: The exposure of human populations to aryl hydrocarbon receptor organochlorinated ligands has been associated with a number of disease outcomes, including cancer, chloracne, cardiovascular disease, diabetes, endometriosis, neurocognitive deficits, immunotoxicity and especially, cardiac developmental abnormalities. Two central questions that need to be addressed in this context are, (i), what are the mechanisms responsible for the causation of these diseases, and (ii) what are the long-term health consequences of developmental exposure on the susceptibility to environmental disease in the adult. By focusing this renewal application on the potential consequences of fetal exposure on congenital cardiac malformations and incidence of adult disease, we will address these two most critical issues of environmental health research today.