Effect of Organophoshate Exposure on Cholesteryl Ester Hydrolase

描述（由申请人提供）：各种形式的心血管疾病（CVD）是美国的主要死亡原因。胆固醇逆向转运是存在于动脉壁内的动脉粥样硬化斑块中的胆固醇通过高密度脂蛋白颗粒转运到肝脏以在胆汁中排泄的机制。最近的研究表明，人胆固醇酯水解酶（CEH），一种代谢胆固醇酯的酶，在胆固醇逆向转运的调节中起着重要作用。这种酶与羧酸酯酶CES1相同。我们的长期目标是了解环境毒物，如农业化学品在人类疾病中的作用。三种常用的有机磷（OP）杀虫剂将用于这项拟议的研究。待检验的假设是暴露于OP杀虫剂将抑制CEH/CES1催化的胆固醇酯代谢，因此可能增加发生动脉粥样硬化的风险。提出了三个目标：（1）测定抑制重组CEH/CES1酶胆固醇酯水解活性的毒死蜱、毒死蜱和甲基毒死蜱的氧磷的剂量反应曲线;（2）测定这些相同氧磷在人单核细胞/巨噬细胞细胞系（THP 1）中抑制CEH/CES1活性的剂量反应曲线;（3）用毒死蜱处理重组CEH/CES1和THP 1细胞后，鉴定CEH/CES1蛋白加合物的形成。我们将确定三种环境相关OP杀虫剂的活性代谢物（oxons）在无细胞系统和培养细胞中抑制CEH/CES1催化的胆固醇酯水解活性的效力。此外，我们将确定蛋白质的共价加合物，使酶功能失活。一些环境因素可能会增加人类CVD的发病率。这项研究的结果将提供OP oxon代谢物是否可以直接改变参与胆固醇代谢的酶的结构-功能的初步见解，从而导致病理结果（即动脉粥样硬化）的可能性增加。这些研究将确定三种与环境相关的有机磷杀虫剂的活性代谢物（氧杂环己基）是否会干扰胆固醇代谢。

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) in its various forms is the leading cause of death in the United States. Reverse cholesterol transport is a mechanism by which cholesterol present in atherosclerotic plaques within arterial walls is transported to the liver via high density lipoprotein particles for excretion in bile. Recent studies have suggested that human cholesteryl ester hydrolase (CEH), an enzyme that metabolizes cholesteryl esters, plays an important role in the regulation of reverse cholesterol transport. This enzyme is identical to the carboxylesterase CES1. Our long term goal is to understand the role that environmental toxicants such as agricultural chemicals play in human disease. Three commonly used organophosphate (OP) insecticides will be used in this proposed study. The hypothesis to be tested is that exposure to OP insecticides will inhibit the CEH/CES1-catalyzed metabolism of cholesteryl esters, which could therefore increase the risk of developing atherosclerosis. Three aims are proposed: (1) Determine the dose response curve for the oxons of chlorpyrifos, parathion, and methyl parathion that inhibit the cholesterol ester hydrolyzing activity of recombinant CEH/CES1 enzyme; (2) Determine the dose response curve for these same oxons with respect to inhibition of CEH/CES1 activity in a human monocyte/macrophage cell line (THP1); (3) Characterize the CEH/CES1 protein adducts formed after treatment of recombinant CEH/CES1 and THP1 cells with chlorpyrifos oxon. We will determine the potency of the active metabolites (oxons) of three environmentally relevant OP insecticides to inhibit CEH/CES1-catalyzed cholesteryl ester hydrolysis activity in a cell-free system and in cultured cells. Furthermore, we will identify the covalent adduct of the protein that inactivates enzyme function. Several environmental factors may increase the incidence of CVD in humans. The results from this study will provide preliminary insights into whether OP oxon metabolites can directly alter the structure-function of an enzyme involved in cholesterol metabolism, thus leading to an increased probability of a pathological outcome (i.e. atherosclerosis). These studies will determine if active metabolites (oxons) of three environmentally relevant organophosphate insecticides can interfere with cholesterol metabolism.