Effect of Organophoshate Exposure on Cholesteryl Ester Hydrolase

描述(申请人提供)：巨噬细胞反向转运胆固醇的限速步骤是将胆固醇酯水解为游离胆固醇，然后将其从细胞外输送到胆固醇受体。在人巨噬细胞中，负责胆固醇酯水解的酶是羧酸酯酶1(CES1)。家长赠款R15 ES015348的总体假设是，暴露于有机磷(OP)杀虫剂的生物活性代谢物将抑制CES1分解胆固醇酯的能力，从而减缓胆固醇的反向运输过程，并增加动脉粥样硬化的风险。我们在当前资金周期中获得的公布数据表明，CES1可以被对氧磷抑制，对氧磷是OP杀虫剂对硫磷的活性代谢物，这种抑制可以导致胆固醇酯在人THP-1巨噬细胞中的增加积累(Crow等人，2008年)。此外，我们最近的初步数据表明，重组的CES1和CES2可以降解内源性大麻化合物2-花生四烯基甘油(2-AG)及其COX-2衍生的前列腺素样代谢物(称为前列腺素甘油酯或PG-Gs)。用对氧磷预处理人THP-1单核细胞和巨噬细胞可以阻断外源添加的2-AG和PG-Gs的后续降解。这些新的发现表明，表达CES1和/或CES2的细胞暴露于生物活性OP代谢物(如对氧磷)将扰乱由这些细胞组成的组织中的内源性大麻素张力。越来越清楚的是，内源性大麻素与大麻的精神活性成分(？9-THC)结合在同一类受体上，在健康和疾病中具有重要作用。因此，我们假设血管壁巨噬细胞的内源性大麻素张力将受到长期暴露于生物活性OP代谢物的显著干扰，并且激活的内源性大麻素系统可以调节巨噬细胞中的胆固醇代谢。为了探索这种可能性，我们建议通过研究模型细胞培养系统(巨噬细胞泡沫细胞)中的内源性大麻素系统来扩大R15 ES015348的范围，我们已经在母基金中使用了这种系统。为了验证我们的假设，我们提出了两个目标：(1)确定氧化(OX)低密度脂蛋白和OP杀虫剂的生物活性代谢产物联合处理培养的人THP-1巨噬细胞是否调节内源性大麻系统的成分，包括大麻素(CB)受体水平、内源性大麻素生物合成酶、内源性大麻素分解代谢酶(MAGL和CES1)以及内源性大麻素本身的水平(2-AG和AEA)。(2)确定内源性大麻素(2-AG和AEA)是否通过CB1或CB2介导的机制调节人巨噬细胞的胆固醇代谢和外流。 公共卫生相关性：动脉粥样硬化是美国的头号杀手。动脉粥样硬化的潜在病理生理学是复杂的和多因素的。动脉粥样硬化发生/发展的一个关键因素是环境因素，如毒物，可能会加速疾病。我们的研究试图检查有机磷(OP)杀虫剂是否在病因上与动脉粥样硬化有关。我们的研究表明，存在调节胆固醇代谢的酶，这种酶可以被OP杀虫剂的生物活性代谢物抑制。这份竞争性补充资料将使我们能够更详细地研究这些机制。

DESCRIPTION (provided by applicant): The rate-limiting step in reverse cholesterol transport from macrophages is the hydrolysis of cholesteryl esters to free cholesterol, which is subsequently transported out of the cell to cholesterol acceptors. In human macrophages, the enzyme responsible for cholesteryl ester hydrolysis is carboxylesterase 1 (CES1). The overall hypothesis of parent grant R15 ES015348 was that exposure to bioactive metabolites of organophosphate (OP) insecticides will inhibit the ability of CES1 to hydrolyze cholesteryl esters, thus slowing the reverse cholesterol transport process and increasing the risk of atherosclerosis. Our published data, obtained during the current funding cycle, shows that CES1 can be inhibited by treatment with paraoxon, the active metabolite of the OP insecticide parathion, and that this inhibition can cause augmented accumulation of cholesteryl esters in human THP-1 macrophages (Crow et al., 2008). Moreover, our recent preliminary data show that recombinant CES1 and CES2 can hydrolyze the endocannabinoid compound 2-arachidonoylglycerol (2-AG) and its COX-2 derived prostaglandin-like metabolites (termed prostaglandin glyceryl esters or PG-Gs). Pretreatment of human THP-1 monocytes and macrophages with paraoxon can block the subsequent degradation of exogenously added 2-AG and PG-Gs. These are novel findings that suggest that exposure of CES1- and/or CES2-expressing cells to bioactive OP metabolites (e.g., paraoxon) will perturb the endocannabinoid tone in tissues composed of these cells. It has become increasingly clear that endogenous cannabinoids, which bind to the same class of receptors as the psychoactive component of marijuana (?9-THC), have important functions in health and disease. Therefore, we hypothesize that the endocannabinoid tone of vessel wall macrophages will be significantly perturbed by chronic exposure to bioactive OP metabolites and an activated endocannabinoid system can modulate cholesterol metabolism in macrophages. To explore this possibility, we propose to extend the scope of R15 ES015348 by studying the endocannabinoid system in a model cell culture system (macrophage foam cells) that we already use in the parent grant. To test our hypothesis we propose two aims: (1) Determine if combined treatment of cultured human THP-1 macrophages with oxidized (ox)LDL and bioactive metabolites of OP pesticides modulate components of the endocannabinoid system, including cannabinoid (CB) receptor levels, endocannabinoid biosynthetic enzymes, endocannabinoid catabolic enzymes (MAGL and CES1), and the levels of endocannabinoids themselves (2-AG and AEA). (2) Determine if endocannabinoids (2-AG and AEA) modulate cholesterol metabolism and efflux from human macrophages via a CB1- or CB2-mediated mechanism. PUBLIC HEALTH RELEVANCE: Atherosclerosis is the number one killer in the United States. The underlying pathophysiology of atherosclerosis is complex and multifactorial. One critical factor in the development/progression of atherosclerosis is environmental factors, such as toxicants, that may accelerate disease. Our research seeks to examine whether organophosphate (OP) pesticides are etiologically associated with atherosclerosis. Our research indicates that there are enzymes that regulate cholesterol metabolism, which can be inhibited by bioactive metabolites of OP pesticides. This competitive supplement will allow us to study these mechanisms in greater detail.