Ozone, lipid-protein adducts, and biological effects

描述(申请人提供)：臭氧是最常见的环境污染物之一，人类暴露于这种活性分子水平增加显然与促炎反应和呼吸道疾病的恶化有关。不饱和脂类特别容易受到臭氧的影响，一些亲电的醛和环氧化物被认为是脂类臭氧暴露的主要产物。例如，胆固醇很容易与臭氧反应，并通过将甾醇的环-B双键转化为活性羰基和环氧化物官能团而产生氧固醇产物。内源性形成的氧甾醇是众所周知的肝X受体(LXR)的配体，它调节与胆固醇稳态、脂肪酸合成和胆固醇反向运输相关的基因的表达。最近的研究也表明，LXR具有强大的抗炎和免疫调节功能。与内源性形成的氧化甾醇相反，一些臭氧衍生的氧化甾醇抑制LXR功能，但这一机制在臭氧发病机制中的作用是已知的。众所周知，臭氧可以改变细胞功能和激活上皮细胞，但这些过程的生化机制尚未确定。臭氧衍生的氧化甾醇是一种亲电体，与蛋白质中常见的亲核残基反应，形成稳定的加合物。我们认为，臭氧的已知化学反应、其对呼吸道中脂质的假定暴露、这些暴露所导致的氧化应激以及人类疾病与环境侮辱的关联，需要进行协调研究，以发现将脂质、环境暴露和病理生理反应联系在一起的基本化学和生物事件。臭氧衍生氧化甾醇是拟议研究中的一个共同主题，我们在这里概述了旨在：1.提供化学纯的臭氧衍生氧化甾醇，以评估这些化合物及其代谢物对上皮细胞功能的影响，特别是LXR信号通路。2.发展基于“点击化学”和人工合成的炔基甾醇类似物的方法，分离和鉴定氧合甾醇-蛋白质加合物，并确定上皮细胞和巨噬细胞中的氧合甾醇加合蛋白质组。3.利用合成的类固醇和氧化类固醇来追踪和鉴定不同上皮细胞隔间中的脂质。4.在细胞研究的基础上，建立脂-蛋白加合生物标志物的检测方法。范德比尔特大学和北卡罗来纳大学教堂山分校的实验室为研究一个重要的环境问题提供了独特的专业知识组合。这些专门知识包括化学合成、分离和表征方面的技能，以及研究暴露在空气污染物中对健康的不利影响以及调节这些反应的细胞机制的经验。

DESCRIPTION (provided by applicant): Ozone is one of the most commonly encountered environmental pollutants and human exposures to increased levels of this reactive molecule are clearly linked to proinflammatory responses and exacerbation of respiratory illness. Unsaturated lipids are particularly vulnerable to ozone and a number of electrophilic aldehydes and epoxides are known as primary products of lipid ozone exposure. Cholesterol, for example, reacts readily with ozone and gives oxysterol products that result from conversion of the ring- B double bond of the sterol to reactive carbonyl and epoxide functionality. Endogenously formed oxysterols are well-known ligands for the liver-X-receptor (LXR), which regulates the expression of genes involved in cholesterol homeostasis, fatty acid synthesis, and reverse cholesterol transport. More recent studies also implicate LXR in having potent anti-inflammatory and immune regulatory function. In contrast to endogenously formed oxysterols, some of the ozone-derived oxysterols inhibit LXR function, yet the role of this mechanism in ozone pathogenesis is known. Ozone is known to modify cellular function and activate epithelial cells but the biochemical mechanisms of these processes have yet to be defined. Ozone-derived oxysterols are electrophiles that react with common nucleophilic residues present in proteins, forming stable adducts. We contend that the known chemical reactivity of ozone, its presumed exposure to lipids in the airway, the oxidative stress that results from these exposures, and the association of human diseases with environmental insults calls for coordinated studies aimed at discovering the fundamental chemical and biological events linking lipids, environmental exposures and the pathophysiological response. Ozone-derived oxysterols are a common theme in the proposed research and we outline strategies here that are designed to: 1. Provide chemically pure ozone-derived oxysterols to assess the effect of these compounds and their metabolites on the function of epithelial cells, with specific focus on the LXR signaling pathway. 2. Develop methods based on "click chemistry" and synthetic alkynyl sterol analogs that permit the isolation and identification of oxysterol-protein adducts and define the oxysterol adduction proteome in epithelial cells and macrophages. 3. Utilize synthetic sterol and oxysterol analogs to track and identify lipids in various epithelial cellular compartments. 4. Develop assays for biomarkers of lipid-protein adduction based on our cellular studies. The laboratories at Vanderbilt University and University of North Carolina at Chapel Hill provide a unique combination of expertise for the study of an important environmental problem. This expertise includes skills in chemical synthesis, isolation and characterization coupled with experience studying adverse health effects induced upon exposure to air pollutants and the cellular mechanisms mediating these responses.