Proteomics of DEP-induced Oxidative Stress

描述（由申请人提供） 迫切需要对环境空气颗粒物进行毒理学分析。研究人员正在研究DEP和CAPS在过敏性气道炎症和哮喘中的辅助作用。 动物和人类的体内研究表明，DEP在常见环境过敏原的挑战期间增强IgE产生和过敏性炎症。 这种效应涉及氧化应激的产生，并且可以用硫醇抗氧化剂逆转。 DEP的促氧化和促炎作用可以用有机DEP提取物以及从这些颗粒制备并通过硅胶色谱法分级分离的芳香族和极性化学基团在体外再现。 研究人员认为，氧化还原循环的多环芳烃及其氧化衍生物是呼吸树中氧化应激效应的原因，并且可以通过蛋白质组学方法围绕这一原理开发新的生物标志物。 新的蛋白质组展示技术已被开发用于鉴定组织培养细胞和支气管肺泡灌洗液（BAL）中的氧化修饰蛋白。 首席研究员的长期目标是利用加州大学洛杉矶分校全面的颗粒和蛋白质组学基础设施开发新的生物标志物，以跟踪颗粒物（PM）对易感人群的不良健康影响。 为了实现这一目标，研究人员将使用蛋白质组学方法来识别暴露于有机DEP化学品期间巨噬细胞和上皮细胞系中新诱导的以及氧化修饰的蛋白质（具体目标1）。 这些细胞将暴露于粗DEP提取物以及通过硅胶色谱法从这些颗粒中分离的极性和芳香族化学基团。 全细胞提取物将通过2-D电泳解析，然后进行蛋白质图像分析并生成2-D数据库，用于通过凝胶内消化和质谱法鉴定新表达的蛋白质。 氧化应激的贡献将被测试的硫醇抗氧化剂在培养基中的列入，以及通过实施新的技术，通过2-D电泳的蛋白质羰基和硝基酪氨酸的显示。 一种补充方法将是使用蛋白质组学来鉴定来自已建立的小鼠模型的BAL液中的氧化应激和氧化修饰蛋白质，该模型证明了雾化DEP对吸入抗原（卵清蛋白）的佐剂作用（特定目的2）。 研究者将确定用硫醇抗氧化剂治疗这些动物是否可以抑制BAL液中DEP诱导的氧化应激事件。 他们还将使用蛋白质羰基的展示来识别BAL液中的氧化修饰蛋白质。

DESCRIPTION (provided by applicant) An urgent need exists for toxicological profiling of ambient air particulates. The investigators are studying the adjuvant effects of DEP and CAPS in allergic airway inflammation and asthma. In vivo studies in animals and humans have demonstrated that DEP enhance IgE production and allergic inflammation during challenges by common environmental allergens. This effect involves the generation of oxidative stress and can be reversed with thiol antioxidants. The pro-oxidative and pro-inflammatory effects of DEP can be reproduced in vitro with organic DEP extracts as well as aromatic and polar chemical groups prepared from these particles and fractionated by silica gel chromatography. The investigators hvpothesize that redox cycling polycyclic aromatic hydrocarbons and their oxidized derivatives are responsible for oxidative stress effects in the respiratory tree, and that new biomarkers can be developed around this principle with a proteomics approach. Novel proteome display techniques have recently been developed to identify oxidatively modified proteins in tissue culture cells and bronchoalveolar lavage (BAL) fluid. The principal investigator's long-term goal is to use the comprehensive particle and proteomics infrastructure at UCLA to develop new biomarkers to follow the adverse health effects of particulate matter (PM) in susceptible populations. In order to accomplish this goal, the researchers will use a proteomics approach to identify newly induced as well as oxidatively modified proteins in macrophage and epithelial cell lines during exposure to organic DEP chemicals (Specific Aim 1). These cells will be exposed to crude DEP extracts as well as polar and aromatic chemical groups fractionated from these particles by silica gel chromatography. Whole cell extracts will be resolved by 2-D electrophoresis, followed by protein image analysis and generation of a 2-D database for identifying newly expressed proteins by in-gel digestion and mass spectrometry. The contribution of oxidative stress will be tested by the inclusion of thiol antioxidants in the culture medium, as well as by the implementation of novel techniques for the display of protein carbonyls and nitrotyrosines by 2-D electrophoresis. A complementary approach will be to use proteomics to identify oxidative stress and oxidatively modified proteins in the BAL fluid from an established murine model demonstrating the adjuvant effects of aerosolized DEP towards an inhaled antigen (ovalbumin)(Specific Aim 2). The investigators will determine whether treatment of these animals with a thiol antioxidant can suppress DEP-induced oxidative stress events in the BAL fluid. They will also use a display of protein carbonyls to identity oxidatively modified proteins in the BAL fluid.