Proteomics of DEP-induced Oxidative Stress

描述(由申请人提供) 迫切需要对环境空气中的颗粒物进行毒理学分析。研究人员正在研究DEP和CAPS在过敏性呼吸道炎症和哮喘中的佐剂作用。在动物和人类的体内研究表明，在常见环境过敏原的攻击下，DEP可增加IgE的产生和过敏性炎症。这种影响涉及氧化应激的产生，并可被硫醇抗氧化剂逆转。DEP的促氧化和促炎作用可以用有机DEP提取物以及由这些颗粒制备的芳香族和极性化学基团在体外复制，并通过硅胶层析进行分级。研究人员认为，氧化还原环状多环芳烃及其氧化衍生物是呼吸树氧化应激效应的罪魁祸首，并可利用蛋白质组学方法围绕这一原理开发新的生物标志物。最近发展了新的蛋白质组展示技术来鉴定组织培养细胞和支气管肺泡灌洗液(BAL)中的氧化修饰蛋白质。主要研究人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来开发新的生物标记物，以跟踪颗粒物(PM)在易感人群中对健康的不利影响。为了实现这一目标，研究人员将使用蛋白质组学方法来鉴定在接触有机DEP化学品期间巨噬细胞和上皮细胞系中新诱导的和氧化修饰的蛋白质(特定目标1)。这些细胞将暴露在粗DEP提取物以及通过硅胶层析从这些颗粒中分离出来的极性和芳香族化学基团中。整个细胞的提取物将通过二维电泳法进行解析，然后进行蛋白质图像分析，并建立一个二维数据库，通过凝胶内消化和质谱仪来鉴定新表达的蛋白质。氧化应激的贡献将通过在培养基中加入硫醇抗氧化剂，以及通过实施新的技术来通过二维电泳法显示蛋白质羰基和硝基酪氨酸来测试。一种补充方法将是使用蛋白质组学从已建立的小鼠模型中识别BAL液中的氧化应激和氧化修饰蛋白，该模型展示了雾化DEP对吸入性抗原(卵清蛋白)的佐剂作用(特定目标2)。研究人员将确定用硫醇抗氧化剂处理这些动物是否可以抑制DEP诱导的BAL液中的氧化应激事件。他们还将使用蛋白质羰基的展示来识别BAL液中氧化修饰的蛋白质。

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.