Proteomics of DEP-induced Oxidative Stress

DEP 诱导的氧化应激的蛋白质组学

基本信息

批准号：
6929358
负责人：
Andre Elias Nel
金额：
$ 22.88万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-10 至 2007-07-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）迫切需要对环境空气微粒的毒理学分析。研究人员正在研究DEP和CAP在过敏性气道炎症和哮喘中的辅助作用。对动物和人类的体内研究表明，在常见环境过敏原挑战期间，DEP可以增强IgE产生和过敏性炎症。这种作用涉及氧化应激的产生，可以用硫醇抗氧化剂逆转。 DEP的促氧化和促炎作用可以在体外用有机DEP提取物以及由这些颗粒制备的芳香和极性化学基团在体外重现，并通过硅胶色谱法分离。研究者HVPOTHES认为，氧化还原循环多环芳烃及其氧化衍生物负责呼吸树中的氧化应激效应，并且可以通过蛋白质组学方法围绕该原理开发新的生物标志物。最近已经开发出了新型的蛋白质组显示技术，以鉴定组织培养细胞和支气管肺泡灌洗（BAL）流体中氧化修饰的蛋白质。主要研究者的长期目标是使用UCLA的综合粒子和蛋白质组学基础设施来开发新的生物标志物，以遵循易感人群中颗粒物（PM）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鉴定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在暴露于有机DEP化学物质的过程中（特定目标1）。这些细胞将暴露于粗糙的DEP提取物，以及通过硅胶色谱分离这些颗粒的极性和芳香化学基团。全细胞提取物将通过2-D电泳解决，然后进行蛋白质图像分析和2-D数据库的生成，以通过凝胶消化和质谱法鉴定新表达的蛋白质。氧化应激的贡献将通过将硫醇抗氧化剂纳入培养基中，以及通过2-D电泳的蛋白质羰基和硝基酪氨酸的实施来测试。一种互补的方法是使用蛋白质组学从已建立的鼠模型中鉴定出BAL液中的氧化应激和氧化性修饰的蛋白质，该模型证明了雾化DEP对吸入的抗原（Ovalbumin）的辅助作用（特异性AIM 2）。研究人员将确定用硫醇抗氧化剂治疗这些动物是否可以抑制BAL液中的DEP诱导的氧化应激事件。他们还将使用蛋白质羰基的显示，以在BAL液中氧化蛋白质的身份。