Proteomics of DEP-induced Oxidative Stress

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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.