Particulate air pollution increases respiratory disease morbidity and mortality, especially in individuals with inflammatory lung disease. Mechanisms of particle health effects at the cellular level are poorly understood. The general hypothesis of this proposal is that particle toxicity operates through oxidant-dependent mechanisms: 1) derived from particle components, 2) targeted to specific intracellular compartments, and 3) enhanced by pre-existing inflammation. Our strategy is to identify the cellular target(s) of PM mediated oxidant stress and to quantify the biological impact of blocking oxidant production at each one of the affected targets.
Aim 1 will test the hypothesis that particles cause inflammation and toxicity by oxidant-dependent mechanisms. We will measure concentrated particles (CAPs) ability to increase ROS in vivo and in vitro, determine CAPs effects on production and detoxification of oxidants, and evaluate the biological impact of CAPs-induced oxidant stress. Treatment with antioxidants will test the role of oxidant mediators in CAPs biological responses.
Aim 2 will test the hypothesis that particles cause oxidant stress by direct interaction with cellular targets. We will test CAPs effect on H2O2 production in cells treated with specific inhibitors, in subcellular fractions supplemented with their optimal substrates, and in genetically altered mice lacking or overexpressing critical antioxidants in different cellular compartments.
Aim 3 will test the postulate that soluble components of CAPs cause oxidant and biological responses in epithelial cells. Water-soluble, insoluble, and organic fractions of CAPs will be assessed for ability to cause oxidant and biological responses in vitro. The variability in responses of different days' CAPs exposures will be exploited to identify bioactive compositional and source factors.
Aim 4 will test the hypothesis that particles cause enhanced oxidant generation in inflammation-primed epithelial cells and whole lungs. We will quantify CAPs-induced increases in H2O2 and cellular damage in naive and TNF-primed AT II cells. To identify the cellular target of CAPs and TNF we will use specific inhibitors of suspected signaling steps. Treatment with antioxidants will test the role of oxidant mediators. The role of TNF on the response to CAPs in vivo will be directly tested by using knockout mice deficient in TNF receptors.
