Supplemental oxygen administration is used extensively in the treatment of pulmonary insufficiency that is commonly observed in patients with acute respiratory distress syndrome (ARDS) and in preterm and term infants. In premature infants, hyperoxic therapies contribute to the development of bronchopulmonary dysplasia (BPD). The central hypothesis of the research proposed in this application is that cytochrome P450 (CYP)1A enzymes play a protective role against oxygen-mediated lung injury.
The Specific Aims are: (1) To characterize the mechanisms of modulation of CYP1A enzymes in lungs and liver of rats exposed to hyperoxia. Adult rats will be exposed to hyperoxia or maintained in room air, and rates of hepatic and pulmonary CYP1AI/1A2 gene (run-on transcription and Northern/RT-PCR) and protein expression (Western blotting and catalytic activities) will be studied at selected time points. 2. To test the hypothesis that sustained induction of CYP1A1 will decrease the susceptibility of animals to lung injury. Rats will be pretreated with 3-methylcholanthrene, which persistently induces pulmonary and hepatic CYPIA1 enzymes, followed by exposure to hyperoxia. Parameters of CYP1A, phase II enzymes, and lung injury will be analyzed. Levels of lipid peroxidation products (e.g., isofurans) will also be studied. 3. To determine the cell-specific expression of CYP1A in lungs and liver of rats exposed to hyperoxia. The effects of hyperoxia on cell-specific localization of hepatic/pulmonary CYP1A proteins (immunohistochemistry) and their corresponding mRNAs (in situ hybridization) will be determined. 4.To characterize the molecular mechanisms of modulation of CYP1A1 gene by hyperoxia in cultured cells. Cell lines of rat (H411E) or human (H441) origin will be exposed to hyperoxia, and CYP1A expression will be studied. Regulatory elements responsible for CYP1A1 gene induction by hyperoxia will be identified by transfection experiments involving CYP1A1 promoter. In order to determine if CYP1A1 induction will translate into protection against hyperoxia, H441 cells overexpressing CYP1A1 will be exposed to hyperoxia and parameters of cell injury will be studied. The proposed studies should provide critical and conceptual foundation(s) for the development of rational strategies for the prevention and treatment of chronic lung disease associated with hyperoxic exposures in adults and premature infants. ? ?
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