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. However, hyperoxia causes lung damage in animals and humans. The central hypothesis of the research proposed in this application is that hyperoxia induces cytochrome P4501A (CYP1 A) enzymes in vivo through mechanisms involving transcriptional activation of the corresponding promoters, and that the induced CYP1A enzymes play protective roles against hyperoxic lung injury by catalyzing the detoxication of ROS-generated endogenous molecules (e.g., F2isoprostanes and isofurans), whose levels are elevated in the lungs of hyperoxic animals.
The specific aims are: (1) to test the hypothesis that hyperoxia induces CYP1A1 and 1A2 gene expression through mechanisms involving transcriptional activation of the CYP1A1 or 1A2 promoter, presumably by generating endogenous ligands for the Ah receptor (AHR). We will use a transgenic mouse carrying a 10 kb human CYP1A1 promoter or an 8 Kb mouse promoter and luciferase reporter gene. These mice will be maintained in room air or exposed to hyperoxia for selected time points, and luciferase activities will be determined by in vivo bioluminescent imaging. (2) To characterize the molecular mechanisms of modulation of CYP1A1 gene by hyperoxia in cultured mouse lung cells. Mouse transformed Clara cells (mtCC) will be exposed to hyperoxia and time dependent effects on CYP1A1 parameters'will be determined. (3) To test the hypothesis that mice lacking the gene for CYP1A1, CYP1A2, or both genes (CYP1A1/1A2 double knockouts) will be more susceptible to hyperoxia-induced lung injury than similarly exposed wild type mice, and that the CYP1A enzymes play 'protective roles against hyperoxic lung injury by catalyzing the detoxication of ROS-generated endogenous molecules (e.g., F2 isoprostanes) in the lungs of hyperoxic animals. The mice will be exposed to hyperoxia for selected time points, and parameters of lung injury and those of pulmonary and hepatic CYP1A1/1A2 expression (run-on transcription, real time RT-PCR/Northern, Western, enzyme activities) will be studied. Levels of F2 isoprostanes and isofurans will be measured in lung and liver by gas chromatography/ mass spectrometry (GC-MS). (4). to test the hypothesis that liver P450s contribute to hyperoxic lung injury in vivo. We will expose wild type, and liver- specific P450 reductase (CPR) null mice to hyperoxia, and determine if hyperoxic responses are altered in the CPR-null mice. The long-term of this proposal is to develop rational strategies for the prevention and/or treatment of chronic lung diseases in infants and adults suffering from respiratory distress and lung dysfunction.
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