Oxygen exposure increases lung oxidant production and causes acute microvascular injury which can lead to fatal pulmonary edema. Although much is known about the regulation of anti-oxidant defenses, the mechanisms, sites and regulation of oxidant production in the hyperoxic lung are uncertain. In previous work, we defined the physiologic and biochemical changes which occur in the lamb lung during oxygen exposure. We demonstrated that oxygen exposure increases lung cytochrome P450 amount and activity of two potential microsomal oxidant sources, P450 IA1 and IIB1. Treatment of lambs with either of two chemically dissimilar P450 inhibitors significantly reduced hyperoxic lung injury in vivo. Immunocytochemical data indicate that lamb lung endothelial cells contain cytochrome P450 enzymes. These data strongly support the general hypothesis that cytochrome P450-derived oxidants are involved in the pathogenesis of acute pulmonary oxygen toxicity in lambs. The objective of this proposal is to extend our physiologic and biochemical studies by applying the techniques of molecular and cell biology to understand the mechanisms by which oxygen exposure increases lung P450. Preliminary data are presented which indicate that the hyperoxia-induced increases in lung P450 IIB1 and IA1 activities in vivo are preceded by an increase in their respective RNA's. Liver RNA levels for P450 are unaffected by oxygen exposure in vivo. We also demonstrate positive regulation of P450 IA1 by increased oxygen tension using cultured cells which have been stably transfected with a fusion gene containing the full- length mouse P450 IA1 5' flanking region. The proposal outlines a series of experiments in vivo and in cultured lung cells using rodent nucleotide probes to measure changes in steady-state lung P450 RNA levels following treatment with varying concentrations of oxygen, alone and in the presence of agents which influence lung P450 levels. We will also isolate full-length cDNA clones encoding lamb lung P450 genes IA1 and IIB1 and use them to identify their 5' flanking regions in genomic DNA. Then, fusion genes containing the 5' flanking regions linked to reporter genes will be constructed and used in transfection experiments to test the hypothesis that lamb lung P450 gene expression is positively regulated in specific lung cells either directly or indirectly by increases in ambient oxygen tension. We will also construct homologous nucleotide probes and use them to localize P450-specific RNA in situ. These studies will lead to important new insights into the regulation of genes which encode oxidant-producing enzymes in the developing lung and may also serve as a model system to study how small molecules regulate gene expression in higher eukaryotes. In addition, these studies may lead to the development of safe and effective methods to reduce the oxidant stress of oxygen exposure in patients with lung diseases who require treatment with high concentrations of oxygen.
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