MECHANISM OF CYTOCHROME P450 GENE EXPRESSION IN THE HYPEROXIC DEVELOPING LUNG

氧气暴露会增加肺部氧化剂的产生，并导致急性 微血管损伤，可导致致命的肺水肿。虽然 关于抗氧化防御的调节，我们知道得很多 高氧血症中氧化剂产生的机制、部位及调控 肺是不确定的。在以前的工作中，我们定义了生理和 氧暴露期间羔羊肺内发生的生化变化。 我们发现氧气暴露增加了肺细胞色素P450的含量。 和两个潜在的微粒体氧化剂来源P450 IA1和P450的活性 IIB1。两种化学性质不同的P450对羔羊的处理 抑制剂能显著减轻体内高氧性肺损伤。 免疫细胞化学数据表明，羔羊肺内皮细胞含有 细胞色素P450酶。这些数据有力地支持了一般 假设细胞色素P450衍生的氧化剂参与了 羔羊急性肺氧中毒的发病机制。 这项提议的目的是扩大我们的生理和生化能力 将分子和细胞生物学技术应用于 了解氧气暴露增加肺P450的机制。 提供的初步数据表明，高氧诱导 体内肺P450 IIB1和IA1活性增加之前是一种 它们各自的RNA水平增加。肝脏P450的RNA水平是 在体内不受氧气暴露的影响。我们还展示了积极的一面 培养细胞氧分压升高对P450 IA1的调节作用 它们已经被稳定地转染了含有完整的- 小鼠P450IA15‘侧翼区的长度。 该提案概述了在活体和培养肺中进行的一系列实验 使用啮齿动物核苷酸探针测量稳态变化的细胞 不同浓度的黄曲霉毒素对肺组织P450 RNA表达的影响 氧气，单独和在影响肺P450的试剂存在的情况下 级别。我们还将分离编码羔羊肺的全长cDNA克隆 P450基因IA1和IIB1及其5‘侧翼区的鉴定 在基因组DNA中。然后，包含5‘侧翼区的融合基因 与报告基因相连的基因将被构建并用于转染 验证羔羊肺P450基因表达的假设的实验 直接或间接地在特定的肺细胞中正调控 通过增加环境中的氧分压。我们还将构建相应的 核苷酸探针，并用它们来原位定位P450特异性RNA。 这些研究将导致对监管的重要新见解 在发育中的肺中编码产生氧化剂的酶的基因，可能 也可以作为研究小分子如何调控基因的模型系统 在高等真核生物中的表达。此外，这些研究可能会导致 降低氧化应激安全有效方法的研究进展 需要治疗的肺部疾病患者的氧气暴露 含氧量很高。

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.