Role of cytochrome P4501B1 in oxygen-mediated pulmonary injury

描述(申请人提供)：高氧血症通常用于治疗早产儿和足月儿的呼吸窘迫和肺功能不全。然而，过量的氧气会导致慢性肺部疾病(CLD)的发展，这也被称为支气管肺发育不良(BPD)。高氧介导的肺损伤的分子机制尚不清楚，但也是由环境化学品产生的活性氧物种(ROS)是最有可能的候选。这项研究的中心假设是，肺细胞色素P450(CYP)1b1在高氧性肺损伤中起关键作用，它通过(I)作为促氧化剂，导致脂质过氧化产物(例如，F2-异前列腺素、异呋喃)的形成，进而介导肺损伤；(Ii)灭活新的内源性AHR配体，其通过诱导CyP1a酶来保护肺损伤；以及(Iii)加剧ROS介导的8-5‘环嘌呤或脂质过氧化产物产生的氧化DNA加合物的形成，从而导致氧介导的损伤。我们提出了以下具体目标。1.为了验证这一假设，缺乏CYP1B1基因的小鼠将比野生型小鼠更不容易受到氧损伤的影响，并且在高氧暴露之前用CYP1A/1B诱导剂2-萘黄酮预处理可以增强CyP1B1缺失的有益效果。2.验证肺内皮细胞和Clara细胞中CYP1B1缺失将导致氧介导的肺损伤易感性不同的假说。需要检验的具体假设是，有条件地删除CYP1B1将提供有关特定肺细胞类型的机制信息，这些细胞类型有助于保护肺免受高氧性肺损伤。3.验证氧化DNA加合物在高氧性肺损伤中的作用机制，以及这些加合物是否可作为高氧性肺损伤和BPD的新生物标志物。需要检验的假设是，与WT小鼠相比，CYP1B1基因缺失的高氧小鼠的肺部表现出较轻的DNA氧化损伤，而CYP1B1基因缺失小鼠中CYP1A酶的增强表达在一定程度上有助于改善CYP1B1基因缺失小鼠的DNA氧化损伤。环嘌呤二核苷酸，即ACA或GCA，或由F2-异前列腺素产生的直接加合物将作为BPD的早期生物标志物的假设将得到检验。我们还将检验这一假设，即CYP1B1基因多态是婴儿BPD发生的危险因素。拟议的研究应有助于开发预防/治疗人类肺部疾病(如BPD和ARDS)的新战略。如果CYP1B1在高氧性肺损伤中起到促氧化剂的作用，那么CYP1B1抑制剂可能被开发为人类BPD和其他由补充氧介导的肺部疾病(如ARDS)的潜在预防/治疗候选药物。这些研究也适用于环境化学物质引起的ROS介导的疾病。 公共卫生相关性：高氧通常用于治疗早产儿和足月儿的呼吸窘迫和肺功能不全，以及患有ARDS的成年人。然而，高氧导致慢性肺部疾病(CLD)，也称为支气管肺发育不良(BPD)。该项目旨在开发预防/治疗人类BPD和ARDS等肺部疾病的新战略。

DESCRIPTION (provided by applicant): Hyperoxia is routinely used to treat respiratory distress and lung inadequacy in preterm and term infants. However, excess oxygen contributes to the development of chronic lung disease (CLD), which is also called bronchopulmonary dysplasia (BPD). The molecular mechanisms of hyperoxia-mediated pulmonary injury are not understood, but reactive oxygen species (ROS), which are also produced by environmental chemicals, are the most likely candidates. The central hypothesis of the proposed research is that pulmonary cytochrome P450 (CYP)1B1 plays a key role in hyperoxic lung injury by (i) acting as a pro-oxidant, leading to enhanced formation of lipid peroxidation products (e.g., F2-isoprostanes, isofurans) that in turn mediate lung injury; (ii) inactivating novel endogenous AHR ligands, which protect against lung injury by inducing CYP1A enzymes; and (iii) exacerbating enhanced formation of ROS-mediated oxidative DNA adducts derived from 8- 5'cyclopurines or lipid peroxidation products, resulting in oxygen-mediated injury. We propose the following Specific Aims. 1. To test the hypothesis mice lacking the gene for Cyp1b1 will be less susceptible to oxygen injury than wild type mice, and that the beneficial effects of Cyp1b1 deletion is augmented by pre-treatment of the mice with the CYP1A/1B inducer, 2-naphthoflavone (BNF) prior to hyperoxic exposures. 2. To test the hypothesis that Cyp1b1-deletion in pulmonary endothelial cells or Clara cells will result in differential susceptibilities to oxygen-mediated lung injury. The specific hypothesis to be tested is that conditional deletion of Cyp1b1 will offer mechanistic information regarding the specific lung cell types that contribute to the protection against hyperoxic lung injury. 3. To test the hypothesis that oxidative DNA adducts contributes mechanistically to lung injury mediated by hyperoxia, and that these adducts will serve as novel biomarkers of hyperoxic lung injury and BPD. The hypothesis to be tested is that lungs of hyperoxic mice deficient in CYP1B1 will display lesser oxidative DNA damage than WT mice, and augmented expression of CYP1A enzymes in the Cyp1b1-null mouse in part contributes to the amelioration of oxidative DNA damage in the Cyp1b1-null mouse. The hypothesis that cyclopurine dinucleotides, i.e. AcA or GcA, or direct adducts resulting from F2-isoprostanes will serve as early biomarkers of BPD will be tested. We will also test the hypothesis that genetic polymorphisms in CYP1B1 are risk factors for the development of BPD in infants. The proposed studies should help in the development of novel strategies for the prevention/treatment of lung diseases (e.g. BPD and ARDS) in humans. Should CYP1B1 play a pro-oxidant roie in hyperoxic lung injury, then CYP1B1 inhibitors could be developed as potential preventive/therapeutic candidates against BPD and other lung diseases mediated by supplemental oxygen (e.g. ARDS) in humans. These studies are also applicable to ROS-mediated disorders caused by environmental chemicals. PUBLIC HEALTH RELEVANCE: Hyperoxia is routinely used in the treatment of respiratory distress and pulmonary insufficiency in preterm and term infants, and in adults with ARDS. However, hyperoxia contributes to the development of chronic lung disease (CLD), also known as bronchopulmonary dysplasia (BPD). This project is aimed at developing novel strategies for the prevention/treatment of lung diseases such as BPD and ARDS in humans.