Role of Cyt P450-Mediated Oxidation in Chemical-Induced

Cyt P450 介导的氧化在化学诱导的氧化中的作用

• 批准号: 7327217
• 负责人: BURHAN I GHANAYEM
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7327217
• 关键词: Role; Cyt; P450; Mediated; Oxidation

Human exposure to environmental chemicals is considered a contributing factor to increased susceptibility to chemical induced toxicity, genotoxicity, and carcinogenicity. The mechanism(s) of action of these chemicals are not well understood, however, a great number of them are metabolized to epoxide intermediates via CYP2E1. We therefore hypothesized that epoxidation of these chemicals is a pre-requisite for the induction of toxicity, genotoxicity, and carcinogenicity. Using trichloroethylene (TCE) as a model chemical, studies were undertaken to investigate the metabolic and molecular basis for the induction of lung damage by epoxide-forming chemicals. Trichloroethylene (TCE) elicits lung cytotoxicity that selectively damages bronchiolar Clara cells. Current study was undertaken to test the hypothesis that bronchiolar damage is associated with TCE bioactivation by CYP2E1 and CYP2F2 within the Clara cells. Histopathology confirmed dose-dependent Clara cell injury and disintegration of the bronchiolar epithelium in mice treated with TCE doses of 200 to 1000 mg/kg. Immunohistochemical studies, using an antibody that recognizes dichloroacetyl lysine adducts, revealed dose-dependent formation of adducts in the bronchiolar epithelium. The localization of dichloroacetyl adducts in the Clara cells coincided with bronchiolar damage in the lungs of TCE-treated mice. Pretreatment of mice with diallyl sulfone, an inhibitor of CYP2E1 and CYP2F2, abrogated formation of the dichloroacetyl protein adducts and protected against TCE-induced bronchiolar damage. Treatment of wild-type and Cyp2e1-/- mice also induced bronchiolar damage that correlated with the presence of adducts in the bronchiolar epithelium. Immunoblotting, using lung microsomes from TCE-treated mice, showed dose-dependent production of dichloroacetyl adducts that co-migrated with CYP2E1 and CYP2F2. However, TCE treatment resulted in loss of CYP2E1 and CYP2F2 proteins and p-nitrophenol hydroxylation, a catalytic activity associated with both P450 enzymes. The TCE metabolite, chloral hydrate, was formed in incubations of TCE with lung microsomes from CD-1, wild-type and Cyp2e1-/- mice. Levels of chloral hydrate were higher in CD-1 than in either wild-type or Cyp2e1-/- mice; no difference was detected between the wild-type and Cyp2e1-/- mice. These findings indicated that dichloroacetyl adducts formed within the Clara cells and involving CYP2F2 and CYP2E1 are associated with TCE-induced bronchiolar cytotoxicity. Public Health or Environmental Health Significance: Cytochrome P450 2E1 (CYP2E1) is responsible for the bioactivation of a wide variety of environmentally important xenobiotics including 1,3-butadiene, acetaldehyde, acetaminophen, aniline, benzene, carbon tetrachloride, trichloroethylene, dichloroethylene, ethylene glycol, vinyl chloride, acrylonitrile, and nitrosamines. Most of these chemicals undergo oxidative metabolism via the cytochrome P450 enzymes to form epoxide intermediates that are thought to play a role in increased human susceptibilities to various diseases. Epoxides are three membered cyclic ethers and are among the most potent known acute toxicants, mutagens, reproductive toxins, and carcinogens. Environmental chemicals that are metabolized to epoxides share a similar pattern of activity. Further, genetic polymorphisms in CYP2E1 were considered important risk factors in the development of xenobiotic-induced diseases in humans. Once the mechanism of action of epoxides is established, the present approach may be used to predict the effects of chemicals that are metabolized to epoxides. Further, once the role of epoxides in environmentally caused human diseases is established, a more accurate assessment of human risks to epoxide-forming chemicals may be accomplished and the connection between metabolism of chemicals,toxicity, and CYP polymorphisms in humans may be established. CYP2E1 polymorphisms and variability in CYP2E1 activity associated with, for example: diabetes, obesity, starvation, and alcohol consumption, may result in increased metabolic efficiencies leading to differential susceptibilities to the epoxide-forming chemicals in humans. In addition, many of the model chemicals that we use in our research are of interest to the National Toxicology Program and therefore our work will have the additional advantage of complementing the National Toxicology Program mission. Finally, current work suggest that CYP-null mice are an appropriate model for the investigation of the role of oxidative metabolism in the toxicity, mutagenicity, and carcinogenicity of environmental chemicals.

人类接触环境化学品被认为是增加化学毒性、遗传毒性和致癌性的一个因素。这些化学物质的作用机制尚不清楚，然而，其中大量通过 CYP2E1 代谢为环氧化物中间体。因此，我们假设这些化学物质的环氧化是诱导毒性、遗传毒性和致癌性的先决条件。使用三氯乙烯（TCE）作为模型化学品，进行了研究以调查环氧化物形成化学品诱导肺损伤的代谢和分子基础。 三氯乙烯 (TCE) 会引起肺细胞毒性，选择性损害细支气管克拉拉细胞。目前的研究旨在检验以下假设：细支气管损伤与 Clara 细胞内 CYP2E1 和 CYP2F2 的 TCE 生物激活有关。组织病理学证实，在接受 200 至 1000 mg/kg TCE 剂量治疗的小鼠中，存在剂量依赖性克拉拉细胞损伤和细支气管上皮崩解。使用识别二氯乙酰基赖氨酸加合物的抗体进行的免疫组织化学研究揭示了细支气管上皮中加合物的形成呈剂量依赖性。 Clara 细胞中二氯乙酰加合物的定位与 TCE 治疗小鼠肺部细支气管损伤一致。用二烯丙基砜（CYP2E1 和 CYP2F2 的抑制剂）对小鼠进行预处理，消除了二氯乙酰蛋白加合物的形成，并防止 TCE 诱导的细支气管损伤。对野生型和 Cyp2e1-/- 小鼠的治疗也诱导了细支气管损伤，这与细支气管上皮中加合物的存在相关。使用 TCE 处理小鼠的肺微粒体进行的免疫印迹显示，二氯乙酰加合物的产生呈剂量依赖性，并与 CYP2E1 和 CYP2F2 共同迁移。然而，TCE 处理导致 CYP2E1 和 CYP2F2 蛋白丢失以及对硝基苯酚羟基化，这是与两种 P450 酶相关的催化活性。 TCE 代谢物水合氯醛是在 TCE 与 CD-1、野生型和 Cyp2e1-/- 小鼠的肺微粒体孵育时形成的。 CD-1 中的水合氯醛水平高于野生型或 Cyp2e1-/- 小鼠；野生型小鼠和 Cyp2e1-/- 小鼠之间没有检测到差异。这些发现表明，Clara 细胞内形成的二氯乙酰基加合物涉及 CYP2F2 和 CYP2E1，与 TCE 诱导的细支气管细胞毒性有关。 公共卫生或环境健康意义：细胞色素 P450 2E1 (CYP2E1) 负责多种对环境重要的异生物质的生物活化，包括 1,3-丁二烯、乙醛、对乙酰氨基酚、苯胺、苯、四氯化碳、三氯乙烯、二氯乙烯、乙二醇、氯乙烯、丙烯腈和亚硝胺。大多数这些化学物质通过细胞色素 P450 酶进行氧化代谢，形成环氧化物中间体，这些中间体被认为在增加人类对各种疾病的易感性方面发挥了作用。环氧化物是三元环醚，是已知最有效的急性毒物、诱变剂、生殖毒素和致癌物。代谢为环氧化物的环境化学物质具有相似的活性模式。此外，CYP2E1 的遗传多态性被认为是人类外源性疾病发生的重要危险因素。一旦建立了环氧化物的作用机制，本方法可用于预测代谢为环氧化物的化学物质的作用。此外，一旦确定了环氧化物在环境引起的人类疾病中的作用，就可以对人类对环氧化物形成化学品的风险进行更准确的评估，并且可以建立化学品代谢、毒性和人类CYP多态性之间的联系。 CYP2E1 多态性和 CYP2E1 活性的变异性与糖尿病、肥胖、饥饿和饮酒等相关，可能会导致代谢效率增加，从而导致人类对环氧化物形成化学物质的敏感性不同。此外，我们在研究中使用的许多模型化学品引起了国家毒理学计划的兴趣，因此我们的工作将具有补充国家毒理学计划任务的额外优势。最后，目前的工作表明，CYP 缺失小鼠是研究氧化代谢在环境化学物质的毒性、致突变性和致癌性中的作用的合适模型。