CYP2A5 and alcoholic liver disease

描述（由申请人提供）：酒精性肝损伤是一个重大的全球健康问题，也是世界范围内死亡的主要原因。了解这种损伤的机制对于设计新的治疗方法和改善许多患者的生活质量至关重要。乙醇处理导致细胞死亡的机制尚不清楚。乙醇的许多肝毒性作用都与它在肝脏中的代谢有关。CYP2E1可被乙醇诱导，并能代谢乙醇，被认为是酒精性肝病（ALD）的危险因素之一。据报道，人肝脏CYP2A6表达在ALD患者中升高。在小鼠模型中，我们发现小鼠CYP2A5 （CYP2A6的同源物）被慢性乙醇喂养诱导的程度远远大于CYP2E1，而且非常有趣的是，小鼠体内对CYP2A5的乙醇诱导是依赖于CYP2E1的。CYP2A5/6代谢香豆素、尼古丁、硝基胺等重要临床药物，因此乙醇诱导其产生具有重要的毒理学意义。在本应用中，我们将进一步了解小鼠CYP2A5是否可能参与ALD的发病机制。我们假设CYP2E1介导的ROS激活氧化还原敏感核因子-类红细胞2相关因子2 (Nrf2)， Nrf2上调CYP2A5，其可能代谢乙醇并增加氧化应激，从而促进ALD的发病，即CYP2E1至少部分通过CYP2A5促进ALD。AIM 1旨在通过体内乙醇饲养模型和体外报告基因转染模型，研究ROS和Nrf2在乙醇诱导CYP2A5中的可能作用。AIM 2旨在检测CYP2A5是否与酒精性肝损伤有关。Cyp2a5-/-小鼠将被用于研究Cyp2a5在ALD发病机制中的可能作用，Cyp2a5 SiRNA将被用于确定Cyp2a5在乙醇代谢和ROS产生以及cyp2e1介导的肝损伤中的作用。从WT和cyp2a5-/-小鼠的慢性乙醇液体饲料中分离的微粒体将被用来测定肝脏微粒体中cyp2a5的乙醇代谢和ROS的产生。乙醇诱导CYP2A5和CYP2E1的作用是一个新的发现，因为大多数关于微粒体乙醇代谢的研究都是在大鼠微粒体中进行的，而大鼠微粒体缺乏小鼠CYP2A5的同源物CYP2A3。我们认为本申请中提出的实验符合R21机制的范畴，支持探索性研究。希望本研究能够回答上述问题，为乙醇代谢和ALD的研究打开一扇新的窗口。

DESCRIPTION (provided by applicant): Alcohol-induced liver injury is a significant global health problem and a leading cause of death worldwide. Understanding the mechanisms of this injury is critical for designing new therapies and improving the quality of life of many patients. The mechanisms by which ethanol treatment causes cell death are still not clear. Many of the hepatic toxic effects of ethanol have been linked to its metabolism in the liver. CYP2E1 can be induced by ethanol and is able to metabolize ethanol and is considered as one of the risk factors of alcoholic liver disease (ALD). It was reported that human hepatic CYP2A6 expression was increased in patients with ALD. In a mouse model, we found that mouse CYP2A5, the ortholog of CYP2A6, can be induced by chronic ethanol feeding to a much greater extent than CYP2E1, and very interestingly, ethanol induction of CYP2A5 in mice is CYP2E1-dependent. CYP2A5/6 metabolizes important clinical drugs such as coumarin, nicotine, nitroamines so its induction by ethanol is of major toxicological significance. In this Application, we move towards understanding whether mouse CYP2A5 may be involved in the pathogenesis of ALD. We hypothesize that CYP2E1-mediated ROS activates redox sensitive nuclear factor-eythroid 2-related factor 2 (Nrf2), and Nrf2 upregulates CYP2A5, which may metabolize ethanol and increase oxidative stress which contributes to pathogenesis of ALD i.e., CYP2E1 promotes ALD at least partially through CYP2A5. AIM 1 is designed to examine the possible role of ROS and Nrf2 in the induction of CYP2A5 by ethanol by using an in vivo ethanol feeding model and in vitro reporter gene transfection model. AIM 2 is designed to examine if CYP2A5 contributes to alcohol-induced liver injury. Cyp2a5-/- mice will be used to examine the possible role of CYP2A5 in pathogenesis of ALD, and cyp2a5 SiRNA will be applied to define the role of CYP2A5 in ethanol metabolism and ROS production and CYP2E1-mediated liver injury. Micosomes isolated from chronic ethanol liquid diet fed WT and cyp2a5-/- mice will be used to define hepatic microsomal ethanol metabolism and ROS production by CYP2A5. Ethanol induction of CYP2A5 and the role of CYP2E1 is a novel new finding, because most studies about microsomal ethanol metabolism were carried with rat microsomes, which lack CYP2A3, the ortholog of mouse CYP2A5. We think the experiments proposed in this Application fit in the category of the R21 mechanism, which supports exploratory studies. Hopefully, this study will answer the above questions and open a new window for ethanol metabolism and ALD study.