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.

Mechanisms responsible for alcoholic liver disease are not fully understood. CYP2A5 can be induced by ethanol to a much greater extent than CYP2E1, and CYP2A5 induction by ethanol was CYP2E1 dependent. Identification of CYP2A5 as a potential enzyme to metabolize alcohol and generate reactive oxygen species would be very informative in developing therapeutic interventions against alcohol-induced liver injury.