Alcohol-induced liver injury is a significant global health problem and a leading cause of death. The mechanisms by which ethanol treatment causes cell death are not clear. CYP2E1 is induced by ethanol, is an active producer of reactive oxygen species and plays a role in ethanol-induced liver injury. Autophagy is a lysosomal-mediated pathway for removal and recycling of long-lived proteins, cellular organelles and lipid droplets. The goal of this R21 application is to evaluate whether autophagy can modulate CYP2E1-dependent ethanol toxicity in vitro and in vivo after acute and chronic ethanol treatment. The rationale is that CYP2E1 plays a role in ethanol-induced oxidant stress, fatty liver and liver injury. Autophagy, in some settings is protective against cell injury, while in other settings autophagy can promote cell toxicity. If autophagy is protective against ethanol/CYP2E1 toxicity, attempts to stimulate autophagy may prove to be helpful in lowering ethanol-induced liver injury. If autophagy promotes ethanol/CYP2E1 toxicity, inhibitors of autophagy may help to ameliorate ethanol hepatotoxicity. We will treat HepG2 cells which express CYP2E1 (E47 cells) or do not (C34 cells) with ethanol (0-100mM, 1-10 days) in the absence and presence of inhibitors of autophagy or activators of autophagy and assay the following: cell viability, apoptosis, oxidant stress, levels and activity of CYP2E1, mitochondrial dysfunction, steatosis, activation of mitogen activated protein kinases, hepatoprotective defense, autophagy and autophagy regulators such as Bcl-2, AMPK, mTOR. For in- vivo studies, wild type SV129 mice, SV129 CYP2E1 knockout (KO) mice, and SV129 CYP2E1 knockin (KI) mice in which human CYP2E1 has been "knocked" in will be treated acutely with ethanol (3g/kg, body wt. twice a day for 1, 2 and 4 days) or saline or be fed the high fat Lieber-DeCarli diet containing ethanol or isocaloric dextrose for 2 to 8 weeks. Some mice will also be treated with the autophagy inhibitor 3-methyladenine or the autophagy activator rapamycin. The effects of acute and chronic ethanol in the absence and presence of modifiers of autophagy in WT mouse with "normal" levels of mouse CYP2E1, in KO mice without CYP2E1 and in KI mice with elevated levels of human CYP2E1 on reactions described above will be evaluated. Time course experiments are designed to help define the sequence of events from the interactions between ethanol and CYP2E1 when autophagy is inhibited or activated. Time course experiments may also be informative as to whether the acute or chronic ethanol feeding may initially activate autophagy as an adaptive response to ethanol, which subsequently is not sustained with more prolonged acute ethanol treatment or chronic ethanol feeding.

Public Health Relevance

CYP2E1 plays a major role in ethanol-induced liver injury. Autophagy can protect or promote cell toxicity. We will evaluate whether autophagy can modulate CYP2E1-dependent ethanol toxicity after acute and chronic ethanol treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AA021362-02
Application #
8508151
Study Section
Health Services Research Review Subcommittee (AA)
Program Officer
Wang, Joe
Project Start
2012-07-10
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
2
Fiscal Year
2013
Total Cost
$187,192
Indirect Cost
$76,754
Name
Icahn School of Medicine at Mount Sinai
Department
Pharmacology
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
Zhou, Richard; Lin, Jianjun; Wu, Defeng (2014) Sulforaphane induces Nrf2 and protects against CYP2E1-dependent binge alcohol-induced liver steatosis. Biochim Biophys Acta 1840:209-18
Wang, Yuan; Kou, Yan; Wang, Xiaodong et al. (2014) Multifactorial comparative proteomic study of cytochrome P450 2E1 function in chronic alcohol administration. PLoS One 9:e92504
Yang, Lili; Rozenfeld, Raphael; Wu, Defeng et al. (2014) Cannabidiol protects liver from binge alcohol-induced steatosis by mechanisms including inhibition of oxidative stress and increase in autophagy. Free Radic Biol Med 68:260-7
Wang, Xiaodong; Wu, Defeng; Yang, Lili et al. (2013) Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1* in vivo. Free Radic Biol Med 63:175-86
Lu, Yongke; Ward, Stephen C; Cederbaum, Arthur I (2013) Nicotine enhances ethanol-induced fat accumulation and collagen deposition but not inflammation in mouse liver. Alcohol 47:353-7