The mechanism(s) by which alcohol causes cell injury are still not clear. A major mechanism that is a focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a key role on how ethanol induces """"""""oxidative stress"""""""". We hypothesize that increased oxidative stress from CYP2E1 induction in vivo sensitizes hepatocytes to chronic ethanol induced hepatotoxicity and steatosis. We propose that oxidants such as peroxynitrite, activation of MAP kinases such as JNK and/or p38 MAPK, a decline in activation of NF-:B and synthesis of survival factors, and mitochondrial dysfunction are downstream mediators of the CYP2E1 potentiated hepatotoxicity. We propose that such downstream mediators from CYP2E1 in association with ethanol-induced elevation of TNF1, homocysteine and ER stress activate lipogenic transcription factors and enzymes while decreasing activation of lipolytic transcription factors and enzymes, thereby resulting in fatty liver. Preliminary data in support of these hypotheses are the findings that ethanol-induced fatty liver is blunted in CYP2E1 knockout mice and restored in humanized CYP2E1 knockin mice. Hepatotoxicity and elevated oxidative/nitrosative stress in found in the CYP2E1 knockin mice with high levels of human CYP2E1 after chronic ethanol feeding. The ethanol-fed CYP2E1 knockin mice appear to be an effective ORAL model of alcohol-induced hepatotoxicity. Wild type, CYP2E1 knockout and CYP2E1 knockin mice will be fed the high fat Lieber-DeCarli diet for varying times, e.g. 1 to 6 weeks. Controls will be pair-fed with dextrose.
AIM1 is designed to evaluate mechanisms by which elevated expression of CYP2E1 causes hepatotoxicity in the ethanol-fed CYP2E1 knockin mice and will include assays of oxidative/nitrosative stress, mitochondrial dysfunction, CYP2E1, iNOS, TNF1 , MAPK and NF:B.
AIM 2 is designed to evaluate pathways by which CYP2E1 contributes to alcohol-induced fatty liver, including assays of levels of transcription factors such as SREBP-1c and PPAR1, content of key and rate- limiting lipogenic and lipolytic enzymes;rates of fatty acid oxidation will be determined in the wild type, CYP2E1 knockout and CYP2E1 knockin mice fed ethanol or dextrose. The time course of changes in the downstream CYP2E1 effectors will be determined relative to that for ethanol-induced steatosis and hepatotoxicity. The effect of inhibitors of CYP2E1, iNOS, oxidative/nitrosative stress, MAPK, mitochondrial dysfunction, TNF1 production on ethanol-induced steatosis and liver injury in wild type and CYP2E1 knockin mice will be determined. We believe these experiments will provide molecular mechanisms by which CYP2E1 promotes ethanol-induced liver injury and fatty liver and may further the development of new therapeutic designs to treat or minimize progression of ethanol-induced liver injury, a stated goal of RFA-AA-006.

Public Health Relevance

Mechanisms responsible for alcohol-induced hepatotoxicity and fatty liver are not fully understood. With the novel use of CYP2E1 knockout mice and humanized CYP2E1 knockin mice, a role for CYP2E1 in these toxic actions of alcohol has been defined. Identification of downstream effectors of alcohol/CYP2E1 actions would be very informative in developing therapeutic interventions against alcohol-induced liver injury and fatty liver.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
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Special Emphasis Panel (ZAA1-JJ (06))
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Gentry, Thomas
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Icahn School of Medicine at Mount Sinai
Schools of Medicine
New York
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Lu, Yongke; Cederbaum, Arthur I (2018) Cytochrome P450s and Alcoholic Liver Disease. Curr Pharm Des 24:1502-1517
Lu, Yongke; Cederbaum, Arthur I (2015) Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity. Biomolecules 5:2659-74
Cederbaum, Arthur I; Lu, Yongke; Wang, Xiaodong et al. (2015) Synergistic toxic interactions between CYP2E1, LPS/TNF?, and JNK/p38 MAP kinase and their implications in alcohol-induced liver injury. Adv Exp Med Biol 815:145-72
Cederbaum, Arthur I (2015) Molecular mechanisms of the microsomal mixed function oxidases and biological and pathological implications. Redox Biol 4:60-73
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
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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
Cederbaum, Arthur I (2014) Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction. Redox Biol 2:1048-54
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

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