Excessive alcohol consumption leading to alcohol liver disease is a major health issue world-wide, and if left untreated, can lead to liver cancer. CYP2E1, one of the two major enzymes involved in alcohol metabolism is known to contribute to alcohol liver injury possibly by inducing oxidative tissue damage. More recent studies suggest that mitochondrial DNA/membrane damage and oxidative damage to mitochondrial proteins are contributing factors in alcohol liver disease (ALD). A major objective here is to investigate the mechanisms of alcohol induced mitochondrial dysfunction and develop antioxidants and enzyme inhibitors to minimize alcohol induced liver damage. Recent studies show that mitochondria targeted CYP2E1 (mt-CYP2E1) modulates more severe alcohol toxicity than the ER targeted CYP2E1 (mc-CYP2E1). Analysis of human liver samples also identified unique human variant forms of CYP2E1 with altered subcellular targeting that result in a skewing of the mt-CYP2E1:mc-CYP2E1 content ratio. Results strongly suggest that carriers of more robust mt-targeting CYP2E1 mutations are more prone to alcohol induced toxicity. Our collaborative studies also suggest that steady state levels of cytochrome c oxidase (CcO), mitochondrial ALDH2 (aldehyde dehydrogenase) and mtTFA (mitochondrial transcription factor A) are critical targets of mt-CYP2E1-potentiated alcohol damage. The present dual investigator proposal is to test the major hypothesis that human mt-CYP2E1 plays a critical role in potentiating ALD and that CcO, ALDH2 and mtTFA are some of the immediate targets as outlined in the following Aims: 1) We propose to use hepatocytes generated by iPSC (induced pluripotent stem cell) technology for defining the role of metabolic activity of mtCYP2E1 in alcohol toxicity and induction of oxidative stress. We will use a combination of genetically modified hepatocytes, state of the art ROS detection systems and mt-targeted antioxidants (Mito-CP and Mito-B) and CYP2E1 inhibitor (Mito-diallylsulfide, Mito-D) for documenting the role of mt-CYP2E1 in toxicity. 2) The in vitro data in Aim1 will be complemented by in vivo studies using recently developed humanized mouse models expressing human variant forms of CYP2E1 (L32N and W23/30R) with the aim of establishing a link for human genetic susceptibility to alcohol liver toxicity. The ability of Mito-CP, Mito-B and Mito-D in alleviating or reversing alcohol toxicity will be investigated. Livers from alcoholic patients will be analyzed to correlate mtCYP2E1 contents with the extent of disease and rate of progression. 3) Using iPSC hepatocytes and humanized mouse models, we will define the mechanism of alcohol mediated mitochondrial PKA activation, and LON protease mediated degradation of CcO, ALDH2 and mtTFA proteins. These results should document novel mechanisms of mitochondrial ROS production under various disease conditions and effective means of preventing or alleviating the stress conditions.

Public Health Relevance

Alcohol consumption has been implicated in a multitude of human diseases including alcoholic liver disease (ALD), liver cancer, myocardial fibrosis/infarction, pancreatitis, and disorders of the immune, endocrine and reproductive systems and estimated to cost annually over 1.5 billion dollars in the U.S. in terms of lost productivity and cost of health management. This study will advance our understanding of ALD and critical molecular targets affected. A combination of subcellular targeting of antioxidants and enzyme inhibitors and use of humanized mouse models should provide novel insights into the mechanism of the disease and also lead to development of clinically important drugs for treating or reversing the alcohol liver damage.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA022986-04
Application #
9404927
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Orosz, Andras
Project Start
2015-02-05
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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