There is interest in the role of oxidative stress and generation of reactive radical species in the mechanism(s) by which ethanol is toxic. Induction of CYP2E1 by ethanol is one central pathway by which ethanol generates oxidative stress. Understanding the biochemical and toxicological properties of CYP2E1 will be important in providing mechanistic information on the actions of CYP2E1 in intact cells. A1M1 will evaluate the role of mitogen activated protein kinases (MAPK) in CYP2E1-dependent toxicity in HepG2 cells which express CYP2E1 and in pyrazole hepatocytes with high levels of CYP2E1. The cells will be treated with ethanol, arachidonic acid, iron or depleted of GSH and activation of MAPK-ERK, JNK, P38 evaluated. The ability of specific inhibitors of different MAPK to prevent CYP2E1 toxicity and oxidative stress will be determined. Experiments will be carried out to evaluate if NF-kappaB is activated in CYP2E1 expressing cells exposed to the above toxic agents and whether this activation protects or increases sensitivity to the toxin.
AIM2 will characterize the role of calcium in CYP2E1-dependent toxicity. Using the above models, toxicity will be evaluated in Ca2+ -containing and Ca2+-free medium, and in the presence of intracellular Ca2+ chelating agents or inhibitors of Ca2+ import into the cell. Intracellular Ca2+ levels will be determined, as will possible linkage between changes in Ca2+ homeostasis, oxidative stress, cell viability and mitochondrial function. Downstream possible mediators such as the Ca2+-activated hydrolases calpain and phospholipase A2 will be studied for their role in CYP2E1-dependent toxicity.
AIM3 -Since oxidative stress plays an important role in alcoholic liver injury, we will evaluate whether such injury occurs in mice deficient in critical antioxidant enzymes such as copper-zinc SOD-1, manganese SOD-2 and GPX-1 knockout mice. These mice will be fed an ethanol diet for varying times and liver injury evaluated. Oxidative stress parameters will be measured, as will mitochondrial function, CYP2E1 and TNFalpha. Toxicity that is associated with binge alcohol treatment or after induction of CYP2E1 in these knockouts and controls will be evaluated. It is hoped that such studies in hepatocyte and HepG2 cell culture models and in-vivo models, will help to define further the biochemical and toxicological actions of CYP2E1 and its role in alcohol-induced liver injury.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA006610-19
Application #
6807569
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Gentry, Thomas
Project Start
1989-07-01
Project End
2007-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
19
Fiscal Year
2004
Total Cost
$423,750
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
Caro, Andres A; Evans, Kerry L; Cederbaum, Arthur I (2009) CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells. Toxicology 255:171-6
Zhuge, Jian; Cederbaum, Arthur I (2009) Inhibition of the mitochondrial permeability transition by cyclosporin A prevents pyrazole plus lipopolysaccharide-induced liver injury in mice. Free Radic Biol Med 46:406-13
Lu, Yongke; Cederbaum, Arthur I (2008) CYP2E1 and oxidative liver injury by alcohol. Free Radic Biol Med 44:723-38
Wu, Defeng; Cederbaum, Arthur (2008) Cytochrome P4502E1 sensitizes to tumor necrosis factor alpha-induced liver injury through activation of mitogen-activated protein kinases in mice. Hepatology 47:1005-17
Jimenez-Lopez, Jose M; Wu, Defeng; Cederbaum, Arthur I (2008) Synergistic toxicity induced by prolonged glutathione depletion and inhibition of nuclear factor-kappaB signaling in liver cells. Toxicol In Vitro 22:106-15
Lu, Yongke; Gong, Pengfei; Cederbaum, Arthur I (2008) Pyrazole induced oxidative liver injury independent of CYP2E1/2A5 induction due to Nrf2 deficiency. Toxicology 252:9-16
Lu, Yongke; Zhuge, Jian; Wang, Xiaodong et al. (2008) Cytochrome P450 2E1 contributes to ethanol-induced fatty liver in mice. Hepatology 47:1483-94
Lu, Yongke; Cederbaum, Arthur (2007) The mode of cisplatin-induced cell death in CYP2E1-overexpressing HepG2 cells: modulation by ERK, ROS, glutathione, and thioredoxin. Free Radic Biol Med 43:1061-75
Dey, Aparajita; Cederbaum, Arthur I (2007) Geldanamycin, an inhibitor of Hsp90 increases cytochrome P450 2E1 mediated toxicity in HepG2 cells through sustained activation of the p38MAPK pathway. Arch Biochem Biophys 461:275-86
Dey, Aparajita; Cederbaum, Arthur I (2007) Induction of cytochrome P450 2E1 [corrected] promotes liver injury in ob/ob mice. Hepatology 45:1355-65

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