There is much current interest in the role of CYP2E1 in alcohol-induced liver injury. We propose to characterize CYP2E1- and ethanol-mediated cytotoxicity in HepG2 cell models either transduced or transfected to express human CYP2E1. We have recently developed a new HepG2 cell line which over-expresses CYP2E1 at levels 5 to 10-fold greater than previously established cell lines. The CYP2E1 over-expressing E47 cells grow at a slower rate than control cells but remain viable. When GSH is depleted, marked toxicity is observed with the E47 cells but not control cells. Sp.
Aim I is designed to characterize and evaluate mechanisms involved in this growth inhibition effect caused by over-expression of CYP2E1 and in the cytotoxic effect observed when GSH is depleted. The ability of antioxidants and iron chelators to prevent the growth inhibition and cytotoxicity, and the ability of iron and polyunsaturated fatty acids to exacerbate these effects will be determined. Impairment of mitochondrial function and development of oxidative stress will be studied. Whether injury is apoptotic in nature and the ability of bcl-2 to """"""""rescue"""""""" the cells from CYP2E1-catalyzed toxicity will be evaluated. Calcium plays a critical role in cell injury produced by oxidative stress and hepatotoxins.
Aim II will evaluate the ability of metabolites derived from CYP2E1 oxidation of ethanol and other substrates and CYP2E1-catalyzed formation of reactive oxygen species to activate Ca2+ channels in microsomes and promote release of Ca2+. The role of Ca2+ in toxicity exhibited by ethanol, PUFA or over-expressed CYP2E1 in HepG2 cells will be evaluated.
Aim III will evaluate whether oxidative stress mediated by CYP2E1 itself or CYP2E1 interactions with ethanol, PUFA, acetaminophen activates the transcription factor NF-kB, and if so, whether the HepG2 cells can initially be protected against injury by upregulation of NF-kB-activated genes. Special emphasis will be on cellular GSH levels. TNF-CYP2E1 interactions and induction of IL-8, a powerful chemoattractant will be evaluated. Selected experiments will be carried out with cultures containing hepatocytes isolated from chronic ethanol-fed rats and pair-fed controls to allow extrapolation of results with the HepG2 cells to normal hepatocytes. It is hoped that these studies will help to define the role of CYP2E1 and CYP2E1-derived reactive oxygen species in the hepatotoxic actions of ethanol.
|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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