Relationship between oxidative stress and hepatic glutathione levels in ethanol-mediated apoptosis of polarized hepatic cells.
OBJECTIVE
To investigate the role of reactive oxygen species (ROS) in ethanol-mediated cell death of polarized hepatic (WIF-B) cells.


METHODS
In this work, WIF-B cultures were treated with pyrazole (inducer of cytochrome P4502E1, CYP2E1) and/or L-buthionine sulfoximine (BSO), a known inhibitor of hepatic glutathione (GSH), followed by evaluation of ROS production, antioxidant levels, and measures of cell injury (apoptosis and necrosis).


RESULTS
The results revealed that ethanol treatment alone caused a significant two-fold increase in the activation of caspase-3 as well as a similar doubling in ROS. When the activity of the CYP2E1 was increased by pyrazole pretreatment, an additional two-fold elevation in ROS was detected. However, the CYP2E1-related ROS elevation was not accompanied with a correlative increase in apoptotic cell injury, but rather was found to be associated with an increase in necrotic cell death. Interestingly, when the thiol status of the cells was manipulated using BSO, the ethanol-induced activation of caspase-3 was abrogated. Additionally, ethanol-treated cells displayed enhanced susceptibility to Fas-mediated apoptosis that was blocked by GSH depletion as a result of diminished caspase-8 activity.


CONCLUSIONS
Apoptotic cell death induced as a consequence of ethanol metabolism is not completely dependent upon ROS status but is dependent on sustained GSH levels.
McVicker
Benita L
BL
Liver Study Unit, Department of Veterans Affairs Medical Center, Research Service (151), 4101 Woolworth Avenue, Omaha, NE 68105, USA. bmcvicker@unmc.edu
Tuma
Pamela L
PL
Kharbanda
Kusum K
KK
Lee
Serene M L
SM
Tuma
Dean J
DJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D000431:Ethanol / Q000494:pharmacology*; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism
D004791:Enzyme Inhibitors; D011720:Pyrazoles; D017382:Reactive Oxygen Species; D019014:fas Receptor; D000431:Ethanol; C031280:pyrazole; D019328:Buthionine Sulfoximine; D019392:Cytochrome P-450 CYP2E1; D053148:Caspase 3; D005978:Glutathione