Apoptosis

Reduced Glutathione Depletion Causes Necrosis and Sensitization to Tumor Necrosis Factor- –Induced Apoptosis in Cultured Mouse Hepatocytes
Hidenari Nagai, Katsuhiko Matsumaru, Guoping Feng, and Neil Kaplowitz
The effect of reduced glutathione (GSH) depletion by acetaminophen (APAP), diethylmaleate (DEM), or phorone on the mode of cell death and susceptibility to tumor necrosis factor(TNF)-induced cell death was studied in cultured mouse hepatocytes. Dose-dependent necrosis was the exclusive mode of cell death with APAP alone, but the addition of TNFinduced a switch to about half apoptosis without changing total loss of viability. This effect was seen at 1 and 5 mmol/L but was inhibited at 10 and 20 mmol/L APAP. The switch to apoptosis was associated with increased caspase activities,release of cytochrome c, and DNA laddering and was inhibited by caspase inhibitors. DEM and phorone also induced dosedependent necrosis. Treatment with TNF- under these conditions lead to incremental cell death in the form of apoptosis at 0.25 and 0.5 mmol/L DEM and 0.1 and 0.2 mmol/L phorone. At 1.0 and 2.0 mmol/L DEM and 0.5 mmol/L phorone, 90% to 100% necrosis was observed with resistance toTNF- effects. The apoptosis with TNF- plus DEM was confirmed by DNA laddering and inhibition by caspase inhibitors. However, in the presence of caspase inhibitors, the increment in cell death induced by TNF- persisted as an increase in necrosis. A combination of antioxidants, vitamin E, and butylated hydroxytoluene (BHT) markedly inhibited necrosis induced by APAP or DEM alone, but the sensitizationto TNF- –induced apoptosis was unaffected. GSH monoethylester (GSH-EE) protected against necrosis and apoptosis. In conclusion, depletion of GSH by APAP, DEM, or phorone causes oxidative stress-induced necrosis and sensitizes to an oxidative stress independent TNF- –induced apoptosis. (HEPATOLOGY 2002;36:55-64.) wo distinct modes of cell death have been described, apoptosis and necrosis. The formerinvolves the activation of a program of proteolytic enzymes, caspases, and can be triggered by extrinsic (death receptors) or intrinsic (intracellular stress) mechanisms leading to nuclear fragmentation, cell shrinkage, and phagocytosis.1,2 Necrosis involves loss of adenosine
Abbreviations: ATP, adenosine triphosphate; APAP, acetaminophen; GSH, reduced glutathione; TNF, tumor necrosis factor;DEM, diethylmaleate; BHT, butylated hydroxytoluene; GSH-EE, GSH-monoethylester; NF- B, nuclear factor- B. From the USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic and Pancreatic Disease, Keck School of Medicine, University of Southern California, Los Angeles, CA. Received December 3, 2001; accepted April 7, 2002. Supported by the USC-UCLA Research Center AlcoholicLiver and Pancreatic Disease (Project I) grant P50 AA11999. Isolation and culture of mouse hepatocytes were performed by the Cell Culture Core of the USC Research Center for Liver Diseases PO1 DK48522. Address reprint requests to: Neil Kaplowitz, M.D., Keck School of Medicine, University of Southern California, 2011 Zonal Ave., HMR 101, Los Angeles, CA 90033. E-mail: kaplowit@hsc.usc.edu; fax:323-442-5425. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3601-0009$35.00/0 doi:10.1053/jhep.2002.33995

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triphosphate (ATP), cell swelling, lysis, and inflammation.1,2 However, examples have been recognized that identify overlap between these 2 modes of cell death, pointing to a common, irreversible event, with the shape of cell death being determinedby the metabolic status of the cell and the status of caspase activity.3-5 Numerous drugs have been implicated in causing hepatotoxicity with destruction of parenchymal cells. However, little is known about the mode of cell death in drug-induced hepatotoxicity. Among drug toxicities, acetaminophen (APAP) toxicity has been extensively studied for decades, although controversy still continues...