Ethanol potentiates tumor necrosis factor-alpha cytotoxicity in hepatoma cells and primary rat hepatocytes by promoting induction of the mitochondrial permeability transition

In the present study, tumor necrosis factor-alpha (TNF-alpha) cytotoxicity is shown to be potentiated by ethanol exposure in vitro in the human hepato ma cell line, HepG2, and in rat primary hepatocytes. Exposure of HepG2 cell s and primary hepatocytes for 48 hours to concentrations of ethanol ranging between 50 and 100 mmol/L significantly increased TNF-alpha cytotoxicity c ompared with cells treated with TNF-alpha alone. The cell killing was assoc iated with, and dependent on, the development of the mitochondrial permeabi lity transition (MPT), Two inhibitors of MPT pore opening, cyclosporin A an d bongkrekic acid, prevented TNF-alpha cytotoxicity in the presence of etha nol. In addition to inhibiting cell death caused by TNF-alpha, blockade of MPT pore opening prevented mitochondrial depolarization, cytochrome c redis tribution from the mitochondria to the cytosol, caspase 3 activation, and o ligonucleosomal DNA fragmentation. Unlike the potentiation of TNF-alpha cyt otoxicity by the translational inhibitor cycloheximide, ethanol promoted TN F-alpha-induced cell killing by a mechanism that was independent of caspase -8 activity. HepG2 cells overexpressing cytochrome-P4502EI were even more s ensitized by ethanol to induction of the MPT by TNP-alpha and the resultant cytotoxicity than wild-type HepG2 cells. In addition, primary hepatocytes isolated from chronically ethanol-fed rats showed enhanced susceptibility t o TNF-alpha cytotoxicity compared with their isocalorically matched control s. Again as with the HepG2 cells, inhibiting MPT pore opening prevented the cytotoxicity of TNF-alpha in the primary hepatocytes isolated from ethanol -fed animals.