known to induce hepatocellular injury. 5,6 Although ceramides Although ceramide signaling pathways have been imare well known to cause cell death by inducing apoptosis, plicated in cell death, neither their role in hepatocellular they can also cause cell death by necrosis. [3][4][5]7 Based on these death nor the cellular mechanisms mediating ceramidedata, we generated the hypothesis that the ceramide signalinduced cell death are known. The mitochondrial meming pathway induces cell death in hepatocytes, either by causbrane permeability transition (MMPT) has been proing apoptosis or necrosis. posed as a common final pathway in cell death. Thus the Induction of the mitochondrial membrane permeability aims of our study were to determine if ceramides cause transition (MMPT), an abrupt increase in the permeability of hepatocellular death by inducing the MMPT. Ceramides the inner mitochondrial membrane to small molecular weight induce hepatocellular death by necrosis and not solutes, has been implicated as a final common pathway causapoptosis as confirmed by morphology and the absence ing hepatocellular injury. 1,[8][9][10] The MMPT is characterized by of internucleosomal DNA cleavage. Ceramide-mediated mitochondrial depolarization, failure of oxidative phosphoryhepatocyte necrosis was acyl chain-length, concentralation with ATP depletion, and ultimately cell death. 1 The tion, and time-dependent. Ceramide induced cell necroincreased permeability of the inner mitochondrial membrane sis was associated with adenosine triphosphate (ATP) is thought to be facilitated by a specific proteinaceous ''megadepletion and mitochondrial depolarization suggesting pore'' located on the inner mitochondrial membrane. The that ceramides caused mitochondrial dysfunction. In opening of this pore is partially inhibited by cyclosporine A isolated mitochondria, ceramides induced the cycloplus trifluoperazine (CyA/TFP). 8-11sporine A-sensitive MMPT in an acyl chain-length and The overall objective of our study was to determine if ceraconcentration dependent manner. Ceramide toxicity mide causes hepatocyte necrosis by inducing the MMPT. Our was specific as the less potent dihydro form did not inspecific aims were to answer the following questions: 1) Do duce cell necrosis, significant ATP depletion, mitochonceramides induce cell death, and if so, does cell death occur by drial depolarization nor the MMPT. 1 However, we have suggested that signaling ley rats (200-300 g) as previously described.12 For those experiments cascades may induce cell death. 2 For example, we have pro-employing multiparameter digitized video microscopy, the hepatoposed that phospholipase-mediated calpain activation contri-cytes were cultured on collagen-coated glass coverslips. [13][14][15] butes to hepatocellular death during anoxia.2 Another potenDetermination of Cell Viability and ATP. In cells suspensions, cell tial signaling mechanism for hepatocellular death maybe the viability was determined by propidium iodide fluorometry.12 ATP was quantified using the luciferin...