Chronic ethanol-initiated apoptosis in hepatocytes is induced by changes in membrane biogenesis and intracellular transport.

The outcome of chronic ethanol consumption recorded in liver by in situ staining of the genomic DNA in fragmented nuclei indicates the course of cellular events that has been coined as apoptosis or programmed cell death. Hence, we designed the study to determine which ethanol-induced modification of the cellular make-up is responsible for the hastening the cell damage. The in vitro assays were performed with cellular organelles and cytosol prepared from hepatocytes derived from rats subjected to 9 weeks of chronic alcohol consumption. The results were compared with the pair-fed controls receiving isocaloric liquid diet. In the initial phase of the studies, we established that the process of apoptosis was not triggered by the aberrant activity of neutral or acidic sphingomyelinase. The hepatocytes derived from alcohol and control diets manifested equal enzymatic activity. The de novo synthesis of sphingoid bases and ceramides in the alcohol-derived sample of endoplasmic reticulum was reduced, but the in situ apoptosis was up to 36-fold higher than in the control. Also, the isolated hepatocytes contained a 2- to 4-fold higher amount of nucleosomal fragments in the cytosolic extracts. The endosomes from liver hepatocytes of ethanol-consuming rats, in the presence of the cytosol and mitochondria from pair-fed controls, disclosed 2 to 3 times higher apoptotic potential than sample consisting of ethanol-derived fractions only, and 3 to 5 times higher than the control-derived fractions. The substantial increase in apoptosis, as recorded in the amount of DNA fragments released to the cytosol from the fresh nuclei, was also recorded when the microsomal membranes of endoplasmic reticulum and Golgi were incubated in the conditions with preserved intracellular transport. The maximal 20-fold increase of apoptotic activity was recorded in the incubation mixtures of ethanol-derived endoplasmic reticulum-Golgi membranes with control-derived cytosol in the presence of the ATP generating system. Results infer that the intracellular transport vesicles, generated from ethanol-modified membranes in the presence of the substrates that are available in the cytosol of the control hepatocytes, activate the apoptotic activity in the in vitro system. This interpretation is supported by the results of analysis of the clathrin-coated transport vesicles that, in contrast to nonclathrin transport vesicles, contain a sizable accumulation of ceramides that are known to induce apoptosis.