Cold-induced apoptosis in isolated rat hepatocytes: Protective role of glutathione

Liver conservation for transplantation is usually made at 2-4 degreesC. We studied the effect of rewarming to 37 degreesC for up to 3 h of rat hepatoc ytes kept at 4 degreesC for 20 h, modulating intracellular glutathione (GSH ) concentration either with a GSH precursor (N-acetyl-L-cysteine, NAC), or with GSH depleting agents (diethylmaleate and buthionine sulfoximine, DEM/B SO). Untreated hepatocytes showed time-dependent production of reactive oxy gen species (ROS), lipid peroxidation, chromatin condensation and membrane blebbing, decrease in GSH concentration, and protein sulfhydryl groups. Flu orochromatization with Propidium Iodide (PI) and Annexin V (AnxV) of cells rewarmed for I h caused an increase of AnxV-positive cells without PI stain ing and any observed lactate dehydrogenase leakage. TUNEL and DNA-laddering tests were negative for all times and treatments, indicating that apoptosi s may occur without DNA fragmentation. Cold preservation and rewarming in t he presence of NAC induced a significant improvement in the morphology, les s oxidative stress and apoptosis. Conversely, DEM/BSO caused a marked deter ioration of morphology, increase of oxidative stress and apoptosis. These r esults suggested that marked changes in GSH status might play a critical ro le in triggering apoptosis during cold preservation of isolated rat hepatoc ytes. NAC, added before rewarming, might represent a therapeutic approach f or preventing the early events of apoptosis during cold storage. (C) 2001 E lsevier Science Inc.