Cold-induced apoptosis in isolated rat hepatocytes: protective role of glutathione.
Liver conservation for transplantation is usually made at 2-4 degrees C. We studied the effect of rewarming to 37 degrees C for up to 3 h of rat hepatocytes kept at 4 degrees C 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/BSO). Untreated hepatocytes showed time-dependent production of reactive oxygen species (ROS), lipid peroxidation, chromatin condensation and membrane blebbing, decrease in GSH concentration, and protein sulfhydryl groups. Fluorochromatization with Propidium Iodide (PI) and Annexin V (AnxV) of cells rewarmed for 1 h caused an increase of AnxV-positive cells without PI staining and any observed lactate dehydrogenase leakage. TUNEL and DNA-laddering tests were negative for all times and treatments, indicating that apoptosis may occur without DNA fragmentation. Cold preservation and rewarming in the presence of NAC induced a significant improvement in the morphology, less oxidative stress and apoptosis. Conversely, DEM/BSO caused a marked deterioration of morphology, increase of oxidative stress and apoptosis. These results suggested that marked changes in GSH status might play a critical role in triggering apoptosis during cold preservation of isolated rat hepatocytes. NAC, added before rewarming, might represent a therapeutic approach for preventing the early events of apoptosis during cold storage.
Vairetti
M
M
Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy. vairetti@botta.unipv.it
Griffini
P
P
Pietrocola
G
G
Richelmi
P
P
Freitas
I
I
D000111:Acetylcysteine / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019328:Buthionine Sulfoximine / Q000378:metabolism / Q000494:pharmacology; D003080:Cold Temperature; D005978:Glutathione / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D007035:Hypothermia / Q000378:metabolism*; D015227:Lipid Peroxidation / Q000502:physiology; D008297:Male; D008298:Maleates / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D017694:Rewarming
D008298:Maleates; D017382:Reactive Oxygen Species; D019328:Buthionine Sulfoximine; C014476:diethyl maleate; D005978:Glutathione; D000111:Acetylcysteine