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

Free Radic Biol Med. 2001 Oct 15;31(8):954-61. doi: 10.1016/s0891-5849(01)00670-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / metabolism*
  • Acetylcysteine / pharmacology
  • Animals
  • Apoptosis / physiology*
  • Buthionine Sulfoximine / metabolism
  • Buthionine Sulfoximine / pharmacology
  • Cold Temperature
  • Glutathione / agonists
  • Glutathione / antagonists & inhibitors
  • Glutathione / metabolism*
  • Hepatocytes / metabolism*
  • Hypothermia / metabolism*
  • Lipid Peroxidation / physiology
  • Male
  • Maleates / metabolism
  • Maleates / pharmacology
  • Rats
  • Rats, Wistar
  • Reactive Oxygen Species / metabolism
  • Rewarming

Substances

  • Maleates
  • Reactive Oxygen Species
  • Buthionine Sulfoximine
  • diethyl maleate
  • Glutathione
  • Acetylcysteine