Oxygen-free radical-mediated injury to cultured rat hepatocytes during cold incubation in preservation solutions

✍ Scribed by U Rauen; I Reuters; A Fuchs; H de Groot

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 224 KB
• Volume: 26
• Category: Article
• ISSN: 0270-9139
• DOI: 10.1002/hep.510260215

✦ Synopsis

ther studies have shown that this energy-dependent injury We have previously shown that the injury to cultured liver to cultured liver endothelial cells is mediated by reactive endothelial cells during cold incubation in University of Wisoxygen species 3 : the injury was inhibited by hypoxia, by consin (UW) solution is energy-dependent and is mediated adding either the spin trap 5,5-dimethyl-1-pyrroline N-oxide by reactive oxygen species. Here we demonstrate that this (DMPO), the hydroxyl radical scavenger dimethyl sulfoxide reactive oxygen-mediated injury is specific neither to endothe-(DMSO), or the flavonoid silibinin to the UW solution, or by lial cells nor to UW solution: cultured hepatocytes incubated preincubating the cells with the iron chelator deferoxamine in cold (4ЊC) UW solution or histidine-tryptophan-ketoglutabefore the hypothermic incubation. The endothelial cell inrate (HTK) solution were injured under normoxic conditions jury thus appears to be mediated by an iron-dependent re-(loss of viability, 63% { 10% after 48 hours of cold incubation lease of reactive oxygen species, most likely hydroxyl radicals in UW solution and 82% { 11% after 24 hours of cold incubaor related ferryl species. tion in HTK solution), whereas hypoxia was protective (loss In contrast to liver endothelial cells, cultured hepatocytes of viability, 29% { 12% [UW] and 13% { 3% [HTK] after are damaged by the addition of cyanide to UW solution. 4

the same cold incubation times). The injury under normoxic

This, at the first glance, suggests a different mechanism of conditions was also largely decreased by adding either the hepatocyte injury. However, we observed that the injury ocspin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or the curring to cultured hepatocytes during cold incubation in flavonoid silibinin to the solutions, or by preincubating the UW solution can, like endothelial cell injury, be inhibited cells with the iron chelator deferoxamine before the hypotherby hypoxic conditions. This protection by hypoxia suggests mic incubation. Marked lipid peroxidation was observed durthat reactive oxygen species are also involved in the hepatoing cold incubation in both preservation solutions. These recyte injury, a hypothesis we pursued further in this study. sults suggest that the injury to cultured hepatocytes during cold incubation in UW and HTK solutions is mediated by MATERIALS AND METHODS reactive oxygen species as is the injury to cultured liver endothelial cells. (HEPATOLOGY 1997;26:351-357.)

Animals. Male Wistar rats (range, 180-240 g) were obtained from the Zentrales Tierlaboratorium (Universita ¨tsklinikum Essen). Animals were kept under standard conditions with free access to food Previously, we described an energy-dependent injury to and water. All animals received humane care in compliance with cultured liver endothelial cells during cold incubation in Uniinstitutional guidelines. versity of Wisconsin (UW) and histidine-tryptophan-keto-Chemicals. UW solution was obtained from DuPont (Bad Homglutarate (HTK) solutions. 1,2 These cells, incubated in the burg, Germany); HTK solution was from Ko ¨hler (Alsbach, Ger- many), and Leibovitz L-15 medium was from Gibco (Eggenstein, preservation solutions at 4ЊC, died rapidly under normoxic Germany). UW solution was supplemented with 200,000 IU/L bencontrol conditions, but the injury was largely decreased by zylpenicillin (Gru ¨nenthal, Stolberg, Germany), 40 IU/L insulin inhibition of the mitochondrial respiratory chain, e.g., with (Merck, Darmstadt, Germany) and 16 mg/L dexamethasone (Serva, cyanide. The protective effect of cyanide was partly elimi-Heidelberg, Germany). Trichloroacetic acid, 2-thiobarbituric acid, nated when glucose, which allows energy generation by anperchloric acid, potassium cyanide and dimethyl sulfoxide (DMSO) aerobic glycolysis, was added to the incubation media. Furwere purchased from Merck (Darmstadt, Germany), DMPO, 4-hy- droxy-(2,2,6,6-tetramethylpiperidine-N-oxyl) (4-hydroxy-TEMPO), salicylate and dodecyltrimethylammonium bromide from Sigma (Deisenhofen, Germany) and superoxide dismutase (SOD) and Abbreviations: UW solution, University of Wisconsin solution; HTK solution, histicatalase from Boehringer (Mannheim, Germany). 1,1,3,3-Tetradine-tryptophan-ketoglutarate solution; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; methoxy-propane was obtained from Fluka (Neu-Ulm, Germany) DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase; TBARS, thiobarbituric acidand deferoxamine mesylate (Desferal) from Ciba-Geigy (Wehr, Gerreactive substances; ATP, adenosine-5-triphosphate; SOD, superoxide dismutase; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl. many). Silibinin was a gift from Madaus (Cologne, Germany).