The toxic effect of thioacetamide on rat liver in vitro.
Thioacetamide (TAA) is a hepatotoxin frequently used for experimental purposes which produces centrilobular necrosis after a single dose administration. In spite of the fact that oxidative stress seems to play a very important role in the mechanism of TAA-induced injury, the effect of TAA on hepatocytes in primary culture with respect to the influence on mitochondria has yet to be verified. Hepatocytes were incubated for 24h in a medium containing TAA (0-70 mmol/l). Glutathione content (GSH/GSSG), reactive oxygen species and malondialdehyde formation were assessed as markers of cell redox state. Toxicity was determined by lactate dehydrogenase leakage and WST-1 assay. The functional capacity of hepatocytes was evaluated from albumin and urea production. Mitochondrial metabolism was assessed by measuring mitochondrial membrane potential and oxygen consumption. Our results show that a profound decrease in the GSH level in hepatocytes precedes a sharp rise in endogenous ROS production. ROS production correlates with an increase in lipoperoxidation. Mitochondria are affected by TAA secondarily as a consequence of oxidative stress. Oxidation of the NADH-dependent substrates of respiratory Complex I is significantly more sensitive to the toxic action of TAA than oxidation of the flavoprotein-dependent substrate of Complex II. Mitochondria can also maintain their membrane potential better when they utilize succinate as a respiratory substrate. It appears that GSH should be depleted below a certain critical level in order to cause a marked increase in lipid peroxidation. Mitochondrial injury can then occur and cell death develops.
Staňková
Pavla
P
Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Physiology, Šimkova 870, 50038 Hradec Králové, Czech Republic. krivakovap@lfhk.cuni.cz
Kučera
Otto
O
Lotková
Halka
H
Roušar
Tomáš
T
Endlicher
René
R
Cervinková
Zuzana
Z
D000818:Animals; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015386:Hazardous Substances / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013853:Thioacetamide / Q000633:toxicity*
D015386:Hazardous Substances; D017382:Reactive Oxygen Species; D013853:Thioacetamide; D008315:Malondialdehyde; D007770:L-Lactate Dehydrogenase