René Endlicher

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.

The Effect of tert-Butyl Hydroperoxide-Induced Oxidative Stress on Lean and Steatotic Rat Hepatocytes In Vitro

Oxidative stress and mitochondrial dysfunction play an important role in the pathogenesis of nonalcoholic fatty liver disease and toxic liver injury. The present study was designed to evaluate the effect of exogenous inducer of oxidative stress (tert-butyl hydroperoxide, tBHP) on nonfatty and steatotic hepatocytes isolated from the liver of rats fed by standard and high-fat diet, respectively. In control steatotic hepatocytes, we found higher generation of ROS, increased lipoperoxidation, an altered redox state of glutathione, and decreased ADP-stimulated respiration using NADH-linked substrates, as compared to intact lean hepatocytes. Fatty hepatocytes exposed to tBHP exert more severe damage, lower reduced glutathione to total glutathione ratio, and higher formation of ROS and production of malondialdehyde and are more susceptible to tBHP-induced decrease in mitochondrial membrane potential. Respiratory control ratio of complex I was significantly reduced by tBHP in both lean and steatotic hepatocytes, but reduction in NADH-dependent state 3 respiration was more severe in fatty cells. In summary, our results collectively indicate that steatotic rat hepatocytes occur under conditions of enhanced oxidative stress and are more sensitive to the exogenous source of oxidative injury. This confirms the hypothesis of steatosis being the first hit sensitizing hepatocytes to further damage.

In Vitro Toxicity of Epigallocatechin Gallate in Rat Liver Mitochondria and Hepatocytes

Epigallocatechin-3-gallate (EGCG) is the main compound of green tea with well-described antioxidant, anti-inflammatory, and tumor-suppressing properties. However, EGCG at high doses was reported to cause liver injury. In this study, we evaluated the effect of EGCG on primary culture of rat hepatocytes and on rat liver mitochondria in permeabilized hepatocytes. The 24-hour incubation with EGCG in concentrations of 10 μmol/L and higher led to signs of cellular injury and to a decrease in hepatocyte functions. The effect of EGCG on the formation of reactive oxygen species (ROS) was biphasic. While low doses of EGCG decreased ROS production, the highest tested dose induced a significant increase in ROS formation. Furthermore, we observed a decline in mitochondrial membrane potential in cells exposed to EGCG when compared to control cells. In permeabilized hepatocytes, EGCG caused damage of the outer mitochondrial membrane and an uncoupling of oxidative phosphorylation. EGCG in concentrations lower than 10 μmol/L was recognized as safe for hepatocytes in vitro.

Effects of Epigallocatechin Gallate on Tert-Butyl Hydroperoxide-Induced Mitochondrial Dysfunction in Rat Liver Mitochondria and Hepatocytes

Epigallocatechin gallate (EGCG) is a green tea antioxidant with adverse effects on rat liver mitochondria and hepatocytes at high doses. Here, we assessed whether low doses of EGCG would protect these systems from damage induced by tert-butyl hydroperoxide (tBHP). Rat liver mitochondria or permeabilized rat hepatocytes were pretreated with EGCG and then exposed to tBHP. Oxygen consumption, mitochondrial membrane potential (MMP), and mitochondrial retention capacity for calcium were measured. First, 50 μM EGCG or 0.25 mM tBHP alone increased State 4 Complex I-driven respiration, thus demonstrating uncoupling effects; tBHP also inhibited State 3 ADP-stimulated respiration. Then, the coexposure to 0.25 mM tBHP and 50 μM EGCG induced a trend of further decline in the respiratory control ratio beyond that observed upon tBHP exposure alone. EGCG had no effect on MMP and no effect, in concentrations up to 50 μM, on mitochondrial calcium retention capacity. tBHP led to a decline in both MMP and mitochondrial retention capacity for calcium; these effects were not changed by pretreatment with EGCG. In addition, EGCG dose-dependently enhanced hydrogen peroxide formation in a cell- and mitochondria-free medium. Conclusion. Moderate nontoxic doses of EGCG were not able to protect rat liver mitochondria and hepatocytes from tBHP-induced mitochondrial dysfunction.

Tissue Specific Sensitivity of Mitochondrial Permeability Transition Pore to Ca2+ Ions.

Ca2+-induced opening of the mitochondrial permeability transition pore (MPTP) is involved in induction of apoptotic and necrotic processes. We studied sensitivity of MPTP to calcium using the model of Ca2+-induced, cyclosporine A-sensitive mitochondrial swelling. Presented data indicate that the extent of mitochondrial swelling (dA520/4 min) induced by addition of 25 μM Ca2+ is seven-fold higher in liver than in heart mitochondria (0.564 ± 0.08/0.077± 0.01). The extent of swelling induced by 100 μM Ca2+ was in liver tree times higher than in heart mitochondria (0.508±0.05/ 0.173±0.02). Cyclosporine A sensitivity showed that opening of the MPTP is involved. We may thus conclude that especially at low Ca2+ concentration heart mitochondria are more resistant to damaging effect of Ca2+ than liver mitochondria. These finding thus support hypothesis that there exist tissue specific strategies of cell protection against induction of the apoptotic and necrotic processes.

Acetaminophen toxicity in rat and mouse hepatocytes in vitro

Abstract Context: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP in vitro. Objectives: The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures. Materials and methods: Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24 h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1). Results: APAP from concentrations of 2.5 and 0.75 mmol/L induced a decrease in viability of rat (p < 0.001) and mouse (p < 0.001) hepatocytes (WST-1), respectively. In contrast to rat hepatocytes, there was no activation of caspase-3 in mouse hepatocytes after APAP treatment. Earlier damage to plasma membrane and faster depletion of reduced glutathione were detected in mouse hepatocytes. Mouse hepatocytes showed increased glutamate + malate-driven respiration in state 4 and higher susceptibility of the outer mitochondrial membrane (OMM) to APAP-induced injury. Conclusion: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.