Mechanisms participating in oxidative damage of isolated rat hepatocytes.

The aim of the study was to evaluate time course and dose dependence of peroxidative damage induced by tert-butyl hydroperoxide (tBHP) in rat hepatocytes cultured in suspension and in monolayer. At the lowest (0.1 mM) concentration, decrease of cytosolic glutathione and discharge of mitochondrial membrane potential (MMP) could be detected. Significant increases in leakage of lactate dehydrogenase and in malondialdehyde concentrations together with decrease of pyruvate-dependent respiration were detected at higher tBHP concentrations (above 0.5 mM) and after longer periods of incubation. Changes in plasma membrane integrity were observed at 1 mM concentration of tBHP. Succinate-dependent oxidation was most resistant to peroxidative damages. Opening of the mitochondrial permeability transition pore was responsible for the discharge of mitochondria membrane potential. In the presence of cyclosporine A and succinate, the membrane potential could be restored. Our data showed that the most sensitive indicators of the peroxidative damage are changes of cytosolic glutathione concentration and MMP.

Tissue specific sensitivity of mitochondrial permeability transition pore to Ca2+ ions.

Ca(2+)-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 Ca(2+)-induced, cyclosporine A-sensitive mitochondrial swelling. Presented data indicate that the extent of mitochondrial swelling (dA520/4 min) induced by addition of 25 microM 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 microM 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.

S-adenosylmethionine exerts a protective effect against thioacetamide-induced injury in primary cultures of rat hepatocytes.

S-adenosylmethionine (SAMe) has been shown to protect hepatocytes from toxic injury, both experimentally-induced in animals and in isolated hepatocytes. The mechanisms by which SAMe protects hepatocytes from injury can result from the pathways of SAMe metabolism. Unfortunately, data documenting the protective effect of SAMe against mitochondrial damage from toxic injury are not widely available. Thioacetamide is frequently used as a model hepatotoxin, which causes in vivo centrilobular necrosis. Even though thioacetamide-induced liver necrosis in rats was alleviated by SAMe, the mechanisms of this protective effect remain to be verified. The aim of our study was to determine the protective mechanisms of SAMe on thioacetamide-induced hepatocyte injury by using primary hepatocyte cultures. The release of lactate dehydrogenase (LDH) from cells incubated with thioacetamide for 24 hours, was lowered by simultaneous treatment with SAMe, in a dose-dependent manner. The inhibitory effect of SAMe on thioacetamide-induced lipid peroxidation paralleled the effect on cytotoxicity. A decrease in the mitochondrial membrane potential, as determined by Rhodamine 123 accumulation, was also prevented. The attenuation by SAMe of thioacetamide-induced glutathione depletion was determined after subsequent incubation periods of 48 and 72 hours. SAMe protects both cytoplasmic and mitochondrial membranes. This effect was more pronounced during the development of thioacetamide-induced hepatocyte injury that was mediated by lipid peroxidation. Continuation of the SAMe treatment then led to a reduction in glutathione depletion, as a potential consequence of an increase in glutathione production, for which SAMe is a precursor.

Evaluation of mitochondrial function in isolated rat hepatocytes and mitochondria during oxidative stress.

The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.

Protective effect of S-adenosylmethionine on cellular and mitochondrial membranes of rat hepatocytes against tert-butylhydroperoxide-induced injury in primary culture.

Accumulating evidence that administration of S-adenosylmethionine (SAMe) protects hepatocytes against oxidative stress-mediated injury led us to evaluate the effect of SAMe on hepatocyte injury induced in culture by oxidant substance tert-butylhydroperoxide (1.5 mM tBHP) with regard to prevent mitochondrial injury. The pretreatment of hepatocyte culture with SAMe in doses of 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 mg/l for 30 min prevented the release of LDH from cells incubated for 30 min with tBHP in a dose dependent manner. The inhibitory effect of SAMe on lipid peroxidation paralleled the effect on cell viability. SAMe also moderated the decrease of the mitochondrial membrane potential induced by tBHP. Our results indicate that the inhibition of lipid peroxidation by SAMe can contribute to the prevention of disruption of both cellular and mitochondrial membranes. While the protective effect of SAMe against tBHP-induced GSH depletion was not confirmed, probably the most potent effect of SAMe on membranes by phospholipid methylation should be verified.

[Mitochondria and their role in cell metabolism]. / Mitochondrie a jejich úloha v bunecném metabolismu.

Mitochondria are subcellular organelles of the endosymbiotic origin. They are bounded by double membrane and contain their own DNA. Recent advance in 3D microscopy have contributed a better understanding of mitochondrial structure. Mitochondria are highly dynamic organelles with a very complex structure of the inner membrane. In cells, mitochondria create an interconnected reticulum. Beyond a fundamental role in energy production, they also play key roles in thermogenesis, maintenance of cellular redox potential, Ca2+ homeostasis, ROS production, cell signaling and cell death. Disturbances in mitochondrial metabolism are known to play a role not only in rare genetics disorders, but have also been implicated in many common diseases of aging. Conventional studies of mitochondrial metabolism are based on the isolation of intact organelles. Because of mitochondrial complex roles rises a need to assay mitochondrial functions in situ. The activity of respiration and oxidative phosphorylation in intact and permeabilized cells can be measured by using high resolution respirometry. We can estimate various mitochondrial functions in living cells by using fluorescent cation dyes.

[Model systems for study of toxic injury of hepatocytes in vitro]. / Modelové systémy pro studium toxického poskození hepatocytu in vitro.

The liver is a common target of toxic effect of a number of xenobiotics, which is in particular a result of its central role in intermediary and energetic metabolism and in biotransformation processes. Ethical, economic, legislative, research and other reasons do not allow testing all of newly-synthesized compounds in in vivo conditions. Hence new methods and approaches for hepatotoxicity testing in vitro have been developing. The most important systems for study of toxicity and metabolic activity in vitro are isolated perfused liver, liver slices, isolated liver cells in suspensions or in primary cultures including co-culture methods and special 3D techniques, various subcellular fractions and stabilised cell lines. These models can be used for cytotoxicity and genotoxicity screening, evaluation of potential hepatoprotective capacity of different compounds, study of toxic injury and characterization of hepatotoxicity mechanisms. Currently there is no an ideal in vitro liver model system for testing of hepatotoxic substances in vitro, nevertheless use of these model systems reduces economic costs and ethic and legislative problems. Model systems in vitro afford opportunity to study in detail mechanisms of hepatotoxicity in comparison with in vivo conditions. Definition of their actual advantages and disadvantages allows choosing a suitable model system for study of particular problem. We cannot imagine current research of liver toxicity without using these model sytems.

Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode.

Mitochondrial membrane potential (Deltapsi(m)) plays important roles in the normal function of cells and in pathobiochemical situations. The application of ion-selective electrodes for the measurement of Deltapsi(m) is important for studying normal biological reactions and pathways and mitochondrial diseases. We constructed and optimized a computerized device for real-time monitoring of the Deltapsi(m), which included modification of tetraphenylphosphonium (TPP(+))-selective membrane that improved reproducibility of the TPP(+)-selective electrode. Application of MATLAB software increased the sensitivity of the system. We tested our improved device for membrane potential measurements of isolated mitochondria (in absolute scale of millivolts). In addition, we assessed relative changes of Deltapsi(m) (as changes in TPP(+) concentration) of digitonin-permeabilized cells (hepatocytes, control transmitochondrial cybrids, HeLa G and BSC-40) after addition of substrates, inhibitors, and uncoupler of respiratory chain. Our system can be successfully used for studies of many aspects of the regulation of mitochondrial bioenergetics and as a diagnostic tool for mitochondrial oxidative phosphorylation disorders.