Adaptation of Mitochondrial Substrate Flux in a Mouse Model of Nonalcoholic Fatty Liver Disease.

Maladaptation of mitochondrial oxidative flux seems to be a considerable feature of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to induce NAFLD in mice fed a Western-style diet (WD) and to evaluate liver mitochondrial functions. Experiments were performed on male C57BL/6J mice fed with a control diet or a WD for 24 weeks. Histological changes in liver and adipose tissue as well as hepatic expression of fibrotic and inflammatory genes and proteins were evaluated. The mitochondrial respiration was assessed by high-resolution respirometry. Oxidative stress was evaluated by measuring lipoperoxidation, glutathione, and reactive oxygen species level. Feeding mice a WD induced adipose tissue inflammation and massive liver steatosis accompanied by mild inflammation and fibrosis. We found decreased succinate-activated mitochondrial respiration and decreased succinate dehydrogenase (SDH) activity in the mice fed a WD. The oxidative flux with other substrates was not affected. We observed increased ketogenic capacity, but no impact on the capacity for fatty acid oxidation. We did not confirm the presence of oxidative stress. Mitochondria in this stage of the disease are adapted to increased substrate flux. However, inhibition of SDH can lead to the accumulation of succinate, an important signaling molecule associated with inflammation, fibrosis, and carcinogenesis.

Acetaminophen toxicity in rat and mouse hepatocytes in vitro.

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.

Does Simple Steatosis Affect Liver Regeneration after Partial Hepatectomy in Rats?

AIM: The aim of our study was to assess whether simple steatosis impairs liver regeneration after partial hepatectomy (PHx) in rats. METHODS: Male Sprague-Dawley rats were fed a standard diet (ST-1, 10% kcal fat) and high-fat diet (HFD, 71% kcal fat) for 6 weeks. Then the rats were submitted to 2/3 PHx and animals were sacrificed 24, 48 or 72 h after PHx. Serum biochemistry, respiration of mitochondria in liver homogenate, hepatic oxidative stress markers, selected cytokines and DNA content were measured, and histopathological samples were prepared. Liver regeneration was evaluated by incorporation of bromodeoxyuridine (BrdU) to hepatocyte DNA. RESULTS: HFD induced simple microvesicular liver steatosis. PHx caused elevation of serum markers of liver injury in both groups; however, an increase in these parameters was delayed in HFD group. Hepatic content of reduced glutathione was significantly increased in both groups after PHx. There were no significant changes in activities of respiratory complexes I and II (state 3). Relative and absolute liver weights, total DNA content, and DNA synthesis exerted very similar changes in both ST-1 and HFD groups after PHx. CONCLUSION: PHx-induced regeneration of the rat liver with simple steatosis was not significantly affected when compared to the lean liver.

Antioxidative effect of epigallocatechin gallate against D-galactosamine-induced injury in primary culture of rat hepatocytes.

Literature data support that green tea and its major component epigallocatechin gallate (EGCG) have powerful antioxidant effects. Contrary, hepatotoxicity can be induced by high-dose EGCG. The timing of exposure to green tea in relation to administration of hepatotoxic agent plays an import role too. The aim of our work was a verification of antioxidative effect of EGCG on D-galactosamine-induced injury in primary culture of rat hepatocytes. Hepatocytes were incubated with EGCG at concentrations of 1.25-10 µM and toxic D-galactosamine (GalN) for 24 hrs. Alternatively, hepatocytes were pretreated with EGCG for 24 hrs, and then incubated with EGCG and GalN for further 24 hrs. Cytotoxicity was analysed by lactate dehydrogenase activity, functional capacity by albumin production. Oxidative stress was evaluated from a production of malondialdehyde and glutathione content in the cells. EGCG protected hepatocytes against GalN-induced cytotoxicity but preventive treatment of intact hepatocytes with EGCG was required to diminish the development of hepatocyte injury. Oxidative stress induced in our study seems to overcome the ability of hepatocytes to improve GSH depletion and albumin production. Prolongation of the pretreatment with EGCG could be a promising strategy leading to amelioration of its hepatoprotective effect.

Collagen I Increases Palmitate-Induced Lipotoxicity in HepG2 Cells via Integrin-Mediated Death.

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2ß1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.

Comparison of HepaRG and HepG2 cell lines to model mitochondrial respiratory adaptations in non­alcoholic fatty liver disease.

Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non­alcoholic steatohepatitis (NASH), there is a lack of in vitro models of non­alcoholic fatty liver disease (NAFLD) with similar findings. Despite being the most commonly used immortalized cell line for in vitro models of NAFLD, HepG2 cells exposed to free fatty acids (FFAs) exhibit a decreased mitochondrial respiration. On the other hand, the use of HepaRG cells to study mitochondrial respiratory changes following exposure to FFAs has not yet been fully explored. Therefore, the present study aimed to assess cellular energy metabolism, particularly mitochondrial respiration, and lipotoxicity in FFA­treated HepaRG and HepG2 cells. HepaRG and HepG2 cells were exposed to FFAs, followed by comparative analyses that examained cellular metabolism, mitochondrial respiratory enzyme activities, mitochondrial morphology, lipotoxicity, the mRNA expression of selected genes and triacylglycerol (TAG) accumulation. FFAs stimulated mitochondrial respiration and glycolysis in HepaRG cells, but not in HepG2 cells. Stimulated complex I, II­driven respiration and ß­oxidation were linked to increased complex I and II activities in FFA­treated HepaRG cells, but not in FFA­treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA­treated HepaRG cells than in FFA­treated HepG2 cells. TAG accumulation was less prominent in HepaRG cells than in HepG2 cells. On the whole, the present study demonstrates that stimulated mitochondrial respiration is associated with lipotoxicity in FFA­treated HepaRG cells, but not in FFA­treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed in vitro model of early­stage NASH.

Assessment of reduced glutathione: comparison of an optimized fluorometric assay with enzymatic recycling method.

Glutathione is an important tripeptide involved in a variety of cellular processes. Thus, precise knowledge of its levels is essential. Glutathione exists in two free forms-reduced and oxidized-and a number of methods exist to measure its levels. The aim of our work was to optimize a spectrofluorometric assay for reduced glutathione based on the reaction between glutathione and o-phthalaldehyde. We found that a change of excitation wavelength to 340 nm and modification of pH to 6.0 enhance sensitivity and specificity of the method (intraassay coefficient of variation CV < 3%, interassay CV = 5.1%, recovery = 98-102%, linearity = 0-1000 µM GSH, calibration R2 = 1.00). We also anticipated possible effect of various amino acids on the fluorescence signal, but no interference was found. We compared the optimized fluorometric method with a popular enzymatic recycling glutathione assay and found very strong correlation of results (r = 0.99, n = 45). We introduce here an optimized fluorometric method possessing sufficient sensitivity and specificity that is comparable to the enzymatic glutathione assay. Because the fluorometric assay procedure is faster and lower in cost, it could be ideal for routine analysis of reduced glutathione levels in a large number of samples.

Susceptibility of rat non-alcoholic fatty liver to the acute toxic effect of acetaminophen.

BACKGROUND AND AIM: Acetaminophen overdose is the most frequent cause of acute liver failure. Non-alcoholic fatty liver disease is the most common chronic condition of the liver. The aim was to assess whether non-alcoholic steatosis sensitizes rat liver to acute toxic effect of acetaminophen. METHODS: Male Sprague-Dawley rats were fed a standard diet (ST-1, 10% kcal fat) and high-fat gelled diet (HFGD, 71% kcal fat) for 6 weeks and then acetaminophen was applied in a single dose (1 g/kg body weight). Animals were killed 24, 48 and 72 h after acetaminophen administration. Serum biochemistry, activities of mitochondrial complexes, hepatic malondialdehyde, reduced and oxidized glutathione, triacylglycerol and cholesterol contents, and concentrations of serum and liver cytokines (TNF-α, TGF-ß1) were measured and histopathological samples were prepared. RESULTS: The degree of liver inflammation and hepatocellular necrosis were significantly higher in HFGD fed animals after acetaminophen administration. Serum markers of liver injury were elevated only in acetaminophen treated HFGD fed animals. Concentration of hepatic reduced glutathione and ratio of reduced/oxidized glutathione were decreased in both ST-1 and HFGD groups at 24 h after acetaminophen application. Mild oxidative stress induced by acetaminophen was confirmed by measurement of malondialdehyde. Liver content of TNF-α was not significantly altered, but hepatic TGF-ß1 was elevated in acetaminophen treated HFGD rats. We did not observe acetaminophen-induced changes in activities of respiratory complexes I, II, and IV and activity of caspase-3. CONCLUSION: Liver from rats fed HFGD is more susceptible to acute toxic effect of acetaminophen, compared to non-steatotic liver.

Is rat liver affected by non-alcoholic steatosis more susceptible to the acute toxic effect of thioacetamide?

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic condition of the liver in the western world. There is only little evidence about altered sensitivity of steatotic liver to acute toxic injury. The aim of this project was to test whether hepatic steatosis sensitizes rat liver to acute toxic injury induced by thioacetamide (TAA). Male Sprague-Dawley rats were fed ad libitum a standard pelleted diet (ST-1, 10% energy fat) and high-fat gelled diet (HFGD, 71% energy fat) for 6 weeks and then TAA was applied intraperitoneally in one dose of 100 mg/kg. Animals were sacrificed in 24-, 48- and 72-h interval after TAA administration. We assessed the serum biochemistry, the hepatic reduced glutathione, thiobarbituric acid reactive substances, cytokine concentration, the respiration of isolated liver mitochondria and histopathological samples (H+E, Sudan III, bromodeoxyuridine [BrdU] incorporation). Activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase and concentration of serum bilirubin were significantly higher in HFGD groups after application of TAA, compared to ST-1. There were no differences in activities of respiratory complexes I and II. Serum tumour necrosis factor alpha at 24 and 48 h, liver tissue interleukin-6 at 72 h and transforming growth factor ß1 at 24 and 48 h were elevated in TAA-administrated rats fed with HFGD, but not ST-1. TAA-induced centrilobular necrosis and subsequent regenerative response of the liver were higher in HFGD-fed rats in comparison with ST-1. Liver affected by NAFLD, compared to non-steatotic liver, is more sensitive to toxic effect of TAA.

Pravastatin modulates liver bile acid and cholesterol homeostasis in rats with chronic cholestasis.

BACKGROUND AND AIM: The administration of pravastatin to patients with cholestatic liver disease has suggested the potential of the drug with regard to reducing raised plasma cholesterol and bile acid levels. Information about the mechanisms associated with this effect is lacking. Thus, the aim of the present study is to evaluate pravastatin effects on the liver bile acid and cholesterol homeostasis in healthy and cholestatic rats. METHODS: Control sham-operated and reversibly bile duct-obstructed (BDO) rats were treated with pravastatin (1 or 5 mg/kg) or the vehicle alone for 7 days after surgery. RESULTS: Lower doses of pravastatin reduced bile acid plasma concentrations in cholestatic animals. The effect was associated with reduced liver mRNA expression of Cyp7a1, Cyp8b1, Mrp2, Ugt1a1 and the increased expression of Bsep. In addition, BDO-induced increase in the liver content of cholesterol was normalized by pravastatin. The change was accompanied by the reduced liver expression of Hmg-CoA reductase, LDL receptor, and Acat2, and induced the expression of Abca1 and Mdr2. These changes corresponded with the upregulation of nuclear receptors LXRα and PPARα, and the downregulation of FXR, CAR, SREBP-2 and HNF1α. High doses of pravastatin lacked any positive effects on bile acids and cholesterol homeostasis, and blocked bile formation through the reduction of the biliary excretion of bile acids. CONCLUSIONS: Pravastatin rendered a positive reduction in BDO-induced increases in plasma bile acid concentrations and cholesterol liver content, mainly through the transcriptionally-mediated downregulation of genes involved in the synthesis of these compounds in the liver.

Deteriorating effect of fluvastatin on the cholestatic liver injury induced by bile duct ligation in rats.

Antiinflammatory effect of statins mediated by the reduction of cytokine IL-6 in hepatocytes have been reported. Contrary to beneficial effect, statins can increase susceptibility to mitochondrial dysfunction. Extrahepatic biliary obstruction is associated with oxidative stress, pro-inflammatory response and hepatocyte mitochondrial dysfunction. The aim of our study was to verify the effect of fluvastatin on cholestatic liver injury. Cholestasis was induced in Wistar rats by bile duct ligation. Fluvastatin (1 or 5 mg/kg) was administered after surgery and then daily for 7 days. The dose of 5 mg/kg led to the deterioration of hepatocellular injury. Despite lower production of IL-6, decrease in GSH content, rise of TGFß and inhibition of respiratory complex I in mitochondria were determined. The mRNA expressions of canalicular transporter Mdr1b and basolateral transporter Mrp3 increased in cholestatic liver. Fluvastatin administration then led to the attenuation of this change. Analogously, mRNA expression of conjugative enzyme Ugt1a1 was diminished by fluvastatin administration to cholestatic rats. We can conclude that decrease in the antioxidative status and mitochondrial dysfunction could at least in part participate on the deteriorating effect of fluvastatin. Whether these processes can be a consequence of the alteration in metabolism and transport of potentially toxic substances remains to verify.

Effect of S-adenosylmethionine on liver regeneration induced by partial hepatectomy.

S-adenosylmethionine (SAMe) is a key metabolite regulating growth, differentiation and death of hepatocytes. Experimentally, exogenous SAMe has been documented to attenuate hepatocarcinogenesis. The aim of our study was to evaluate the effect of SAMe on proliferation of hepatocytes that are not cancerously transformed. Partial 2/3 hepatectomy (PH) was performed in rats, control animals underwent laparotomy. SAMe was injected immediately after the surgery and then at 24 h intervals for two days at 10 or 40 mg/kg. The animals were sacrificed 24, 48 and 72 h after operation and the intensity of liver regeneration was evaluated. SAMe treatment at 10 mg/kg was associated with decrease in the synthesis of liver DNA 48 h after PH, however, it was not reflected in DNA content. SAMe treatment at 40 mg/kg led to the reduction of DNA synthesis 72 h after PH followed by the diminution of DNA content. The results have documented the inhibition of the liver regeneration by SAMe that may be mediated by the suppression of liver fat accumulation. Cell GSH level correlating with the growth rate was not affected by SAMe. Prevention from the decrease in the intracellular content of SAMe, as a factor attenuating regeneration remains to be verified.

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.

Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection.

A HPLC method for determination of both reduced (GSH) and oxidized (GSSG) glutathione in plasma, whole blood and rat hepatocytes has been developed and evaluated. Reduced glutathione reacts with orthophthaldehyde (OPA) to form a stable, highly fluorescent tricyclic derivate at pH 8, while GSSG reacts with OPA at pH 12. At measurement of GSSG, GSH was complexed to N-ethylmaleimide. For the separation, reverse phase column Discovery C(18), 150 mm x 4 mm, 5 microm, was used. The mixture of methanol and 25 mM sodium hydrogenphosphate (15:85, v/v), pH 6.0, was used as mobile phase. The analytical performance of this method is satisfactory for both GSH and GSSG. The intra-assay coefficients of variation were 1.8 and 2.1% for whole blood, 2.0 and 1.9% for rat hepatocytes, 4.3 and 5.2% for plasma. The inter-assay coefficients of variation were 5.8 and 6.2% for whole blood, 6.6 and 7.1% for rat hepatocytes, 6.9 and 7.8% for plasma. The recoveries were as follows: 98.2% (CV 3.5%) and 101.5% (CV 4.2%) for whole blood, 99.1% (2.5%) and 102.3 (4.4%) for rat hepatocytes, 94.1% (CV 7.5%) and 103.5 (CV 8.5%) for plasma. The calibration curve was linear in the whole range tested. The limit of detection was 14.0 and 5.6 fmol, respectively. The preliminary reference ranges of reduced and oxidized glutathione in a group of blood donors are (4.69+/-0.93) and (0.28+/-0.12)micromol/gHb for whole blood, (1.82+/-0.55) and (0.154+/-0.044)microM for plasma.

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.

The model of D-galactosamine-induced injury of rat hepatocytes in primary culture.

D-galactosamine (GalN) is a highly selective hepatotoxin that causes liver damage similar to human viral hepatitis via depletion of uridine nucleotides, which subsequently diminishes synthesis of RNA and proteins. Model of galactosamine hepatotoxicity is frequently used in animal experiments in vitro. The purpose of our study was to establish the model of GalN-induced hepatocyte injury in in vitro conditions using primocultures of rat hepatocytes as an important pre-requisite for further experiments in which we would like to study potential hepatoprotective effect of various substances. Rate of hepatocyte injury was evaluated by morphological changes, changes in cell viability, albumin production, mitochondrial membrane potential, activity of mitochondrial dehydrogenases and glutathione content. Marked dose dependent hepatocyte injury was found after 24-hour incubation with GalN. Based on the results we suggest as an optimal model for short-term toxicity test exposure to GalN for 24 hours in dose of 40 mM.

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.