Halka Lotková

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.

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.

Proteomic analysis to display the effect of low doses of erythropoietin on rat liver regeneration.

AIMS Several groups found different impact of erythropoietin (EPO) on liver regeneration. Both pro-proliferative as well as anti-proliferative and non-proliferative activities have been reported using high dosage of EPO. Systemic administration of high doses of this cytokine is a clinical concern due to risk of thrombosis. Herein, we applied EPO in low dosages and investigated whether it can stimulate liver regeneration after liver resection. MAIN METHODS Parameters of liver regeneration were assessed 3 days after 70% hepatectomy by means of immunochemistry and proteomics. EPO was given twice in low dosages (200 and 600 IU/kg BW). KEY FINDINGS We showed that EPO facilitated hepatic regeneration in rats. Enhanced hepatocyte proliferation (Ki67, BrdU-positive cells) was observed in all EPO-treated groups. By performing Differential Proteomic analysis, we identified two proteins which resulted sensitive to EPO treatment after hepatectomy: Peroxiredoxin-1 and glutathione S-transferase Mu 1. SIGNIFICANCE Based on our results, low doses of rhEPO increase the hepatic regenerative capacity after partial hepatectomy in rats by enhancing hepatocyte proliferation and acting on antioxidant enzymes. Both proteins identified by proteomic analysis have not previously been associated with liver regeneration and will aid in the understanding of EPOs regenerative response having clinical implications to treat liver failure.

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 β1 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.

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.

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.

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.

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.