The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP - 1, 2. 5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST-1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS prod uction was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation., Tomáš Roušar ... [et al.]., and Obsahuje seznam literatury
The aim of the present work was to investigate a new mechanism likely contributing to the toxic action of acetaminophen, especially to explore the possible inhibition of glutathione reductase through an acetaminophen-glutathione conjugate (APAP-SG). APAP-SG conjugate was synthesized by organic synthesis and purified by column chromatography. The inhibitory effect of the conjugate on two types of glutathione reductase (from yeasts and rat hepatocytes) was tested spectrophotometrically. We found that the enzyme activity was reduced similarly after the treatment with 2.96 mM acetaminophenglutathione conjugate in both yeast and hepatocyte glutathione reductases (GR); the enzyme activity was inhibited to 52.7±1.5 % (2.4±0.3 mU/ml) in yeast GR (control activity was 5.6±0.3 mU/ml) and to 48.1±8.8 % (2.2±0.2 mU/ml) in rat hepatocytes lysate GR (control activity was 5.2±0.2 mU/ml). In addition, the enzyme activity (from hepatocytes lysate) was decreased to 79±7 %, 67±2 % and 39±7 %, in 0.37, 1.48 and 3.7 mM concentration of the conjugate, respectively. We found that glutathione reductase, the essential enzyme of the antioxidant system, was dose-dependently inhibited by the product of acetaminophen metabolism - the conjugate of acetaminophen and glutathione., T. Roušar ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy
Acetaminophen overdose is the most often cause of acute liver injury. The toxic mechanism is linked to formation of an active metabolite that reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). This compound has been recognized to be non-toxic generally. Our preliminary results showed, however, that APAP-SG could possess a toxic effect too. Therefore, the aim of our study was to prepare, purify and to test possible toxicity of APAP-SG. We prepared APAP-SG using organic synthesis. The conjugate was purified by preparative HPLC and its structure was confirmed using mass spectrometry. Final purity of APAP-SG was >98 %. We estimated a toxic effect of APAP-SG in isolated rat liver mitochondria using a fluorescent ROS probe. We assessed ROS production in presence of complex I or complex II substrates. The increase of ROS-dependent fluorescence in presence of glutamate/malate was 104±13 % and 130±10 % in 1 mM and 5 mM APAP-SG, respectively, in comparison with controls. ROS production related to presence of complex II substrate was enhanced 4-times in APAP-SG (5 mM) treated mitochondria (compared to controls). We conclude, we proved our hypothesis that APAP-SG conjugate is able to induce a mitochondrial impairment leading to enhanced ROS production., T. Roušar, ... [et al.]., and Obsahuje seznam literatury
Acetaminophen (APAP) overdose is the most common cause of acute liver failure in humans. Non-alcoholic fatty liver disease is the most frequent chronic liver disease in developed countries. The aim of our work was to compare the effect of APAP on intact rat hepatocytes and hepatocytes isolated from steatotic liver in primary cultures. Male Wistar rats were fed with standard diet (10 % energy from fat) and high-fat diet (71 % energy from fat) for 6 weeks and then hepatocytes were isolated. After cell attachment, APAP (1; 2.5; 3.75 and 5 mM) was added to culture media (William´s E medium) and hepatocytes were cultured for up to 24 hours. APAP caused more severe dose-dependent damage of steatotic hepatocytes as documented by increased release of lactate dehydrogenase (LDH) and LDH leakage, decreased activity of cellular dehydrogenases (WST-1 test) and reduced albumin production. Intact steatotic hepatocytes contained lower amount of reduced glutathione (GSH). Treatment with APAP (1 and 2.5 mmol/l) caused more pronounced decrease in GSH in steatotic hepatocytes. ROS (reactive oxygen species) formation after 24-hour incubation was significantly higher in fatty hepatocytes using APAP at concentration of 3.75 and 5 mmol/l. Interleukin 6 (IL-6) production was elevated in 2.5 mM APAP-treated nonsteatotic and steatotic hepatocyte cultures at 8 hours, compared to appropriate controls. In conclusions, our results indicate that steatotic hepatocytes exert higher sensitivity to the toxic action of APAP. This sensitivity may be caused by lower content of GSH in intact steatotic hepatocytes and by more pronounced APAPinduced decrease in intracellular concentration of GSH., O. Kučera, ... [et al.]., and Obsahuje seznam literatury
The aim of our work was to compare the effect of D-galactosamine (GalN) on primary cultures of lean and steatotic rat hepatocytes isolated from intact and fatty liver, respectively. GalN caused more severe injury to steatotic hepatocytes than to lean cells as documented by lactate dehydrogenase leakage. Necrotic mode of cell death strongly prevails over apoptosis since we did not observe any significant increase in activities of caspase 3, 8 and 9 in any group of hepatocytes treated with GalN. Reactive oxygen species (ROS) formation and lipid peroxidation were elevated in a dose-dependent manner by GalN and were significantly more pronounced in fatty hepatocytes. A decrease in the percentage of hepatocytes with energized mitochondria was observed from 30 mM and 10 mM GalN in lean and steatotic hepatocytes, respectively. Our results undoubtedly indicate that steatotic hepatocytes exert higher sensitivity to the toxic effect of GalN. This sensitivity may be caused by more intensive GalN-induced ROS production and lipid peroxidation and by higher susceptibility of mitochondria to loss of mitochondrial membrane potential in steatotic hepatocytes. In our experimental arrangement, apoptosis does not seem to participate considerably on hepatotoxic action of GalN in either group of hepatocytes., O. Kučera, H. Lotková, O. Sobotka Z. Červinková., and Obsahuje bibliografii
In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone., A. Moravcová, Z. Červinková, O. Kučera, V. Mezera, D. Rychtrmoc, H. Lotková., and Obsahuje bibliografii
Non-alcoholic fatty liver disease (NAFLD) is an important cause of liver-related morbidity and mortality. The aim of this work was to establish and characterize a nutr itional model of NAFLD in rats. Wistar or Sprague-Dawley male rats were fed ad libitum a standard diet (ST-1, 10 % kcal fat), a medium-fat gelled diet (MFGD, 35 % kcal fat) and a high-fat gelled diet (HFGD, 71 % kcal fat) for 3 or 6 weeks. We examined the serum biochemistry, the hepatic malondialdehyde, reduced glut athione (GSH) and cytokine concentration, the respiration of liver mitochondria, the expression of uncoupling protein-2 (UCP-2) mRNA in the liver and histopathological samples. Feeding with MFGD and HFGD in Wistar rats or HFGD in Sprague-Dawley rats induced small-droplet or mixed steatosis without focal infl ammation or necrosis. Compared to the standard diet, there were no significant differences in serum biochemical parameters, except lower concentrations of triacylglycerols in HFGD and MFGD groups. Liver GSH was decreased in rats fed HFGD for 3 weeks in comparison with ST-1. Higher hepatic malondialdehyde was found in both strains of rats fed HFGD for 6 weeks and in Sprague-Dawley groups using MFGD or HFGD for 3 weeks vs. the standard diet. Expression of UCP-2 mRNA was increased in Wistar rats fed MFGD and HFGD for 6 weeks and in Sprague-Dawley rats using HFGD for 6 weeks compared to ST-1. The present stud y showed that male Wistar and Sprague-Dawley rats fed by HFGD developed comparable simple steatosis without signs of progression to non-alcoholic steatohepatitis under our experimental conditions., O. Kučera ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy