D-Galactosamine/Lipopolysaccharide (D-GalN/LPS) is a well known model of hepatotoxicity that closely resembles acute liver failure (ALF) seen clinically. The role of sirtuin 1 in this model has not yet been documented. However, there have been a number of studies about the cytoprotective effects of resveratrol, a SIRT1 activator, in the liver. This study was aimed at elucidating the roles of SIRT1 protein expression or catalytic activity in DGalN/ LPS model of hepatotoxicity. ALF was induced in male Wistar rats by intraperitoneal injection of D-GalN and LPS. Some groups of animals were pretreated with resveratrol and/or EX-527 (SIRT1 inhibitor). The effects of these treatments were evaluated by biochemical and Western blot studies. D-GalN/LPS treatment was able to induce hepatotoxicity and significantly increase all markers of liver damage and lipid peroxidation. A dramatic decrease of SIRT1 levels in response to D-GalN/LPS treatment was also documented. Resveratrol pretreatment attenuated D-GalN/LPS-induced hepatotoxicity. EX-527 blocked the cytoprotective effects of resveratrol. However, both resveratrol and EX-527 pretreatments did not exhibit any significant effect on SIRT1 protein expression. Collectively, these results suggest that downregulation of SIRT1 expression is involved in the cytotoxic effects of D-GalN/LPS model and SIRT1 activity contributes to the cytoprotective effects of resveratrol in the liver., M. K. Kemelo, L. Wojnarová, N. Kutinová Canová, H. Farghali., and Obsahuje bibliografii
Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. This study is focused on addressing the mutual relationship and early signals of these mechanisms in the D-galactosamine and lipopolysaccharide (D-GalN/LPS) hepatotoxicity model, with the help of standard liver function and biochemistry tests, histology, and measurement of gene expression by RT-PCR. Intraperitoneal injection of 400 mg/kg D-GalN and 50 μg/kg LPS was able to induce hepatotoxicity in rats, as evidenced by significant increases in liver enzymes (ALT, AST) and raised bilirubin levels in plasma. Heme oxygenase-1 and nitric oxide synthase-2 gene expressions were significantly increa sed, along with levels of their products, bilirubin and nitrite. Th e gene expression of glutathione peroxidase 1 remained unchanged, whereas a decrease in superoxide dismutase 1 gene expression was noted. Furthermore, the significant increase in the gene expression of apoptotic genes Bid, Bax and caspase-3 indicate early activation of apoptotic pathways, which was confirmed by histological evaluation. In contrast, the measured caspase-3 activity remained unchanged. Overall, the results have revealed differential oxidative stress and apoptotic responses, which deserves further investigations in this hepatotoxicity model., N. Lekić ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy
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 (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