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Changes in the composition and localization of gangliosides in cholestasis associated with other markers of pathological processes in hepatocytes.
Petr, Tomáš
This thesis is focused on the study of glycosphingolipids in the rat liver in different types of cholestasis and the effect of oxidative stress on changes in the composition and localization of gangliosides. First, it was necessary to optimize the immunochemical detection of glycosphingolipids. GM1 ganglioside was selected as a representative of a large glycolipid family. We found that minimum water content in the fixing solution was a key condition for fixation of histological sections. Optimized method of GM1 detection was subsequently used in in vivo experiments. We have demonstrated that estrogen-induced cholestasis characterized by high concentrations of bile acids and increased oxidative stress caused changes in the synthesis and distribution of liver gangliosides. HMOX induction is associated with a reduction in oxidative stress level and accompanied by normalization in GSL content. In experiments with obstructive cholestasis, we found that changes in the distribution and synthesis of gangliosides were not strictly specific to a particular type of cholestasis. We assume that it represents a general mechanism of hepatoprotection. We also confirmed the important role of bilirubin, product of HMOX reaction, in protection of hepatocytes against oxidative damage caused by high concentrations of...
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Alterations in gene expression of hepatobiliary transporters as potential mechanisms for drug-induced cholestasis by amoxicillin and clavulanic acid
Řepová, Veronika ; Pávek, Petr (advisor) ; Mičuda, Stanislav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Řepová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D., Prof. Ramiro Jover Atienza, Ph.D. Title of diploma thesis: Alterations in gene expression of hepatobiliary transporters as potential mechanisms for drug-induced cholestasis by amoxicillin and clavulanic acid The combination of amoxicillin and clavulanic acid (AMO/CLA) represents one of the most frequent causes of the idiosyncratic type of drug-induced liver injury (DILI) nowadays. Despite difficulties in diagnosis and causality assessment, the clinical features have already been reported and in most of the cases categorized as cholestatic damages. Number of descriptions of the molecular mechanisms of drug-induced cholestasis has been rising recently and the role of hepatobiliary transporters has turned out to be crucial in the pathogenesis. However, the mechanisms of AMO/CLA-induced DILI at the molecular level still remain indistinct. In order to investigate the hepatotoxic effects of AMO/CLA and AMO alone in vitro, HepG2 and human Upcyte hepatocytes were used as hepatocellular models. The mRNA levels of key bile acid (BA) transporters, enzymes and nuclear receptors (NRs) were measured by quantitative real-time polymerase chain...
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Alterations in gene expression of hepatobiliary transporters as potential mechanisms for drug-induced cholestasis by amoxicillin and clavulanic acid
Řepová, Veronika ; Pávek, Petr (advisor) ; Mičuda, Stanislav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Řepová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D., Prof. Ramiro Jover Atienza, Ph.D. Title of diploma thesis: Alterations in gene expression of hepatobiliary transporters as potential mechanisms for drug-induced cholestasis by amoxicillin and clavulanic acid The combination of amoxicillin and clavulanic acid (AMO/CLA) represents one of the most frequent causes of the idiosyncratic type of drug-induced liver injury (DILI) nowadays. Despite difficulties in diagnosis and causality assessment, the clinical features have already been reported and in most of the cases categorized as cholestatic damages. Number of descriptions of the molecular mechanisms of drug-induced cholestasis has been rising recently and the role of hepatobiliary transporters has turned out to be crucial in the pathogenesis. However, the mechanisms of AMO/CLA-induced DILI at the molecular level still remain indistinct. In order to investigate the hepatotoxic effects of AMO/CLA and AMO alone in vitro, HepG2 and human Upcyte hepatocytes were used as hepatocellular models. The mRNA levels of key bile acid (BA) transporters, enzymes and nuclear receptors (NRs) were measured by quantitative real-time polymerase chain...
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Bilirubin secretory pathway and its disorders.
Sticová, Eva ; Jirsa, Milan (advisor) ; Bronský, Jiří (referee) ; Jirásek, Tomáš (referee)
Identification and functional characterization of numerous transport systems at the sinusoidal and canalicular membrane of hepatocytes have significantly expanded our understanding of bilirubin metabolism and contributed to elucidation of molecular basis of hereditary jaundice. Moreover, dysregulation of hepatobiliary transport systems could explain jaundice in many acquired liver disorders. This thesis is focused on the new aspects of bilirubin handling in hepatocytes based on elucidation of the molecular basis of Rotor syndrome. The first study is focused on the antioxidative properties of bilirubin in liver tissue in a model of obstructive cholestasis. In the second part of the thesis we present several novel mutations in ABCC2, the gene associated with Dubin-Johnson syndrome, identified in patients selected for the Rotor locus mapping study. In the key third study concerned with Rotor syndrome we demonstrated that biallelic inactivating mutations causing complete absence of transport proteins OATP1B1 and OATP1B3 result in disruption of hepatic reuptake of bilirubin, which is the molecular basis of Rotor-type jaundice. These results indicate that apart from secretion of conjugated bilirubin into bile, a significant fraction of bilirubin glucuronide is secreted via MRP3 into sinusoidal blood and...
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Metabolism of estrogene in UGT1A1-deficient rats
Módos, Anna ; Muchová, Lucie (advisor) ; Brůha, Radan (referee)
Introduction Estrogen-induced cholestasis is a disease characterized by a failure of bile flow and bile production. It can develop in women after oral contraceptives use, hormone replacement therapy or during pregnancy. The estrogen metabolism is a complex process leading to formation of metabolites with different biological activities. It takes place primarily in the liver (Phase I and Phase II including hydroxylation, methylation, sulfation and glucuronidation). The enzymes from UDP-glucuronosyltransferases family , abbreviated UGT, are responsible for the glucuronidation of estrogens. Aims The objective of my work is to define estrogen metabolism and gene expression of UGT1A1, CYP1A2 and SULT1A1 and characterize cholestatic liver damage in the UGT1A1 deficient rat strain (Gunn rats) compared to rats with normal enzyme activity and try to define possible mechanisms responsible for the liver damage. Methods Adult female Gunn and corresponding heterozygous rats were treated with ethinylestradiol (EE, 5 mg/kg body weight SC) for 5 days, while control rats received propanediol (vehicle). Day six, the animals were sacrificed and plasma and liver tissue were collected for analysis. Markers of cholestasis and liver damage ALP, AST, ALT and bilirubin were determined using an automatic analyzer, total...
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Liver gangliosides in cholestasis induced by bile duct ligation.
Hynková, Barbora ; Entlicher, Gustav (advisor) ; Ledvinová, Jana (referee)
Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17α− ethinylestradiol induced cholestasis, but the increase of b-series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatis induced bile duct ligation (HO-1 activator- hemine, HO-1 inhibitor- Sn-mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals, but a decrease of some a- and b- series gangliosides was observed. In group with activated HO-1 total sialic acid increased, but the composition of gangliosides...
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Liver gangliosides in cholestasis induced by bile duct ligation.
Hynková, Barbora ; Entlicher, Gustav (advisor) ; Ledvinová, Jana (referee)
Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17ethinylestradiol induced cholestasis, but the increase of b- series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatic rats (HO-1 activator- hemine, HO- 1 inhibitor- Sn- mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals but without significant changes in gangliosides composition. Lipid sialic acid and gangliosides were not changed in animals with hemine activated HO-1. Expression of mRNA of key...
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Changes in the composition and localization of gangliosides in cholestasis associated with other markers of pathological processes in hepatocytes.
Petr, Tomáš ; Muchová, Lucie (advisor) ; Mičuda, Stanislav (referee) ; Befekadu, Asfaw (referee)
This thesis is focused on the study of glycosphingolipids in the rat liver in different types of cholestasis and the effect of oxidative stress on changes in the composition and localization of gangliosides. First, it was necessary to optimize the immunochemical detection of glycosphingolipids. GM1 ganglioside was selected as a representative of a large glycolipid family. We found that minimum water content in the fixing solution was a key condition for fixation of histological sections. Optimized method of GM1 detection was subsequently used in in vivo experiments. We have demonstrated that estrogen-induced cholestasis characterized by high concentrations of bile acids and increased oxidative stress caused changes in the synthesis and distribution of liver gangliosides. HMOX induction is associated with a reduction in oxidative stress level and accompanied by normalization in GSL content. In experiments with obstructive cholestasis, we found that changes in the distribution and synthesis of gangliosides were not strictly specific to a particular type of cholestasis. We assume that it represents a general mechanism of hepatoprotection. We also confirmed the important role of bilirubin, product of HMOX reaction, in protection of hepatocytes against oxidative damage caused by high concentrations of...
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