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The effect of ethinylestradiol on Na+, K+ - ATPase
Kettnerová, Karolína ; Svoboda, Petr (advisor) ; Driák, Daniel (referee)
This diploma thesis is oriented to analysis of physiological effect of synthetic estrogen ethinylestradiol (EE), which represents the main component of steroid-based substance used in hormonal contraception. From wide range of physiologically important protein molecules, which might be effected by this steroid, thesis focuses to the study of the sodium plus potassium activated, magnesium dependent adenosinetriphosphatase (Na+, K+ - ATPase), which is selectively inhibited by cardiac glycosides such as ouabain (g strophantine). Na+, K+ - ATPase represents an important plasma membrane bound enzyme, which catalyzes the active transport of sodium and potassium across plasma membrane. In the first part of this work, Na+, K+ - ATPase was determined by binding of radioactively labeled selective inhibitor of this enzyme [3H]ouabain, used for this purpose. In the second part of this work, plasma membrane fluidity was analyzed by steady-state fluorescence anisotropy of DPH. The effect of EE on [3H]ouabain binding was studied first under in vitro conditions by using human embryonic kidney cells (HEK293) which were cultivated for 24 hours in the presence of EE in tissue culture medium. Second, the effect of EE was also studied under in vivo conditions, by subcutaneous application of EE to the female rats of Wistar...
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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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The effects of endocrine disruptors on the expression and the activity of cytochromes P450 2B in laboratory rat as a model organism
Měkotová, Barbora ; Dračínská, Helena (advisor) ; Levová, Kateřina (referee)
Endocrine disrupting chemicals are compounds that interfere with natural hormones and they are responsible for functional changes which may lead to damage of the endocrine system. Their presence in the environment is associated with a number of diseases whose extent is hard to predict. As endocrine disrupting chemicals, a wide range of exogenous and endogenous compounds is present in the environment. Important exogenous endocrine disrupting chemicals include benzo[a]pyrene (BaP) and 17α-ethinylestradiol (EE2); the female sex hormone 17β-estradiol (E2) can act like endogenous endocrine disruptor. In this thesis, the effect of these three compounds and their combinations on the expression and the activity of rat biotransformation enzymes cytochromes P450 2B is studied. The gene expression was determined by quantitative PCR, the expression of the protein itself was studied using Western blot method and consecutive immunodetection. The results show that CYP2B expression is almost unchanged after BaP premedication, whereas estrogenic compounds, E2, EE2, their combination and their combinations with BaP, significantly decrease the expression. The enzyme activity of CYP2B was also studied in rat liver microsomes using the marker substrate 7-pentoxyresorufin. EE2, E2 and their combination decrease the...
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The effect of ethinylestradiol on Na+, K+ - ATPase
Kettnerová, Karolína ; Svoboda, Petr (advisor) ; Driák, Daniel (referee)
This diploma thesis is oriented to analysis of physiological effect of synthetic estrogen ethinylestradiol (EE), which represents the main component of steroid-based substance used in hormonal contraception. From wide range of physiologically important protein molecules, which might be effected by this steroid, thesis focuses to the study of the sodium plus potassium activated, magnesium dependent adenosinetriphosphatase (Na+, K+ - ATPase), which is selectively inhibited by cardiac glycosides such as ouabain (g strophantine). Na+, K+ - ATPase represents an important plasma membrane bound enzyme, which catalyzes the active transport of sodium and potassium across plasma membrane. In the first part of this work, Na+, K+ - ATPase was determined by binding of radioactively labeled selective inhibitor of this enzyme [3H]ouabain, used for this purpose. In the second part of this work, plasma membrane fluidity was analyzed by steady-state fluorescence anisotropy of DPH. The effect of EE on [3H]ouabain binding was studied first under in vitro conditions by using human embryonic kidney cells (HEK293) which were cultivated for 24 hours in the presence of EE in tissue culture medium. Second, the effect of EE was also studied under in vivo conditions, by subcutaneous application of EE to the female rats of Wistar...
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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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