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Inhibitory effect of endocrine disruptor 17α-ethinylestradiol on cytochrome P450 subfamily 1A
Kurshakova, Evgeniia ; Dračínská, Helena (advisor) ; Ptáčková, Renata (referee)
17-ethinylestradiol (EE2) is a synthetic derivative of the natural estrogen 17-estradiol. It is used in medicine as a key component of oral contraceptives. Due to its ability to modulate the functions of the endocrine system, EE2 belongs to the group of endocrine disruptors. The ability to bioaccumulate and affect the reproduction and development of wildlife makes EE2 a compound that possesses a potential environmental risk. Cytochromes P450 1A1 and 1A2 catalyze the oxidative reactions in metabolism of exogenous compounds, including EE2. 17-Ethinylestradiol is known to have a strong inhibitory effect on the isoform CYP1A1. Within the framework of the present thesis, the inhibition effect of EE2 on the activity of cytochromes P450 of subfamily 1A was studied in vitro via three marker reactions: 7-methoxyresorfin O-demethylation, 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation, which had to be pre-optimised for the following uses. A highly selective inhibitory effect of 17-ethinylestradiol on the human and rat isoform CYP1A1 was confirmed. An inhibitory effect on the activity of the CYP1A2 isoform was not observed by any of marker reactions. Using phenacetin O-deethylation, the concentration of EE2 causing 50% inhibition of CYP1A1 (IC50) was also determined, being of 3,4 M, at a...
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Isolation and characterization of microsomal fraction of fungus Pleurotus ostreatus and its role in the degradation of 17α-ethinylestradiol
Valášková, Petra ; Černá, Věra (advisor) ; Hodek, Petr (referee)
A synthetic hormone 17α-ethinylestradiol (EE2) which is a component of hormonal contraception pills has been identified as a main component of the endocrine-disrupting compounds (EDc). EDc are substances that mimic natural hormones in their action. Recently their amount especially in the groundwater and the surface water has been increased, which results in a negative impact on the hormonal system especially of aquatic organisms. Since it is not easy to replace these substances from the environment by conventional techniques other possibilities of their biodegradation are examined. White rot fungi, which are able to degrade lignin in nature, have promising biodegradation abilities towards many pollutants. These fungi contain a wide range of non-specific extracellular and intracellular enzymes that play an important role in the degradation. This bachelor thesis was targeted on the study of a white rot fungus, Pleurotus ostreatus, and especially on the degradation potential of its intracellular enzymes in the biodegradation of EE2. Initially, the ability of fungi Pleurotus ostreatus to degrade EE2 in vivo was tested. During the 48 hour incubation there was replaced 95,5 % of EE2. However, the role of cytochromes P450 (CYPs) in a metabolism of EE2 was not confirmed in this experiment by reason that an...
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The effect of selected endocrine disruptors on cytochromes P450 1B1 and 3A1/2
Holecová, Jana
Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...
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Inhibitory effect of 17α-ethinylestradiol on the activity of cytochrome P450 subfamilies 2B and 2C
Knapp, Kryštof ; Dračínská, Helena (advisor) ; Kukačka, Zdeněk (referee)
17α-ethinylestradiol (EE2) is a synthetic derivative of endogenous estrogen 17β-estradiol. It is widely used in pharmaceutics as a ingredient of female oral contraceptives and also in hormone replacement therapy for women with postmenopausal syndrome. Due to its high estrogenic potential together with resistance to the chemical degradation and tendency to bio- accumulate in environment it is considered as an important persistent organic pollutant. In organisms, EE2 is oxidatively metabolised by enzymes from a family of cyto- hromes P450. Some studies show that EE2 is also a potent inhibitor of some isoforms from cytochrome P450 family. In this work, we studied inhibitory effect of EE2 on two human isoforms CYP2B6, CYP2C9 and their rat orthologs CYP2B1 and 2C6, respectively. All studied isoforms exhibited reduced enzyme activity in the presence of EE2. The greatest decrease of enzyme activity was observed with CYP2B1 catalyzing pentoxyresoru n O-depentylation with value of IC50 = 9,6 µM. For hydroxylation of bupropion, selective reaction of CYP2B1 and CYP2B6, EE2 behaved as a more potent inhibitor of CYP2B6 with IC50 = 60 µM. The inhibitory effect of EE2 on rat CYP2B1 for similar reaction conditions was signi cantly lower. With 200µM concentration of EE2, CYP2B1 exhibited still more than 50 % of...
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Inhibition of enzyme activity of cytochromes P450 by endocrine disruptor 17α-ethinylestradiol
Otáhalová, Barbora ; Dračínská, Helena (advisor) ; Ryšlavá, Helena (referee)
17α-ethinylestradiol (EE2) is a synthetic hormone, derivative of the natural hormone estradiol. EE2 is one of the the most prescribed drugs in the world. It belongs to the estrogenic endocrine disrupter chemicals. These compounds are able to alter functions of the endocrine system and cause adverse effects in the organism, offspring and (sub)population. In this thesis, there are observed effects of 17α-ethinylestradiol on enzyme activities of main enzymes involved in phase I of xenobiotic biotransformation, i.e. cytochromes P450 (CYP), in vitro. Isoforms of CYP subfamilies 1A, 2B, 2C, 2E and 3A were studied in rats and humans. Each CYP isoform was incubated with EE2 at two concentrations, 10μM EE2 and the concentration corresponding to the substrate concentration in the specific marker reactions of individual CYP isoforms. The results indicate, that in rat liver microsomes the activity of all studied isoforms except CYP1A2 was decreased in the presence of EE2. When EE2 was added to the incubation mixture at the concentration of the reaction substrate, the greatest decrease in enzyme activity was observed for CYP2C6, with the remaining activity only 36%. In human liver microsomes, the activity of CYP2B6, CYP2C9, CYP2E1 and CYP3A4 was also effected by EE2. As in the case of rat model, CYP2C subfamily...
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The inhibitory effect of estrogenic endocrine disruptors on cytochrome P450 activity
Otáhalová, Barbora ; Dračínská, Helena (advisor) ; Hýsková, Veronika (referee)
Endocrine disruptors are exogenous and endogenous compounds that interfere with the production, signaling and metabolism of natural hormones, thereby disturbing the balance of the endocrine system. Exogenous endocrine disruptors include 17α-ethinylestradiol and endogenous endocrine disruptors include 17β-estradiol. This thesis examinates effects of these endocrine disruptors on the specific activities of rat cytochromes P450 1A1 and 3A1. The enzyme specific activity of CYP1A1 is determined by the marker reaction O-deethylation 7-ethoxyresorufin and the activity of CYP3A1 is determined by the marker reaction 6β-hydroxylation of testosterone. It has been confirmed that both estrogens 17β-estradiol and 17α-ethinylestradiol inhibit activity of CYP1A1 and CYP3A1. The stronger inhibitor of CYP1A1 was 17β-estradiol and of CYP3A1 was 17α-ethinylestradiol. The concentration of estrogens causing 50% inhibition (IC50) of enzymes has been determined, for 17β-estradiol it was 4,6 μM and for 17α-ethinylestradiol 7,9 μM. CYP1A1 is a important enzyme for the biotransformation of carcinogens, it can be said that estrogens can modulate the genotoxicity of CYP1A1 activated carcinogens. The results show that CYP1A1 inhibition increased after pre-incubation of estrogens with NADPH (cofactor of cytochromes P450), that...
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The effect of selected endocrine disruptors on cytochromes P450 1B1 and 3A1/2
Holecová, Jana
Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...
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The effect of selected endocrine disruptors on cytochromes P450 1B1 and 3A1/2
Holecová, Jana ; Bořek Dohalská, Lucie (advisor) ; Cajthaml, Tomáš (referee)
Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...
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The influence of endocrine disruptors on the expression of cytochrome P450 1A2
Orlovská, Ľubica ; Dračínská, Helena (advisor) ; Ječmen, Tomáš (referee)
The term endocrine disruptor is used for chemical compounds which imitate or antagonize the effects of endogenic hormones, alter hormone synthesis and metabolism or modify levels of hormonal receptors. Synthetic estrogen 17α-ethinylestradiol (EE2) and carcinogenic substance benzo[a]pyrene (BaP) belong to the group of chemicals described as exogenic compounds with endocrine destruction, while estrogenic hormone 17β- estradiol (EST) figures as natural endogenic endocrine disruptor. The bachelor thesis focuses on study of the influence of these endocrine disruptors and their combinations on expression and specific activity of CYP1A2. RNA was isolated from the lungs of rats treated with the endocrine disruptors and from untreated rats. RNA was converted to cDNA by reverse transcription. Relative amount of CYP1A2 in livers, kidneys and lungs was quantified by real-time PCR. The protein expression of CYP1A2 was studied using the Western blot with consecutive immunodetection. Finally, the specific activity of hepatic CYP1A2 was determined by measuring 7-methoxyresorufin O- demethylation. It was confirmed that BaP induces gene expression of CYP1A2 in livers, kidneys and lungs, even in combination with EE2 and EST. However, both estrogens decrease the induction potential of BaP. When given individually,...
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The effect of selected endocrine disruptors on the cytochromes P450 1A1 and 2C
Klusoňová, Zuzana ; Bořek Dohalská, Lucie (advisor) ; Linhartová, Lucie (referee)
Many currently produced chemicals reveal specific properties which allow them to be referred to as endocrine disruptors (ED). These substances exhibit an exogenic hormone activity and usually act as antagonists or agonists of endogenic hormones. The exogenic EDs studied in this work were 17α-ethinylestradiol (EE2) and benzoapyrene (BaP). 17β-estradiol (E2), a typical endogenic hormone, was also included to the study. In the presented work, the effect of these EDs and their combinations on the expression and specific activities of cytochromes P450 (CYP) 1A1 and 2C was determined. First, the microsomal fraction (MF) of liver, kidney and lung of rats premedicated with these compounds or without premedication was isolated. CYP expression was assessed by the Western blot analyses in these MF samples. Moreover, CYP1A1 and CYP2C specific activities were evaluated. It was found that premedication of rats with BaP increased CYP1A1 expression in all above mentioned organs. Whereas BaP strongly induced rat CYP1A1, EE2 and E2 were almost without this effect. But, when these disruptors were administered to rats with BaP, they supported its potency to induce CYP1A1. Further, CYP2C11 expression and its specific activity were gently increased by premedication of rat with EE2 and its combination with BaP....
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