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Optimization of endocrine disruptors determination in wastewater treatment plant sludge and application of the method in environmental samples.
Medková, Jaroslava ; Cajthaml, Tomáš (advisor) ; Tesařová, Eva (referee)
Hormonaly active compounds in wastewaters represent nowdays a serious problem. Proceses currently used in watewater treatment plants (WWTP) are unefficient in removing these compounds from contaminated wastewaters. The compounds are supposed to sorb onto solid sludge elements and sediments. In this work seven endocrine disruptors were detected in the sludge samples from WWTPs. A new sensitive method for detection of seven selected endocrine disruptors (4-nonylphenol, bisphenol A, estriol, 17β-estradiol, estrone, 17α- ethynylestradiol, irgasan) was developed. The method is based on accelerated solvent extraction (ASE), gel permeation chromatography (GPC) and solid phased extraction. For final extract analysis, gas chromatography coupled with mass spectrometry (GC/MS) was used. The efficiency of this method was tested using artificially contaminated sludge and the method was used to analyse real samples from several WWTPs in Czech Republic. The effect of sludge age on detection of individual analytes was assessed as well. The concentrations of endocrine disruptors measured in the samples reached up to 1 µg/g. The results are comparable or higher then those reported in other works and they show the necessity of further research on endocrine disruptors in the environment.
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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 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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Optimization of endocrine disruptors determination in wastewater treatment plant sludge and application of the method in environmental samples.
Medková, Jaroslava ; Cajthaml, Tomáš (advisor) ; Tesařová, Eva (referee)
Hormonaly active compounds in wastewaters represent nowdays a serious problem. Proceses currently used in watewater treatment plants (WWTP) are unefficient in removing these compounds from contaminated wastewaters. The compounds are supposed to sorb onto solid sludge elements and sediments. In this work seven endocrine disruptors were detected in the sludge samples from WWTPs. A new sensitive method for detection of seven selected endocrine disruptors (4-nonylphenol, bisphenol A, estriol, 17β-estradiol, estrone, 17α- ethynylestradiol, irgasan) was developed. The method is based on accelerated solvent extraction (ASE), gel permeation chromatography (GPC) and solid phased extraction. For final extract analysis, gas chromatography coupled with mass spectrometry (GC/MS) was used. The efficiency of this method was tested using artificially contaminated sludge and the method was used to analyse real samples from several WWTPs in Czech Republic. The effect of sludge age on detection of individual analytes was assessed as well. The concentrations of endocrine disruptors measured in the samples reached up to 1 µg/g. The results are comparable or higher then those reported in other works and they show the necessity of further research on endocrine disruptors in the environment.
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