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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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Hormonal activity in wastewater
Čermáková, Tereza ; Cajthaml, Tomáš (advisor) ; Klusoň, Petr (referee)
Endocrine disruptors are natural or manmade substances which affect hormonal systems of organisms. Biologically relevant concentrations are commonly being detected in the environment. The effluents of wastewater treatment plant present their significant secondary source. Due to their occurence and quantity the interest in mixtures increases. Ecotoxicological assays with genetically modified Saccharomyces cerevisiae were aplied to verify reliability of predictive mathematical models for mixtures of standards (estrone, 17β-estradiol, 17α-ethinylestradiol, estriol, bisphenol A, irgasan, 4-nonylphenol). Chromatographic analysis along with yeast assays were used for the evaluation of real samples of wastewater treatment plant effluents and sediments. Schindler's predictive model and Full logistic model (FLM) were more reliable for predicting the whole dose-response curve compared to Generalized concentration addition (GCA). Predicted values of a parameter EC50 from all three models were comparable to empirical measurements. Three out of four samples exhibited estrogenic activity 0.65 - 1.70 ng/L 17β-estradiol ekvivalent (EEQ) above the limit of detection 0.13 - 0.33 ng/L EEQ. Antiestrogenic activity was detected in one of the samples. Prediction could be carried out only in the case of the sediments...
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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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Reproductive and epigenetic parameters in sperm connected to the disruptive development and early embryo loss.
Flintová, Jennifer ; Hortová, Kateřina (advisor) ; Petelák, Aleš (referee)
DNA methylation, histone modifications and regulation by non-coding RNAs are considered to play vital role in embryonal development and gametogenesis. Epigenetic mechanisms are not only inwardly programmed, but are massively affected by numerous exogenous aspects. Environmetal pollution and unhealthy parental lifestyle are two major factors related to impaired fertility status, abberant embryonal development of progeny. Some altered epigenetic marks can be transmitted to offspring generations, however in some cases these aberrations may be reversed by adequate lifestyle interventions. Since epigenetic regulations and genes responsible for spermatogenesis are conserved among vertebrates, studies based on animal models are highly relevant for understanding mechanisms causing impaired fertility and overall health in humans. Key words: Epigenetics, sperm, endocrine disruptors, hypoxia, lifestyle, transgenerational effect.
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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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Effect of selected pollutants on mammalian organisms in vivo and cells in vitro and preparation of specific monoclonal antibodies for their detection
Dorosh, Andriy ; Pěknicová, Jana (advisor) ; Linhart, Otomar (referee) ; Motlík, Jan (referee)
Environmental pollution and its effect on the living organisms has attracted lots of attention recently. There is a growing body of evidence that we are exposed to environmental pollutants at low concentrations in everyday life. The cells and organisms have tools to identify, neutralize and excrete the majority of the toxic compounds. The most dangerous are those that can escape this process or act at low trace concentrations. Endocrine disruptors (EDs) belong to the latter group. Endocrine disruptors can be of natural and anthropogenic origin. EDs target corresponding hormonal receptors and can act at low concentrations. A wide family of nuclear receptors recognize steroid hormones. The majority of EDs can pass through the cytoplasmic membrane, use the hydrophobic nature of the receptor-ligand binding, trigger hormone response and change the expression of the sensitive genes. By interfering with estrogen and androgen signaling, EDs can have effect on the whole organism, but the reproductive system is influenced most. In the present work, our aim was to develop the methods for ED detection and monitoring, analyze the estrogenic potency of EDs, and evaluate the effects of natural estrogens and EDs on male reproductive functions, including sperm and testicular physiology and endocrine functions. First, we...
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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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Vliv syntetických progestinů na vývoj gonád kapra obecného
PROFANT, Vít
This paper was focused on effect of synthetic progestins on gonadal development in fish. A 160-day chronic test on common carp (Cyprinus carpio), was run underlaboratory conditions. The fish was divided into five experimental groups. The first control group included clear diluted water without any occurrence of progestins. The second control group was kept in diluted water with dissolving agent DMSO (dimethylsulfoxide). The third group of fish was exposed to gestogene at a concentration level of 2 ng.l-1 and the fourth group was exposed to drospirenone at a concentration level of 2 ng.l-1. The last group (Mix) was exposed to gestogene and drospirenone simultaneously, where both substances corresponded to the concentration of 2 ng.l-1. The experimental groups were dosed with progestins dissolved in DMSO, since they have lipophilic nature. The determination of the influence of synthetic progestins on the gonadal development was based on histological examination of the exposed fish samples in toto. There was any signifficant difference in fish sex ratio and in percentage of intersex occurrence between the groups that wwere exposed to individual synthetic progestins and the control groups. However, the exposure of the mixture of drospirenone and gestogene (Mix) showed a significant difference. Primarily, a high percentage of intersex was detected with the number reaching 36.6 %. The occurrence of intersex suggested a twist in sex of exposed fish through ontogenesis.
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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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