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Determination of estrogen pollutants in real water sample by HPLC-UV after solid phase extraction.
Kozlík, Petr ; Bosáková, Zuzana (advisor) ; Coufal, Pavel (referee)
4 Abstract Estrogens are considered to belong to chemicals that negatively affect the endocrine system, even if present at very low concentrations. They are discharged into environment as a result of an increasing application of drugs etc. This work is focused on the separation and quantification of five estrogens, namely estrone (E1), 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2) and estriol (E3) in natural water samples by HPLC-UV method. The chromatographic system consisted of a C18 stationary phase (SunFire® C18, 150 x 4.6 mm, octadecyl bounded to silica gel, particle size 5 µm) and binary mobile phase of acetonitrile/water in various ratios in isocratic separation mode. The effect of acetonitrile content in the mobile phase and flow rate of the mobile phase on retention and separation parameters was tested. Under the optimized separation conditions (acetonitrile/water 40/60 (v/v), 1.3 ml/min), all the compounds were baseline resolved and eluted within 15 min. These experimental conditions were applied to the calibration measurements which were carried out within the concentration range from 0.001 to 1 mg/ml. Limits of detection (LOD) and limits of quantification (LOQ) for the individual estrogens and their mixture (standards dissolved in methanol) were determinated. The detection...
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The effect of estrogens on capacitation and acrosome reaction of boar spermatozoa in vitro.
Dostálová, Pavla ; Pěknicová, Jana (advisor) ; Nedvídek, Josef (referee)
Fertilization is a unique biological event where male and female gametes fuse together to produce a new organism. Before the gametes are able to fuse, however, they must undergo a series of controlled changes. For the male gamete, capacitation and acrosome reaction (AR) must occur, which take place during the sperm migration through the female genital tract. Unfortunately, while the process of capacitation has been known for over half a century, the molecular basis and influential factors behind it are not fully understood. Although estrogens have been considered mainly female reproductive hormones, there is increasing evidence suggesting that these steroids have an important role also in regulation of male reproductive functions. Sperm come into the contact with estrogens during their formation in the male and female genital tract, indicating that the hormone may play an important role in sperm maturation. In this study, we examined the importance of three endogenous estrogens (E1 -estron, E2 - 17β estradiol, E3 - estriol) and one synthetic estrogen (EE2 - 17α ethinylestradiol) on sperm maturation during capacitation and AR. Stimulatory effect were observed with all tested estrogens on both capacitation and zona pellucida induced AR. Moreover, we have determinied that the stimulatory effect on...
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Methods of water samples processing with subsequent in vitro detection of endocrine disrupters
Daniel, Romain Oliver ; Komendová, Renata (referee) ; Bittner,, Michal (advisor)
In recent years, interest in monitoring of estrogenic compounds in water environment due to their adverse effects on human and animal health has been increased. Partiucular attention is paid to analytical techniques used to quantify estrogens in water samples, such as instrumental and biological techniques (in vitro, in vivo). Water samples need to be concentrated due to very low estrogen levels (ng·L-1) and low limit of detection of used analytical methods. Solid phase extraction (SPE) or liquid-liquid extraction (LLE) are comonly used to concentrate water samples. This bachelor thesis represents reverse osmosis (RO) and nanofiltration (NF) as another possible methods for this purpose. This thesis is focused on the preconcentration of water samples spiked with four estrogens (estrone E1, 17-estradiol E2, estriol E3 and 17-ethynylestradiol EE2) and surface water samples taken from three watercourses in city of Brno using RO and SPE and its comparison of their effeciency. This was evaluated by in vitro estrogenicity assay on the stably transfected cell line hER-HeLa-9903. By using RO, estrogens were concentrated with different recovery – the highest recovery was reached for EE2 129.29%, lowest for E2 38.53%. No detectable amount of estrogen has been detected in surface water samples. RO appears to be a suitable method to the water samples preconcentration prior to in vitro analysis of endocrine disrupting compounds. Water samples treated by the SPE method were not measured untill the sunbmission of bachelor thesis, so the RO and SPE efficiency was not performed.
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Mathematic Modelling of Reverse Osmosis System Design for Detection of Estrogens in Water
Siegel, Jan ; Skopalík, Josef (referee) ; Bittner, Michal (advisor)
Estrogen compounds are a subgroup of the environmental pollutants named Endocrine Disrupting Chemicals. It is a large group of chemicals which are capable of causing hormonal imbalance of an organism and affecting its reproductive, developmental or behavioral functions. In the environment, they are found in very low concentrations (below ng/l). This makes their detection and elimination highly problematic. The Research Centre for Toxic Compounds in the Environment (RECETOX) has been developing an experimental device which employes reverse osmosis (RO) to concentrate the trace amounts of micropollutants (e.g. estrogens). The aim is to lower the detection limit of subsequent analytical methods. RO seems to be a promising alternative to commonly used methods which are more time consuming and costly. The aim of this thesis is to create a mathematical model of RO, which would describe the process of estrogen concentration and therefore could contribute to the optimization of experimental device. The mathematical model was created in MATLAB-simulink and verified by comparison to thirteen experimental results. The solutions used to test the model were NaCl at 0.002–2 g/l and drinking water with 17-ethinylestradiol of 25 ng/l. In order to concentrate the retentate on a flat-sheet RO membrane the model achieves a mean relative error in the range of 0.9–4.9 % for NaCl and drinking water. For the concentration of estrogens the deflection is 3.6 %. Comparing the model and experimental concentration of NaCl on a spiral wound RO-membrane, the deviation is in the range of 5.9–8.7 %. So far, ratio of retentate volume from the beginning to the end of the process has been used to determine the process recovery. However, due to the results obtained in the thesis, this approach appears inaccurate. A more accurate recovery would likely be achieved by using the theoretical concentration value obtained by the proposed mathematical model. However, to increase reliability, the further measurements with estrogens are needed, and also to determine the causes of deflection of model and experimental retentate volume values at the end of the concentration
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The influence of estrogens on mouse sperm capacitation and acrosome reaction in vitro
Tejnická, Magda ; Komrsková, Kateřina (advisor) ; Linhart, Otomar (referee)
There are an increasing amount of compounds in the environment that can have a negative effect on reproductive parameters in both male and female organism. There has been a worldwide decline of sperm quality during past decades and this fact lead to an increase of unnatural ways of conception through assisted reproduction techniques in the specialised centres. Natural estrogens are one of these compounds and they get into waste water after being excluded from the body by the urine. They get back into the human body from drinking water or from the food, and they can interfere with function of endogenous hormones in very low concentrations. For these reasons it is up to date to deal with the influence of these compounds on mammalian sperm. For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the development of germ cells, spermatogenesis and processes leading to successful egg fertilization such as a capacitation or acrosomal reaction. Tyrosine phosphorylation is one of the essential steps for the properly ongoing process of capacitation in sperm followed by a...
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The role of peripheral quantitative CT in early diagnostics of osteoporosis in chronically ill children
Souček, Ondřej ; Šumník, Zdeněk (advisor) ; Hoza, Jozef (referee) ; Zapletalová, Jiřina (referee)
Patients with Turner syndrome (TS) have increased fracture risk and decreased bone density, patients with severe hemophilia have low bone density. The etiology of these changes hasn't been completely elucidated in any of the two diseases. Our aimes were to assess bone density and geometry at the radius using a new method peripheral quantitative CT densitometry (pQCT) and to describe associations between densitometry parameters and estrogen treatment in TS and laboratory as well as clinical markers of disease severity in haemophilia. Sixty-seven girls with TS (median age 14.3 years, range 6.0-19.4) and 42 boys with haemophilia (median age 12.7 years, range 6.6-19.2) have been measured using peripheral quantitative CT at the radius. The results have been compared to published reference data. Girls with TS had decreased cortical bone density and thinner cortex. These parameters were positively correlated to the length of estrogen treatment. Trabecular bone density was normal in prepubertal girls but it was decreased after puberty. There was no association between trabecular bone density and the estrogen treatment. Boys with haemophilia had decreased trabecular density and low muscle area at the forearm. Densitometry parameters were influenced neither by the clotting factor VIII/IX level nor by the frequency of...
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Monitoring of selected drugs in surface water
Langová, Jana ; Benešová, Libuše (advisor) ; Křenková, Marcela (referee)
This literature review based on recent works, deals with surface water above all river water, focusing on occurrence and detected concentration of pharmaceuticals. Targeted substances were selected according to four moulds: consumption in the Czech Republic - Nonsteroidal anti-inflammatory drugs, resistance in the environment - antiepileptic carbamazepin, possible environmental risks - estrogens and according to published findings of these drugs. The occurrence of pharmaceuticals was not only note in the surface water but in the drinking water too. Through this review, it appears that the pharmaceutical risk must be considered. Key words: PPCPs, pharmaceuticals, surface water, NSAIDs, carbamazepin, estrogens
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The effect of estrogens on capacitation and acrosome reaction of boar spermatozoa in vitro.
Dostálová, Pavla ; Pěknicová, Jana (advisor) ; Nedvídek, Josef (referee)
Fertilization is a unique biological event where male and female gametes fuse together to produce a new organism. Before the gametes are able to fuse, however, they must undergo a series of controlled changes. For the male gamete, capacitation and acrosome reaction (AR) must occur, which take place during the sperm migration through the female genital tract. Unfortunately, while the process of capacitation has been known for over half a century, the molecular basis and influential factors behind it are not fully understood. Although estrogens have been considered mainly female reproductive hormones, there is increasing evidence suggesting that these steroids have an important role also in regulation of male reproductive functions. Sperm come into the contact with estrogens during their formation in the male and female genital tract, indicating that the hormone may play an important role in sperm maturation. In this study, we examined the importance of three endogenous estrogens (E1 -estron, E2 - 17β estradiol, E3 - estriol) and one synthetic estrogen (EE2 - 17α ethinylestradiol) on sperm maturation during capacitation and AR. Stimulatory effect were observed with all tested estrogens on both capacitation and zona pellucida induced AR. Moreover, we have determinied that the stimulatory effect on...
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Influence of estrogens on mammalian sperm.
Šidlová, Adéla ; Šebková, Nataša (referee) ; Hortová, Kateřina (advisor)
For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction, whether their effect is positive or negative. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the growth and maintenance of bone mass, as well as in the development of germ cells and in the marches of spermatogenesis. Healthy sperm are essential for successful reproduction. Effect of endogenous estrogens is partly involved in the regulation of proper development of sperm. Besides the positive effect of endogenous estrogens, there is also a negative effect of estrogens from the external environment, so-called environmental estrogens. Between environmental estrogens are man-made chemicals, xenoestrogens, which increasingly contaminate the environment. Hazards of these estrogenic substances in nature is hidden in their ability to act as endocrine disruptors, which cause defects in many aspects of mammalian reproduction. With regards to the fact that there are more and more substances in current environment that can interfere with hormonal path of mammalian reproduction, it is important to look at this issue. Reproductive disorders that may be caused by...
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