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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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Endocrine-disruptive effects of bisphenol A and other representatives of bisphenols
Kromková, Lucia ; Cajthaml, Tomáš (advisor) ; Vítků, Jana (referee)
Xenoestrogens are substances of anthropogenic origin that disrupt the human endocrine system. This work deals with bisphenol A, a substance found in plastics all around us, and its effect on the human reproductive system. As the toxicity of bisphenol A is gradually being proven, its production is regulated by legislation, and therefore bisphenol A is being replaced by other derivatives. This work focuses only on the 7 most widespread bisphenol derivatives - S, F, E, B, AP and AF. The endocrine-disrupting effect of bisphenols is discussed from carefully selected relevant studies with an emphasis on the last 5 years (2018-2023) and compiled into clear tables. It turns out that bisphenol AP has the least endocrine disruption per organism, while bisphenol AF and bisphenol S have the highest. In in vitro and in vivo studies, bisphenol A has different concentrations that influence disruptiveness, which indicates the interaction of bisphenol A with other factors in the organism. The greatest risk to humanity is the proven cocktail effect of bisphenol A and transgenerational transmission. Key words: endocrine disruptors, xenoestrogens, bisphenol A, in vitro, bisphenol derivatives, in vivo
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Laccase activity profiling in Trametes versicolor cultures degrading endocrine-disrupting compound Delor 103
Plačková, Martina
In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...
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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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Laccase activity profiling in Trametes versicolor cultures degrading endocrine-disrupting compound Delor 103
Plačková, Martina
In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...
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Laccase activity profiling in Trametes versicolor cultures degrading endocrine-disrupting compound Delor 103
Plačková, Martina ; Svobodová, Kateřina (advisor) ; Mikušová, Gabriela (referee)
In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...
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Microbial degradation of 17alfa-ethinylestradiol and bisphenol A
Křesinová, Zdena ; Svobodová, Kateřina ; Moeder, M. ; Cajthaml, Tomáš
17ethinylestradiol (EE2) and bisphenol A (BA) are endocrine disrupting compounds (ED). ED have a negative influence on animals and human hormonal systems animals, and thus they are serious envronmental contaminants. This work was focused on biodegradtion of EE2 and BA using 8 white rot fungi strains in liquid media. Pleurotus ostreatus, Pycnoporus cinnabarinus and Irpex lacteus were able to remove both ED under detection limit (initial concetration was 10 ppm) during the first three days of cultivation. In contrary, Phanerochaete chrysosporium was not able to degrade both ED durin 2 weaks long cultivation
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