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Utilization of tissue cultures for toxicology of the environment.
Polanská, Daniela ; Cajthaml, Tomáš (advisor) ; Klusoň, Petr (referee)
5 Abstract Five substances from the group of so-called personal care products, known for their low degradability and regular environmental detection, were tested for toxicity using two fish tissue lines (RTgill-W1 a RTG-2) isolated from rainbow trout (Oncorhynchus miykiss). The tested substances were hexadecylpyridinium chloride (HDP), chlorhexidine (CHX), octenidine (OCT), thymol (THM) and triclosan (TCS). A cell viability assay was performed with each of these compounds using Alamar Blue ™ (AB), 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and neutral red (NR) protocols. The results were used to construct dose-response curves along with an EC50 value for each of these substances. The EC50 values ranged from 0,51 (HDP) to 33,75 µg.ml-1 (THM) for RTgill-W1 and from 0,31 (HDP) to 33,37 µg.ml-1 (THM) for RTG-2. The theoretical LC50 estimation was calculated according to Tanneberger et al. (2013). For all substances, cytochrome P450 1A activity was monitored using 7-ethoxyresorufin-o-deethylase (EROD), four out of five tested chemicals were statistically positive for EROD, the highest EROD response was observed for the most toxic compound - HDP. Only TCS did not show statistically significant cytochrome P450 1A activity. In addition, oxidative stress was measured with the fluorescent dye...
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Bioaktivní látky ve vodním prostředí a jejich vliv na ryby - zaměření na cytochromy P450 v rybách
SAKALLI, Sidika
The term "biologically active compounds" covers a wide range of substances originating from natural or synthetic origins. These compounds can enter the aquatic environment through wastewater treatment plants, manufactural discharge or they are naturally present in the aquatic plants or microorganisms. Their adverse effects on fish has been widely studied and accepted. This thesis is focussed on the adverse effect of several bioactive compounds (i.e. pharmaceuticals, phytochemicals, or complex mixture of pollutants) on fish using different experimental design as in vitro, in vivo and in situ. In the first part of this thesis, in vitro effects of pharmaceuticals and phytochemicals or their combinations on fish CYP system were investigated. Moreover, effects of standard carrier solvents used in enzyme activity assays were also investigated. An important finding in this study was the lack of effects of either dexamethasone, quercetin, or indole-3-carbinol on EROD activity; however, when these agents were combined, EROD activity was strongly inhibited. This demonstrates that combination of compounds might exert different effects than single compounds, and the effects of mixture compounds cannot be predicted from the effects of individual compounds. In the second part of the thesis, the effects of chronic exposures of rainbow trout to dexamethasone and clotrimazole were investigated. The results regarding dexamethasone showed similarities with in vitro studies, and both in vitro and in vivo exposure of dexamethasone did not alter the CYP enzyme activities. On the other hand, in vivo exposure of clotrimazole yielded conflicting results with findings from the in vitro studies. Clotrimazole induced EROD activity in chronically exposed rainbow trout, and BFCOD activity showed biphasic pattern in which it was inhibited at environmentally relevant concentrations and induced at high concentrations. Thus, the observed effects suggest that clotrimazole could negatively affect fish CYPs at environmentally relevant concentrations. However, in the environment, the effects of clotrimazole and dexamethasone on fish CYPs system might be modified because of unknown compounds in these mixtures. Therefore, further investigations were done to identify the effects of mixture compounds using an in situ model. The last part of the dissertation addresses the effects of cocktail PPCPs on common carp under natural conditions. In situ studies provide valuable information on both hepatic and intestinal CYP activities. Both EROD and BFCOD activities were affected by the PPCPs that are present in the exposed fish. Moreover, changes in intestinal CYP activities suggest that fish can ingest some of these contaminants through their feed. Therefore, the intestines might be responsible for elimination of some of these pollutants and act as the first barrier of pollutant entry in fish. Despite the extensive studies concerning aquatic pollution, further studies are necessary. Development of new pharmaceuticals, their occurrence in the aquatic environment, and their effects on non-target organisms should be continuously monitored.
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IMPACT OF MUNICIPAL POLLUTION ON FISH LIVING IN A SMALL STREAM
HORA, Vojtěch
Main objective of this thesis is evaluation of the impact of extraneous substances that enter Živný stream through the WWTP Prachatice onto the stream's fish population. Brown trout (Salmo trutta m. fario) was selected as a sampling organism. Sampling was conducted in 2012 using an electrical aggregate at three locations. The sample counted a total of 36 fish. Subsequently there was conducted blood sample and tissue analysis using selected biomarkers (vitellogenin and EROD). Furthermore, an analysis of fish tissue and water samples was made to identify presence of selected extraneous substances.Concentration of vitellogenin in blood plasma of male fish in the controled location was zero, in polluted location the average concentration varied between 275 and 573 ng.ml-1. Concentration of vitellogenin in blood plasma of female fish was the lowest at a location directly downstream the WWTP (7 185 823 ng.ml-1). At other locations, the average concentration ranged between 9 250 302 - 11 614 177 ng.ml-1. The activity of liver detoxifying enzymes (EROD) was the highest at polluted locations, between 63,9 - 87,4 pmol.mg-1protein.min-1. In the controled location was the lowest activity EROD (5,4 pmol.mg-1protein.min-1). The result of identification of selected extraneous substances in fish tissues (liver) and in the water is a sum of all detected pharmaceuticals. In fish tissues was the sum of all detected pharmaceuticals in the range from 20,8 to 138,8 ng.g-1, while in water it varied between 0,07 - 3,00 ?g.l-1. The highest values were again measured in polluted areas. The results of the analyses conducted imply, that the present concentrations of extraneous substances in Živný stream downstream from Prachatice have a negative impact onto live organisms.
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