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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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Vliv vybraných kardiovaskulárních léčiv nalézaných ve vodním prostředí na ryby
STEINBACH, Christoph Antonius
Cardiovascular pharmaceuticals are among the most prescribed drugs. As a result of the high consumption, these pharmaceuticals have been frequently detected in waste and surface waters. Verapamil, diltiazem and atenolol are very important representatives of cardiovascular pharmaceuticals; therefore, the present research focused on their acute and sub-chronic effects, bioconcentration, half-life time and metabolism in fish. Moreover, unified protocol for the quantitative assessment of histopathological alterations on the heart ventricle and coronary blood vessels employing heart index calculation was developed with the aim to better assess histopathological changes in fish heart which is one of the targets of cardiovascular pharmaceuticals and other chemicals. The effects caused by high concentrations of the studied substances, verapamil, diltiazem and atenolol, in fish can be considered similar to the therapeutic effects and side effects that are found in humans. The acute exposure to verapamil at the human therapeutic plasma level reduced the heart rate in common carp embryos and larvae. In addition, the acute and chronic exposure to this substance caused peripheral edema and gastrointestinal haemorrhage in carp. Similarly, the histological changes in heart and the blood vessels of the liver in diltiazem exposed rainbow trout suggested vasodilatation similar to the pharmacological effect of diltiazem in the human body. In rainbow trout sub-chronically exposed to atenolol at a human therapeutic blood plasma concentration, histopathological changes in the cardiovascular system were found. The bioconcentration of verapamil, diltiazem and atenolol in fish can be classified as low. Bioconcentration factor (BCF) of verapamil in whole body homogenates of common carp ranged between 6.6 and 16.6. The BCF of diltiazem was also relatively low (0.5-194) in analysed tissues of trout, following the order kidney liver muscle blood plasma. BCF of atenolol in rainbow trout tissues was the lowest among the tested substances (BCF = 0.002-0.27), following the order of liver > kidney > muscle. In the blood plasma, the concentration of atenolol was below the limit of quantification. Verapamil showed a longer half-life time (10.6 days) in fish compared to the human body, indicating the slow rate of biotransformation and/or elimination of verapamil in fish. Estimated half-life times of diltiazem in liver (1.5 h) and kidney (6.2 h) were in the same order of magnitudes as those determined for the human blood plasma. The half-life time of atenolol in trout was not studied, because of its very low bioconcentration. In diltiazem exposed rainbow trout, 8 groups of metabolites of diltiazem with 17 different isoforms were identified using liquid chromatography/high resolution mass spectrometry method. Diltiazem was found to undergo a biotransformation involving desmethylation, desacethylation and hydroxylation in fish. These results showed that diltiazem was metabolised in fish in a similar way like in the human body by desmethylation and desacethylation. On the other hand, hydroxylation, which was involved to a minor extent, seemed to be species specific. Verapamil had no effect on early life stages of common carp at the environmentally relevant concentration after one month lasting exposure. On the other hand, atenolol and diltiazem in environmentally realistic concentrations caused after 42-day exposure some physiological changes in rainbow trout. Namely, atenolol affected haematological and biochemical parameters of the blood in exposed rainbow trout and diltiazem caused changes in the activity of antioxidant enzymes in trout liver and gills. These data indicated that atenolol and diltiazem, when present in the aquatic environment, could be a source of sub-lethal detrimental effects in fish.
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