|
|
Application of Mass Spectrometry for the Determination of Oxidative Stress Markers and Mycotoxins
Čumová, Martina ; Večeřa, Zbyněk (referee) ; Hajšlová, Jana (referee) ; Vávrová, Milada (referee) ; Čáslavský, Josef (advisor)
The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.
|
|
|
Application of Separation Techniques Connected with Mass Spectrometry for the Analysis of Environmentally Important Compounds
Mácová, Daniela ; Čelechovská, Olga (referee) ; Márová, Ivana (referee) ; Demnerová, Kateřina (referee) ; Čáslavský, Josef (advisor)
The identification of the hydrolysis and photodegradation products of flexible polyurethane foams (PUFs) with addition of biooriginated and biodegradable additive was the first topic of this dissertation work. Separation of polyurethane foam hydrolysis degradation products, designed for ecotoxicological tests, was managed by high-performance liquid chromatography coupled with mass spectrometry (HPLC/MS). The degradations product structure was elucidated by tandem mass spectrometry (MSn). PUF photodegradation products were obtained by exposure of materials by irradiation at 254 nm. Semi-volatile degradation products were isolated from the exposed polyurethane by n-hexane extraction; volatile compounds were collected by solid phase microextraction (SPME). Gas chromatography with mass spectrometry (GC/MS) and complete orthogonal tandem gas chromatography with mass spectrometry (GCxGC/TOF MS) was used for separation and identification of photodegradation products. The influence of the bio-filler on the character of degradation products and the possible effect of PUF degradation products on the environment was discussed at the end of this section. The determination of isoprostanes – markers of oxidative stress in tissues of beadlet anemone (Actinia equina) was the subject of the second topic. F2-isoprostanes were synthesized from the arachidonic acid. With thereby prepared isoprostanes the method of determination by liquid chromatography with tandem mass spectrometry (HPLC/MS/MS) was developed and optimized. The isoprostane isolation process from the Actinia equina tissues was optimized with solid phase extraction (SPE). The resulting methodology was used to quantify isoprostanes in tissues of anemones, which were exposed to both moderate and high temperature changes. The temperature changes were used to initiate the oxidative stress in organisms. In addition, concentration levels of unknown compounds were also monitored. These unknown compounds were extracted from tissues together with F2-isoprostanes and their identity is discussed in this dissertation work too. The possibility of using isoprostane levels in the Anthozoa tissues for the oxidative stress monitoring is discussed in the conclusion of this work.
|
|
|
Evaluation of glutathione content in plants as a marker of heavy metals environmental contamination
Borková, Marie ; Stoupalová, Michaela (referee) ; Opatřilová,, Radka (advisor)
Dependence of glutathione concentration on the amount of thallium in the plant was studied. Observed plant was maize (Zea mays) which was divided to two parts – root and overground. Two culture procedures were elaborated where seeds and young seedlings were cultivated in a solution of thallium of concentration 0, 1, 3, 5, 8, a 10 µmol/l. Extraction agents used during extraction were phosphate buffer and solution of ascorbic acid. Determination of glutathione was realized by capillary electrophoresis (CE) and high performance liquid chromatography (HPLC). Diode array detector (DAD) was used in both methods. Quantification of the thallium amount in the plant was done by method of inductively coupled plasma-atomic emission spectrometry (ICP-AES).
|
|
|
Effect of environmentally relevant concentration of dexamethasone to fish organism
BOŘÍK, Adam
Pharmaceutical pollutants have been aim of many recent aquatic environment studies. Due to human activities surface waters are polluted with plenty of substances, including products of personal care, which are ranked among a number of pharmaceuticals. The main goal of this work is to assess the impact of environmentally relevant concentrations of the synthetic glucocorticoid dexamethasone on aquatic organisms through biomarkers of oxidative stress. The principle of this experiment was chronic in vivo exposure model organisms (rainbow trout) to doses of this drug and the subsequent monitoring activities of antioxidant enzyme system in the liver and gill tissues. Experimental results have demonstrated a response enzymatic protection mechanism on the increased production of reactive oxygen species induced by the drug at environmentally relevant concentrations especially in gill tissue. Our results illustrate a real risk that this xenobiotic represents to aquatic organisms.
|
|
|
The effect of metribuzine on oxidative stress and antioxidant enzymes of signal crayfish
LIDOVÁ, Jaroslava
The aim of this study was to investigate effects of the triazine herbicide metribuzine on oxidative stress level and antioxidant enzymes activity in gills, muscle and hepatopancreas of signal crayfish (Pacifastacus leniusculus Dana) and also extension of knowledge about effect of metribuzine on the environment. The experiment took up 60 days. Crayfish were exposured to metribuzine concentrations of 0.52 micrograms.l-1 (real environmental concentration) and 3.06 mg.l-1 (10% 96hLC50) for the first 30 days. Then a second phase followed depuration without metribuzine (30 days). Changes in the oxidative stress level (TBARS), superoxiddismutase (SOD) activity and catalase (CAT) activity were observed in all examined tissues. Changes in glutathionreductase (GR) activity were observed only in hepatopancreas. Chronic exposure of metribuzine demonstrated an oxidative damage of cell lipids, proteins and also changes in antioxidant activity in examined crayfish tissues.The results of this study suggest that crayfish are a very suitable organisms for toxicological tests and simultaneously extend knowledge about effect of metribuzine on the environment.
|
|
|
Effect of oxidative stress on telomere lenght in \kur{Drosophila melanogaster}
SZAKOSOVÁ, Klára
Oxidative stress is caused by an imbalance between high production of reactive oxygen species and ability of organism to detoxify them or counteract their effects. The chromosomal ends telomeres - are specialized nucleoprotein structures protecting chromosome ends from DNA damage. Telomeres of Drosophila melanogaster are arrays of specific non-long terminal repeated (LTR) retrotransposons HeT-A, TART and TAHRE. This thesis evaluates effects of exposure of paraquat, which is a strong oxidative stress inducer, on telomere length and transcription activity in Drosophila.
|
|
|
Vliv triazinových pesticidů na ryby
STARÁ, Alžběta
Fish and crayfish are widely used as biological monitors of environmental levels of anthropogenic pollutants. The present thesis is a contribution to the assessment of the toxicity and effects of long-term effect of triazines on the different developmental stages of common carp (Cyprinus carpio L.) and adult red swamp crayfish (Procambarus clarkii). The carp was selected as a model fish due to its economic importance, e.g. carp farming contributes about 90% to total fish production in the Czech Republic. Crayfish are easily identified species representing given locality, they are widespread, and they provide a sufficient amount of tissue for individual biochemical and chemical analyses. The results of these studies provide further data on chronic exposure to triazines for consideration in risk assessment. We selected three active substances of triazine herbicides which are the most frequently detected in surface waters such as prometryne, simazine and terbutryn. The findings contribute to knowledge of the toxic potential of triazine herbicides to carp and crayfish at environmentally relevant concentrations in Czech rivers. There is a scarcity of information regarding the toxicity of triazines on freshwater organisms. During the tests we monitored several parameters: behaviour, mortality, biometric, haematological, biochemical blood, histopathology, oxidative stress and antioxidants. The data obtained from all tests performed during my thesis are very valuable for assessment and evaluation of long-term effects xenobiotics on aquatic organisms, especially fish and crayfish. In the future, I would extend the focus of my research for study of possible synergic or amplifying effect of mixtures triazines with other xenobiotics which are often found in the aquatic environment. This approach is recently accented as the aquatic environment is polluted by mixtures of different compounds. Therefore, more research is needed in order to clarify the more detailed effects of xenobiotics on non-target aquatic organisms.
|
|
|
Effect of oxidative stress on antioxidant systems, telomere length and telomerase activity in \kur{Locusta migratoria}
VRBOVÁ, Kristýna
Oxidative stress is caused by an imbalance between oxidants and antioxidants. Oxidative stress generated by reactive oxygen species (ROS) or reactive nitrogen species (RNS) occurs when protection of antioxidants fails or when an amount of ROS is too high. Telomeres, regions of repetitive nucleotide sequences at the end of chromosomes, are especially sensitive to oxidative stress because they contain a lot of guanine which is often oxidised. Antioxidants play an important role in protection against oxidative stress. In this thesis I analysed the effect of oxidative stress caused by paraquat on activity of antioxidant enzymes (catalase and superoxide dismutase) and telomere length in Locusta migratoria. I also studied differences in telomere length between locusts of various development stages and telomerase activity in locusts and other Orthoptera.
|
|
|
Identification and isolation gene encoding peroxiredoxin, detoxification enzyme involved in antioxidant defense of hard tick Ixodes ricinus.
HAVRAN, Jiří
Antioxidant enzymes play an important role in protection against the oxygen radicals generating during aerobic metabolism, as well as in defense against host immune responses. Peroxiredoxin isolated from the hard tick Ixodes ricinus belongs to 1CysPrx group of peroxiredoxins. It is expressed in all stages of hard tick (larva, nymph and adult) and in all tissues examined. The full gene (EU373821) is 756 nt long and consists of 251 amino acids, encoding 27.94 kDa protein. This is the first report of isolation of detoxification enzyme (peroxiredoxin) involved in antioxidant defense of hard tick Ixodes ricinus.
|
|
| |
guest ::
