Identification and application of bile metabolites to assess the exposure of fish to pharmaceuticals in the environment

Thousands of tons of pharmaceuticals are consumed yearly worldwide. Due to the continuous and increasing consumption and their incomplete elimination in wastewater treatment plants (WWTP), pharmaceuticals and their metabolites can be detected in receiving waters, although at low concentrations (ng to low μg/L). As bioactive molecules the presence of pharmaceuticals in the aquatic environment must be considered potentially hazardous for the aquatic organisms. In this thesis, the biotransformation and excretion of pharmaceuticals in fish was studied. The main biotransformation pathways of three anti‐inflammatory drugs, diclofenac, naproxen and ibuprofen, in rainbow trout were glucuronidation and taurine conjugation of the parent compounds and their phase I metabolites. The same metabolites were present in fish bile in aquatic exposures as in fish dosed with intraperitoneal injection. Higher bioconcentration factor in bile (BCFbile) was found for ibuprofen when compared to diclofenac and naproxen. Laboratory exposure studies were followed by a study of uptake of pharmaceuticals in a wild fish population living in lake contaminated with WWTP effluents. Of the analyzed 17 pharmaceuticals and six phase I metabolites, only diclofenac, naproxen and ibuprofen was present in bream and roach bile. It was shown, that diclofenac, naproxen and ibuprofen excreted by the liver can be found in rainbow trout and in two native fish species living in the receiving waters. In the bream and roach bile, the concentrations of diclofenac, naproxen and ibuprofen were roughly 1000 times higher than those found in the lake water, while in the laboratory exposures, the bioconcentration of the compounds and their metabolites in rainbow trout bile were at the same level as in wild fish or an order of magnitude higher. Thus, the parent compounds and their metabolites in fish bile can be used as a reliable biomarker to monitor the exposure of fish to environmental pharmaceuticals present in water receiving discharges from WWTPs.

Capillary electrophoresis for monitoring of carboxylic, phenolic and amino acids in bioprocesses

Bioprocess technology is a multidisciplinary industry that combines knowledge of biology and chemistry with process engineering. It is a growing industry because its applications have an important role in the food, pharmaceutical, diagnostics and chemical industries. In addition, the current pressure to decrease our dependence on fossil fuels motivates new, innovative research in the replacement of petrochemical products. Bioprocesses are processes that utilize cells and/or their components in the production of desired products. Bioprocesses are already used to produce fuels and chemicals, especially ethanol and building-block chemicals such as carboxylic acids. In order to enable more efficient, sustainable and economically feasible bioprocesses, the raw materials must be cheap and the bioprocesses must be operated at optimal conditions. It is essential to measure different parameters that provide information about the process conditions and the main critical process parameters including cell density, substrate concentrations and products. In addition to offline analysis methods, online monitoring tools are becoming increasingly important in the optimization of bioprocesses. Capillary electrophoresis (CE) is a versatile analysis technique with no limitations concerning polar solvents, analytes or samples. Its resolution and efficiency are high in optimized methods creating a great potential for rapid detection and quantification. This work demonstrates the potential and possibilities of CE as a versatile bioprocess monitoring tool. As a part of this study a commercial CE device was modified for use as an online analysis tool for automated monitoring. The work describes three offline CE analysis methods for the determination of carboxylic, phenolic and amino acids that are present in bioprocesses, and an online CE analysis method for the monitoring of carboxylic acid production during bioprocesses. The detection methods were indirect and direct UV, and laser-induced frescence. The results of this work can be used for the optimization of bioprocess conditions, for the development of more robust and tolerant microorganisms, and to study the dynamics of bioprocesses.

Flavonoids and other phenolic compounds: characterization and interactions with lepidopteran and sawfly larvae

This thesis focuses on flavonoids, a subgroup of phenolic compounds produced by plants, and how they affect the herbivorous larvae of lepidopterans and sawflies. The first part of the literature review examines different techniques to analyze the chemical structures of flavonoids and their concentrations in biological samples. These techniques include, for example, ultraviolet-visible spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. The second part of the literature review studies how phenolic compounds function in the metabolism of larvae. The harmful oxidation reactions of phenolic compounds in insect guts are also emphasized. In addition to the negative effects, many insect species have evolved the use of phenolic compounds for their own benefit. In the experimental part of the thesis, high concentrations of complex flavonoid oligoglycosides were found in the hemolymph (the circulatory fluid of insects) of birch and pine sawflies. The larvae produced these compounds from simple flavonoid precursors present in the birch leaves and pine needles. Flavonoid glycosides were also found in the cocoon walls of sawflies, which suggested that flavonoids were used in the construction of cocoons. The second part of the experimental work studied the modifications of phenolic compounds in conditions that mimicked the alkaline guts of lepidopteran larvae. It was found that the 24 plant species studied and their individual phenolic compounds had variable capacities to function as oxidative defenses in alkaline conditions. The excrements of lepidopteran and sawfly species were studied to see how different types of phenolics were processed by the larvae. These results suggested that phenolic compounds were oxidized, hydrolyzed, or modified in other ways during their passage through the digestive tract of the larvae.

Suomalaisten marjojen fenoliyhdisteet ja niiden stabiilisuus prosessoinnin ja säilytyksen aikana

Suomalaiset marjat sisältävät huomattavia määriä erilaisia fenoliyhdisteitä ja ne ovat siksi erinomaisia fenoliyhdisteiden lähteitä moniin muihin elintarvikkeisiin verrattuna. Fenoliyhdisteet ovat kasveissa syntyviä aineenvaihduntatuotteita, joilla on todettu olevan terveyden kannalta positiivia vaikutuksia. Ne antavat kasvikunnan tuotteille niiden tunnusomaisen värin sekä toimivat suoja-aineina taudinaiheuttajia vastaan. Erityisesti elintarvike-, lääke- ja kosmetiikkateollisuus jalostavat marjoja erilaisiksi lopputuotteiksi. Fenoliyhdisteet tuovat näihin tuotteisiin hyödyllisiä terveysvaikutuksia ja ne voivat toimia niissä myös väripigmentteinä. Fenoliyhdisteet ovat kuitenkin suhteellisen epästabiileja yhdisteitä. On havaittu, että prosessointi- ja säilytystavat vaikuttavat huomattavasti niiden stabiilisuuteen ja hajoamiseen. Tämän kandidaatintyön tavoitteena oli selvittää fenoliyhdisteiden stabiilisuuteen vaikuttavia tekijöitä prosessoinnin ja säilytyksen aikana kirjallisuuden pohjalta. Tämän työn tulosten perusteella marjojen prosessointi kannattaa suorittaa mahdollisimman lyhyessä ajassa matalissa lämpötiloissa, sillä monet fenoliyhdisteistä eivät ole pitkään lämpöstabiileja. Säilytys kannattaa tehdä myös matalissa lämpötiloissa; pitkänä säilytysaikana pakastettuna. Korkea pH vaikuttaa usean fenoliyhdisteen hajoamiseen ja se saattaa aiheuttaa tuotteissa värinmuutoksia. Valo, hapen läsnäolo sekä erilaiset muut yhdisteet voivat vaikuttaa heikentävästi fenoliyhdisteiden stabiilisuuteen sekä prosessoinnin että säilytyksen aikana. Fenoliyhdisteiden stabiilisuus on hyvin rakennekohtaista ja siksi prosessoinnin ja säilytyksen tarkkojen vaikutusten yleistäminen kaikkiin fenoliyhdisteisiin on vaikeaa.