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Effekte Des Antidiabetikums Metformin Und Seines Transformationsprodukts Guanylharnstoff Auf Den Gesundheitszustand Von Bachforellen (Salmo Trutta F

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Effekte Des Antidiabetikums Metformin Und Seines Transformationsprodukts Guanylharnstoff Auf Den Gesundheitszustand Von Bachforellen (Salmo Trutta F Effekte des Antidiabetikums Metformin und seines Transformationsprodukts Guanylharnstoff auf den Gesundheitszustand von Bachforellen (Salmo trutta f. fario) und Posthornschnecken (Planorbarius corneus) Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Stefanie Jacob aus Saarlouis Tübingen 2019 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 18.02.2020 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: apl. Prof. Dr. Rita Triebskorn 2. Berichterstatter: Prof. Dr. Christian Zwiener Inhaltsverzeichnis Teil 1: Zusammenfassung ........................................................................................................................ 1 1. Promotionsthema ........................................................................................................................ 1 2. Graphische Zusammenfassung .................................................................................................... 1 3. Einleitung ..................................................................................................................................... 2 4. Übergeordnete Fragestellungen ................................................................................................. 5 5. Material und Methoden .............................................................................................................. 6 6. Ergebnisse und Diskussion ........................................................................................................ 24 7. Synthese .................................................................................................................................... 32 8. Referenzen ................................................................................................................................ 45 Teil 2: Eigenanteil an den durchgeführten Arbeiten in den zur Dissertation eingereichten Publikationen und Manuskripten .......................................................................................................... 57 Teil 3: Wissenschaftliche Arbeiten ........................................................................................................ 59 Kapitel I: Impact of the antidiabetic drug metformin and its transformation product guanylurea on the health of the big ramshorn snail (Planorbarius corneus) ............................................................ 59 Kapitel II: Does the antidiabetic drug metformin affect embryo development and the health of brown trout (Salmo trutta f. fario)? .................................................................................................. 77 Kapitel III: Effects of guanylurea, the transformation product of the antidiabetic drug metformin, on the health of brown trout (Salmo trutta f. fario) ....................................................................... 103 Anhang I ............................................................................................................................................... 122 Anhang II .............................................................................................................................................. 126 Publikationsliste und Konferenzbeiträge ............................................................................................ 129 Danksagung ......................................................................................................................................... 130 Abkürzungsverzeichnis °C Grad Celsius µg Mikrogramm µl Mikroliter µS Mikro-Siemens ADA American diabetes association = Amerikanische Diabetes-Gesellschaft AG Arbeitsgruppe AMP Adenosinmonophosphat AMPK Adenosinmonophosphat-aktivierte Proteinkinase ANOVA Analysis of variance = Varianzanalyse atm/m3/mol Atmosphäre pro Kubikmeter pro Mol ATP Adenosintriphosphat bzw. Beziehungsweise cAMP Cyclisches Adenosinmonophosphat Chemical abstracts service; CAS-Registrierungsnummer = internationaler CAS Bezeichnungsstandard für chemische Stoffe CE Capillary electrophoresis = Kapillarelektrophorese CHP Cumolhydroperoxid cm Zentimeter DNA Desoxyribonukleinsäure EG Europäische Gemeinschaft EMA European medicines agency = Europäische Arzneimittel-Agentur EPA Environmental protection agency = Umweltschutzbehörde EU Europäische Union FOX-Assay Ferrous oxidation xylenol orange = Eisenoxidation-Xylenolorange-Assay g Gramm GLP-1 Glukagon-like Peptid 1 GLUT Glukosetransporter High performance liquid chromatography = HPLC Hochleistungsflüssigkeitschromatographie Hsp70 Hitze-Schock-Protein mit einem Molekulargewicht von 70 kDa im Vgl. Im Vergleich insg. insgesamt kDa Kilo-Dalton KIT Karlsruher Institut für Technologie L Liter Lowest observed test concentration = niedrigste Testkonzentration eines Stoffes, LOEC bei der in einem ökotoxikologischen Test ein signifikanter Effekt zu sehen ist log Kow Logarithmus des Oktanol-Wasser-Verteilungskoeffizienten M Mol pro Liter mA Milli-Ampere mg Milligramm min Minute ml Milliliter mm Millimeter mRNA Messenger Ribonukleinsäure MS 222 Tricain-Methansulfonat nm Nanometer No observed effect concentration = höchste Konzentration eines Stoffes, die in NOEC einem ökotoxikologischen Test gerade noch keinen signifikanten Effekt zeigt Organisation for economic co-operation and development = Organisation für OECD wirtschaftliche Zusammenarbeit und Entwicklung OGewV Oberflächengewässerverordnung P1 Proximaler Tubulus 1 P2 Proximaler Tubulus 2 PAS Periodic acid-Schiff = Periodsäure und Schiffsches Reagenz Predicted environmental concentration = vorausgesagte Umweltkonzentration PEC eines Stoffes pg Pikogramm Predicted no effect concentration = vorausgesagte Konzentration eines Stoffes, bei PNEC der noch kein Effekt auftritt Triple quadropole mass spectrometer= Dreifaches Quadropol- QqQ-MS Massenspektrometer Quadropol time of flight mass spectrometer = Quadropol-Flugzeit- QTOF-MS Massenspektrometer rcf Relative centrifugal force = relative Zentrifugalkraft ROS Reactive oxygen species = reaktive Sauerstoffspezies rpm Rounds per minute = Runden pro Minute rRNA Ribosomale Ribonukleinsäure RTL-W1 Rainbow trout liver -Waterloo 1 = Regenbogenforellen-Leberzellen Waterloo 1 s Sekunden SDS Sodiumdodecylsulfat = Natriumdodecylsulfat t Tonne TBS Tris buffered saline = Tris-gepufferte Salzlösung V Volt vgl. vergleiche WFD Water framework directive = Wasserrahmenrichtlinie WHO World health organization = Weltgesundheitsorganisation w.w. Wet weight = Nassgewicht z.B. Zum Beispiel Teil 1: Zusammenfassung 1. Promotionsthema Effekte des Antidiabetikums Metformin und seines Transformationsprodukts Guanylharnstoff auf den Gesundheitszustand von Bachforellen (Salmo trutta f. fario) und Posthornschnecken (Planorbarius corneus) 2. Graphische Zusammenfassung 1 3. Einleitung Zum Schutz und zur Verbesserung des Zustands von aquatischen Ökosystemen wurde im Jahr 2000 die Wasserrahmenrichtlinie in Kraft gesetzt. Dabei wurde berücksichtigt, dass verschiedene anthropogene Einflüsse eine Bedrohung für aquatische Organismen in Oberflächengewässern darstellen können. Dazu gehören beispielsweise strukturelle Veränderungen von Flüssen, die zu einer schlechteren Durchgängigkeit führen, durch den Klimawandel bedingte höhere Wassertemperaturen, die Einwanderung von Neobiota sowie der Eintrag von Chemikalien. Laut Wasserrahmenrichtlinie soll für Oberflächengewässer ein guter ökologischer und chemischer Zustand erreicht werden (EU, 2000). Untersucht werden biologische (z.B. die Zusammensetzung und Abundanz von Arten), hydromorphologische (z.B. die Struktur der Uferzone) und physikalisch-chemische Komponenten (z.B. Temperatur) sowie prioritäre und national festgelegte flussgebietsspezifische Schadstoffe. Zur Beurteilung der chemischen Belastung werden gemessene Umweltkonzentrationen von momentan 45 prioritären Chemikalien, die ein erhebliches Risiko für die aquatische Umwelt darstellen, mit den EU-weit geltenden Umweltqualitätsnormen für diese Substanzen abgeglichen (EU, 2013). Gleiches gilt für 67 flussgebietsspezifische Schadstoffe, die in Deutschland untersucht werden (OGewV, 2016). Zusätzlich befinden sich auf einer Beobachtungsliste aktuell acht Stoffe bzw. Stoffgruppen, die EU- weit überwacht und analysiert werden sollen, um zu bestimmen, ob sie ebenfalls auf die Liste prioritärer Substanzen aufgenommen werden sollen (EU, 2018). Generell ist im Hinblick auf die Verschmutzung von Gewässern mit Chemikalien eine große Anzahl verschiedenster Substanzen in Konzentrationen von pg/L bis µg/L in Oberflächengewässern zu finden. Beispiele hierfür sind Pestizide, Industriechemikalien, Wasch- und Reinigungsmittel, Körperpflegeprodukte, Biozide, Nahrungsmittelzusatzstoffe und Pharmazeutika sowie deren Metabolite und Transformationsprodukte (Rodriguez-Mozaz et al., 2004; Scheurer et al., 2009a; Bueno et al., 2012; Aus der Beek et al., 2016). Zum einen stiegen Anzahl und Verbrauch dieser Stoffe in den letzten Jahren und Jahrzehnten stetig, zum anderen erlaubten aber auch verbesserte Messmethoden eine genauere Identifizierung und Quantifizierung dieser Spurenstoffe (Zwiener und Frimmel, 2004; Fischer et al., 2012; Agüera et al., 2013). Humanpharmazeutika sind dabei eine Gruppe von Stoffen, die einen spezifischen Effekt im Mensch haben sollen, um therapeutisch wirken zu können (Cleuvers, 2003), und deren Wirkmechanismus zumeist stark konserviert ist.
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