@phdthesis{Arnold2020, author = {Arnold, Charlotte Antonia}, title = {Reduktion des Genomschadens in peripheren Lymphozyten adip{\"o}ser Patienten nach bariatrischer Operation}, doi = {10.25972/OPUS-21174}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211748}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {{\"U}bergewicht, das als Volkskrankheit ein wachsendes globales Problem darstellt, ist mit mehreren folgenreichen Komorbidit{\"a}ten behaftet und die Assoziation der Erkrankung mit nachweisbarer Sch{\"a}digung des Erbguts durch oxidativen Stress ist mittlerweile unangefochten. In der vorliegenden Studie wurden periphere Lymphozyten stark bis morbid adip{\"o}ser Patienten mit Hilfe des Mikrokern-Assays untersucht und es konnte - begleitend zu der zu erwartenden BMI-Abnahme - eine signifikante Reduktion des Genomschadens durch Magenbypass bzw. Sleeve-Gastrektomie 12 Monate postoperativ detektiert werden. Daneben demonstrierte die Analyse zus{\"a}tzlich erhobener Patientendaten, die u. a. N{\"u}chternglucose, HbA1c, Blutdruck und Herzfrequenz sowie ein Blutbild der Patienten (inklusive CRP als Entz{\"u}ndungsmarker, Transaminasen, gGT sowie Lipidprofil) umfasste, eine deutliche Besserung vieler Parameter bis auf teilweise wieder physiologische Normwerte. All diese Ergebnisse st{\"u}tzen die These der metabolischen Wirksamkeit sowohl des Roux-en-Y-Magenbypass als auch der Sleeve- Gastrektomie. Ihre Bedeutung liegt nicht zuletzt in der angenommenen Reduktion des Krebserkrankungsrisikos, da jeweils ein Zusammenhang mit der Adipositas an sich, dem Diabetes und durch oxidativen Stress verursachter DNA-Sch{\"a}digung besteht. Mit Blick auf eine gegenw{\"a}rtig in der Forschung diskutierte potentielle therapeutische Anwendung von Antioxidantien zur Reduktion von Erbgutsch{\"a}digungen, die durch oxidativen Stress zustande kommen, wurden die Substanzen Tricetinidin, Curcumin und Resveratrol im Rahmen des Mikrokerntests an HL60- und NRK-Zellen untersucht, in denen mit der Mischung aus Insulin und Angiotensin II eine gentoxische Wirkung erzielt worden war.}, subject = {Fettsucht}, language = {de} } @phdthesis{Maimari2020, author = {Maimari, Theopisti}, title = {The influence of N-terminal peptides of G-protein coupled receptor kinase (GRK) 2, 3 and 5 on β-adrenergic signaling}, doi = {10.25972/OPUS-19932}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199322}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {G protein coupled receptor kinases (GRK) phosphorylate and thereby desensitize G protein coupled receptors (GPCR) including β-adrenergic receptors (βAR), which are critical regulators of cardiac function. We identified the Raf kinase inhibitor protein (RKIP) as an endogenous inhibitor of GRK2 that leads to increased cardiac contractility via βAR activation. RKIP binds to the N-terminus (aa1-185) of GRK2, which is important for the GRK2/receptor interaction. Thereby it interferes with the GRK2/receptor interaction without interference with cytosolic GRK2 target activation. In this project, the RKIP/GRK interface was investigated to develop strategies that simulate the effects of RKIP on βAR. RKIP binding to different isoforms of GRK expressed in the heart was analyzed by protein interaction assays using full-length and N-termini of GRK2, GRK3 and GRK5: 1-53, 54-185 and 1-185. Co-immunoprecipitation (Co-IPs) and pull-down assays revealed that RKIP binds to the peptides of GRK2 and GRK3 but not to the ones of GRK5, which suggests the existence of several binding sites of RKIP within the N-termini of GRK2 and GRK3. To analyze whether the peptides of GRK2 and GRK3 are able to simulate the RKIP mediated interference of the GRK2/receptor interaction, we analyzed the β2-AR phosphorylation in the absence and presence of the peptides. Interestingly, N-termini (aa1-185) of GRK2 and GRK3 reduced β2AR phosphorylation to a comparable extent as RKIP. In line with reduced receptor phosphorylation, the peptides also reduced isoproterenol-stimulated receptor internalization as shown by [3H] CGP-12177 radioligand binding assay and fluorescence microscopy compared to control cells. Subsequently, these peptides increased downstream signaling of β2AR, i.e. the phosphorylation of the PKA substrate phosducin. In an attempt to elucidate the mechanism behind the observed effects, Co-IPs were performed in order to investigate whether the peptides bind directly to the β2-AR and block its phosphorylation by GRK2. Indeed, GRK2 1-185 and GRK3 1-185 could bind the receptor, suggesting that this way GRK2 is prevented from inhibiting the receptor. To investigate the physiological effect of GRK2 1-185, GRK3 1-185 and GRK5 1-185, their effect on neonatal mouse cardiomyocyte contractility and hypertrophy was analyzed. After long-term isoproterenol stimulation, in the presence of GRK2 1 185 and GRK3 1-185 the cross-sectional area of the cardiomyocytes showed no significant increase in comparison to the unstimulated control cells. In addition, upon isoproterenol stimulation, GRK2 1-185 and GRK3 1-185 increased the beat rate in cardiomyocytes, mimicking RKIP while the base impedance, an indicator of viability, remained stable. The N-termini (1-185) of GRK2 and GRK3 simulated RKIP's function and had a significant influence on β2AR phosphorylation, on its downstream signaling and internalization, could bind β2-AR, increased beat rate and did not significantly induce hypertrophy, suggesting that they may serve as a model for the generation of new and more specific targeting strategies for GRK mediated receptor regulation.}, language = {en} } @phdthesis{Kircher2020, author = {Kircher, Malte Tim}, title = {Neuronale Genotoxizit{\"a}t von Angiotensin II}, doi = {10.25972/OPUS-21427}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214273}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {In recent decades, the acceptance has steadily increased that oxidative stress plays an important role in the development of chronic diseases, malignant neoplasia and the acceleration of the aging process. As one of the most common chronic diseases, hypertension is often associated with a misregulated renin-angiotensin-aldosterone system that causes chronic oxidative stress. Hypertension is a risk factor for neurological diseases such as vascular dementia (VaD) and many neurological disorders, including VaD, have an ROS-associated or inflammatory component in their etiology. Our group has already demonstrated AT-II-induced genotoxicity in kidney and myocardial cells and tissues. The aim of this dissertation was to investigate a possible association between AT-II and neurodegeneration that is triggered by neuronal genotoxicity of AT-II. First, we showed in two neuronal cell lines that AT-II causes dose-dependent genome damage. Subsequent experiments could attribute this toxicity to NOX-produced superoxide generated after AT-II binding to the AT1R. In addition, AT-II-induced depletion of the most important intracellular antioxidant - glutathione - was demonstrated. In vivo, we were able to show that AT1aR knockout mice after AT-II treatment showed significantly more genome damage in the subfornic organ (SFO) than wild-type mice. The SFO is one of the few structures in the brain with an interrupted blood-brain barrier, which makes it accessible and particularly sensitive to circulating AT-II. In the recent literature, these genome damages were also observed in kidney and heart tissues and prove an additional genotoxicity of AT-II independent of AT1aR and consequently independent of blood pressure. In summary, this work shows that increased AT-II levels in neuronal cells cause genome damage due to NOX-produced superoxide. It is hoped that these results will one day help to decipher the complete development of VaD.}, subject = {Angiotensin II}, language = {de} } @phdthesis{Konrad2021, author = {Konrad, Charlotte}, title = {Biochemische Charakterisierung von cAMP-Gradienten - Einfluss von Phosphodiesterasen}, doi = {10.25972/OPUS-20572}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205728}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Cyclisches Adenosinmonophosphat ist ein ubiquit{\"a}rer zweiter Botenstoff zahlreicher Signalwege im menschlichen K{\"o}rper. Auf eine Vielzahl verschiedenster extrazellul{\"a}rer Signale folgt jedoch eine Erh{\"o}hung desselben intrazellul{\"a}ren Botenstoffs - cAMP. Nichtsdestotrotz schafft es die Zelle, Signalspezifit{\"a}t aufrecht zu erhalten. Ein anerkanntes, wenn auch bisher unverstandenes Modell, um dieses zu erm{\"o}glichen, ist das Prinzip der Kompartimentierung. Die Zelle besitzt demnach Areale verschieden hoher cAMP-Konzentrationen, welche lokal begrenzt einzelne Signalkaskaden beeinflussen und somit eine differenzierte Signal{\"u}bertragung erm{\"o}glichen. Eine m{\"o}gliche Ursache f{\"u}r die Ausbildung solcher Bereiche geringerer cAMP- Konzentrationen (hier als Dom{\"a}nen bezeichnet), ist die hydrolytische Aktivit{\"a}t von Phosphodiesterasen (PDEs), welche als einzige Enzyme die F{\"a}higkeiten besitzen, cAMP zu degradieren. In dieser Arbeit wird der Einfluss der cAMP-Hydrolyse verschiedener PDEs auf die Gr{\"o}ße dieser Dom{\"a}nen evaluiert und mit denen der PDE4A1 verglichen, welche bereits durch unsere Arbeitsgruppe aufgrund ihrer Gr{\"o}ße als Nanodom{\"a}nen definiert wurden. Der Fokus wird dabei auf den Einfluss von kinetischen Eigenschaften der Phosphodiesterasen gelegt. So werden eine PDE mit hoher Umsatzgeschwindigkeit (PDE2A3) und eine PDE mit hoher Substrataffinit{\"a}t (PDE8A1) verglichen. Mithilfe sogenannter Linker, Abstandshaltern definierter L{\"a}nge, werden zus{\"a}tzlich die Nanodom{\"a}nen ausgemessen, um einen direkten Zusammenhang zwischen Gr{\"o}ße und kinetischer Eigenschaft anzugeben. Die Zusammenschau der Ergebnisse zeigt, dass die maximale Umsatzgeschwindigkeit der Phosphodiesterasen direkt mit der Gr{\"o}ße der Nanodom{\"a}nen korreliert. Durch den unmittelbaren Vergleich der gesamten PDE mit ihrer katalytischen Dom{\"a}ne wird zus{\"a}tzlich der Einfluss von regulatorischen Dom{\"a}nen evaluiert. Es wird gezeigt, dass diese cAMP-Gradienten modulieren k{\"o}nnen. Bei der PDE2A3 geschieht die Modulation u.a. durch Stimulation mit cGMP, welche h{\"o}chstwahrscheinlich dosisabh{\"a}ngig ist und somit graduell verl{\"a}uft. Hiermit pr{\"a}sentieren sich die Dom{\"a}nen als dynamische Bereiche, d.h. sie k{\"o}nnen in ihrer Auspr{\"a}gung reguliert werden. In dieser Arbeit wird die Hypothese best{\"a}tigt, dass Phosphodiesterasen eine wichtige Rolle in der Kompartimentierung von cAMP spielen, die Gruppe jedoch inhomogener ist, als bislang angenommen. Die Gradienten-Bildung l{\"a}sst sich nicht bei jeder Phosphodiesterase darstellen (PDE8A1). Einige Phosphodiesterasen (PDE2A3) jedoch bilden Kompartimente, die durch externe Stimuli in ihrer Gr{\"o}ße reguliert werden k{\"o}nnen. Die Arbeit legt den Grundstein zur breiteren Charakterisierung des spezifischen Einflusses weiterer PDEs auf cAMP-Kompartimentierung, welches nicht nur das Verst{\"a}ndnis der Kompartimentierungs-Strategien voranbringt, sondern auch essentiell f{\"u}r das Verst{\"a}ndnis der Pathophysiologie zahlreicher Krankheitsbilder, aber auch f{\"u}r das Verst{\"a}ndnis bereits angewandter aber auch potentiell neuer Medikamente ist.}, subject = {Cyclo-AMP}, language = {de} } @phdthesis{Schaefer2020, author = {Sch{\"a}fer, Florian}, title = {Kontraktionsverhalten neonataler Rattenkardiomyozyten bei {\"U}berexpression phosphorylierungsdefizienter RKIP Mutanten S51/S52}, doi = {10.25972/OPUS-21374}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213747}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Durch Phosphorylierung inaktiviert GRK2 kardiale ß-Adrenorezeptoren und vermindert dadurch die Kontraktilit{\"a}t von neonatalen Rattenkardiomyozyten. Als nat{\"u}rlicher Inhibitor der GRK2 beeinflusst RKIP die Signalweiterleitung bei Stimulation von ß-Adrenorezeptoren. In dieser Arbeit konnte gezeigt werden, dass eine {\"U}berexpression von RKIP die Kontraktilit{\"a}t von neonatalen Rattenkardiomyozyten erh{\"o}ht. Es wurde die Anzahl auftretender Spontankontraktionen vor und nach Stimulation mit Isoproterenol erfasst sowie eine zeitliche Analyse der Calciumfreisetzung und -aufnahme nach elektrischer Stimulation durchgef{\"u}hrt. Im unstimulierten Zustand zeigten neonatale Rattenkardiomyozyten, die Wildtyp-RKIP {\"u}berexprimierten, verglichen mit der Kontrollgruppe, keine signifikanten Unterschiede hinsichtlich auftretender Spontankontraktionen. Nach Stimulation mit Isoproterenol zeigten neonatale Rattenkardiomyozyten, in denen Wildtyp-RKIP {\"u}berexprimiert wurde, eine signifikant h{\"o}here Anzahl auftretender Spontankontraktionen. Eine Analyse der Calciumtransienten zeigte bei RKIP-Wildtyp {\"u}berexprimierenden neonatalen Rattenkardiomyozyten eine erh{\"o}hte Calciumfreisetzung w{\"a}hrend der Systole sowie eine beschleunigte Calciumwiederaufnahme w{\"a}hrend der Diastole. Zudem wurden die RKIP-Mutanten RKIPS51V und RKIPS52V im Hinblick auf ihre Kontraktilit{\"a}t untersucht. Neonatale Rattenkardiomyozyten welche RKIPS51V und RKIPS52V {\"u}berexprimierten zeigten weder im Hinblick auf auftretende Spontankontraktionen noch bei der Analyse der Calciumtransienten signifikante Unterschiede zur Kontrollgruppe. Da auch eine Phosphorylierung an Aminos{\"a}ureposition 51 bzw. Aminos{\"a}ureposition 52 ohne direkte Auswirkung auf die Kontraktilit{\"a}t m{\"o}glich ist, wurde in einem in vitro Kinase Assay analysiert, ob neben der bekannten Phosphorylierungsstelle S153 eine weitere Phosphorylierung von RKIP durch PKA oder PKC erfolgt. Bei Einsatz der an Aminos{\"a}ureposition 153 phosphorylierungsdefizienten RKIP Mutante RKIPS153A konnte keine Phosphorylierung beobachtet werden. In der vorliegenden Arbeit konnte neben einer Phosphorylierung an S153 keine weitere Phosphorylierung von RKIP durch PKC oder PKA beobachtet werden.}, subject = {Herzinsuffizienz}, language = {de} } @article{DekantBridges2016, author = {Dekant, Wolfgang and Bridges, James}, title = {Assessment of reproductive and developmental effects of DINP, DnHP and DCHP using quantitative weight of evidence}, series = {Regulatory Toxicology and Pharmacology}, volume = {81}, journal = {Regulatory Toxicology and Pharmacology}, doi = {10.1016/j.yrtph.2016.09.032}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186750}, pages = {397-406}, year = {2016}, abstract = {Quantitative weight of evidence (QWoE) methodology utilizes detailed scoring sheets to assess the quality/reliability of each publication on toxicity of a chemical and gives numerical scores for quality and observed toxicity. This QWoE-methodology was applied to the reproductive toxicity data on diisononylphthalate (DINP), di-n-hexylphthalate (DnHP), and dicyclohexylphthalate (DCHP) to determine if the scientific evidence for adverse effects meets the requirements for classification as reproductive toxicants. The scores for DINP were compared to those when applying the methodology DCHP and DnHP that have harmonized classifications. Based on the quality/reliability scores, application of the QWoE shows that the three databases are of similar quality; but effect scores differ widely. Application of QWoE to DINP studies resulted in an overall score well below the benchmark required to trigger classification. For DCHP, the QWoE also results in low scores. The high scores from the application of the QWoE methodology to the toxicological data for DnHP represent clear evidence for adverse effects and justify a classification of DnHP as category 1B for both development and fertility. The conclusions on classification based on the QWoE are well supported using a narrative assessment of consistency and biological plausibility.}, language = {en} } @phdthesis{Berlin2017, author = {Berlin, Christopher}, title = {Die Untersuchung der kardialen Folgen einer ubiquit{\"a}ren Deletion von RKIP in M{\"a}usen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152882}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die Herzinsuffizienz, eine der h{\"a}ufigsten chronischen Krankheiten in der westlichen Welt, ist als Folge einer Myokardsch{\"a}digung durch eine verschlechterte Pumpfunktion des Herzens charakterisiert, die der K{\"o}rper durch verschiedene Kompensationsmechanismen zur Kontraktilit{\"a}tssteigerung auszugleichen versucht. Wichtiger Mechanismus hierf{\"u}r ist die Kontraktilit{\"a}ts- und Frequenzsteigerung {\"u}ber ß-adrenerge Rezeptorsignale, welche bei langfristiger Stimulation allerdings zu einer Abnahme der Funktionalit{\"a}t und Minderexpression eben dieses Rezeptorsystems, sowie der gleichzeitigen Verschlechterung der Herzinsuffizienz f{\"u}hrt. Interessanterweise wird parallel zur verminderten Rezeptorexpression bei Herzinsuffizienzpatienten eine Zunahme der GRK-Aktivit{\"a}t beobachtet. Diese Kinase ist in der Lage, ß-adrenerge GPCR-Signale durch Phosphorylierung des membranst{\"a}ndigen Rezeptors herunterzuregulieren. Durch einen PKC-abh{\"a}ngigen switch von Raf1 zu GRK2 konnte mit RKIP ein kardialer, endogener Inhibitor der GRK2 identifiziert werden. Es wurde in vitro und in vivo in M{\"a}usen mit myokardialer {\"U}berexpression von RKIP gezeigt, dass RKIP f{\"a}hig ist, die kontraktile Funktion von Herzmuskelzellen zu verbessern, negative kardiale Langzeitfolgen wie eine Verschlechterung der Insuffizienz, Remodeling-Prozesse wie Zunahme der Fibrosierung und eine gesteigerte Apoptoserate, sowie kardiale Rhythmusst{\"o}rungen protektiv zu beeinflussen. Um die endogene Rolle von RKIP weiter zu er{\"o}rtern, wurde in dieser Arbeit der Knockout von RKIP unter basalen Bedingungen, als auch nach transverser Aortenkonstriktion (TAC) untersucht. Zur Untersuchung physiologischer Parameter wie der Verk{\"u}rzungsfraktion, oder dem linksventrikul{\"a}rem diastolischen Durchmesser wurden echokardiographische Verfahren herangezogen. In diesen Untersuchungen zeigte sich nach dreiw{\"o}chiger TAC eine Verschlechterung der Pumpfunktion, sowie eine verst{\"a}rkte Dilatation des linken Ventrikels in RKIP-/--M{\"a}usen. Gest{\"u}tzt wurden diese Ergebnisse durch einen erh{\"o}hten pulmonalen Blutr{\"u}ckstau in RKIP-/--M{\"a}usen nach chronischer Druckbelastung. Zudem wurde an isolierten Kardiomyozyten die Kinetik von Kalzium als f{\"u}r die Kontraktion verantwortlichen Botenstoff durch intrazellul{\"a}re Fluoreszenz-Echtzeit-Messungen, sowie die Kontraktion und Relaxation auf Zell- und Sarkomerebene durch ein optisches Kamerasystem untersucht. Hier zeigte sich ohne den Einfluss β-adrenerger Stimulantien {\"a}quivalent zum basalen Ph{\"a}notyp dieser Tiere in RKIP-/--Kardiomyozyten keine Ver{\"a}nderung der Kalzium-Kinetik, sowie der Kontraktion und Relaxation auf Zell- und Sarkomerebene. Des Weiteren wurden mittels realtime PCR die Expressionslevels von Insuffizienzmarkern wie BNP und ANP, sowie von Kollagen 3 bestimmt. Der Grad der Fibrosierung wurde zus{\"a}tzlich durch Quantifizierung der fibrosierten Areale in histologischen Querschnitten untersucht. Apoptotische Ver{\"a}nderungen wurden mittels TUNEL-Assay auf histologischer Ebene bestimmt. In all diesen Untersuchungen zeigte sich ein fortgeschrittenes kardiales Remodeling in RKIP-/--M{\"a}usen nach TAC im Vergleich zu Wildtyptieren. Hand in Hand mit dem Bild einer fortgeschrittenen Herzinsuffizienz in RKIP-/--M{\"a}usen nach TAC konnte zudem in diesen Tieren eine gesteigerte Mortalit{\"a}t nach chronischer Hochdruckbelastung festgestellt werden. In Kombination mit den protektiven Eigenschaften einer kardialen RKIP-{\"U}berexpression, sowie dem positiven Effekt einer retroviralen RKIP-Transfektion sprechen diese Ergebnisse f{\"u}r RKIP als einen interessanten k{\"o}rpereigenen Angriffspunkt f{\"u}r die kontraktilit{\"a}tssteigernde Therapie der Herzinsuffizienz, den es in weiteren klinischen Studien zu untersuchen gilt.�}, subject = {RKIP}, language = {de} } @phdthesis{Huemmert2020, author = {H{\"u}mmert, Martin W.}, title = {Untersuchung einer monomeren Mutante der extrazellul{\"a}r regulierten Kinase 2 (ERK2) bei kardialer Hypertrophie}, doi = {10.25972/OPUS-15564}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155644}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Die Raf-MEK-ERK1/2-Kaskade spielt eine wichtige Rolle in der Vermittlung von kardialer Hypertrophie und Zell{\"u}berleben. Durch unsere Arbeitsgruppe konnte im Vorfeld gezeigt werden, dass die Dimerisierung von ERK2 eine Voraussetzung f{\"u}r dessen Autophosphorylierung an Thr188 darstellt, welche wiederum f{\"u}r die {\"U}bermittlung der hypertrophen Effekten von ERK1/2 erforderlich ist. Im Rahmen dieser Arbeit wurde daraus abgeleitet die Fragestellung untersucht, ob mit Verhinderung der ERK2-Dimerisierung eine n{\"u}tzliche Strategie zur Inhibition von Hypertrophie vorliegt und welchen Einfluss diese auf das Zell{\"u}berleben hat. Die Auswirkungen der Dimerisierungsdefizienz von ERK2 wurden in neonatalen Kardiomyozyten der Ratte und in transgenen M{\"a}usen mithilfe einer ERK2-Mutante untersucht, der einige Aminos{\"a}uren in der ERK-ERK-Interaktionsfl{\"a}che fehlen und daher keine Dimere bilden kann (ERK2Δ174-177). Eine {\"U}berexpression von ERK2Δ174-177 in neonatalen Kardiomyozyten verringerte signifikant die Antwort auf hypertrophe Stimuli (Phenylephrin, Endothelin 1). Im Anschluss daran wurden die Effekte der Dimerisierungsdefizienz von ERK2 in vivo an transgenen M{\"a}usen mit kardialer {\"U}berexpression von ERK2Δ174-177 erforscht. Diese M{\"a}use zeigten unter basalen Bedingungen keine Unterschiede gegen{\"u}ber Wildtyp-M{\"a}usen hinsichtlich Kardiomyozytengr{\"o}ße, Ventrikelwanddicke und kardialer Funktion. Unter chronischer Druckbelastung mittels TAC ließ sich hingegen ein signifikant vermindertes Ausmaß an Hypertrophie im Vergleich zu Wildtyp quantifizieren. Da der ERK1/2-Signalweg auch am {\"U}berleben von Kardiomyozyten beteiligt ist, wurde die Apoptose an histologischen Schnitten von Mausherzen analysiert. Interessanterweise fand sich bei Herzen, die das dimerisierungsdefiziente ERK2-Protein {\"u}berexprimierten, eine mit Wildtyp vergleichbare Anzahl TUNEL-positiver Zellen. Ein {\"a}hnliches Ergebnis konnte bei der Messung des Fibrosegrades an Sirius-Rot gef{\"a}rbten histologischen Schnitten beobachtet werden. Zuletzt wurden die Folgen der ERK2-Dimerisierungsdefizienz auf physiologische Hypertrophie mit einem Laufrad-Versuchsaufbau evaluiert. Transgene ERK2Δ174-177- und Wildtyp-M{\"a}use zeigten unter diesem physiologischen Stimulus keine Unterschiede im Hinblick auf die Zunahme an kardialer Hypertrophie. Da die Dimerisierungsdefizienz von ERK2 zu einer reduzierten pathologischen Hypertrophie, ohne negative Auswirkungen auf ERK1/2-vermittelte anti-apoptotische Effekte noch auf kardiale Funktion oder physiologische Hypertrophieprozesse f{\"u}hrt, stellt die Hemmung der ERK-Dimerisierung ein attraktives Ziel zur Therapie pathologischer Hypertrophie sowie potentiell auch anderer auf den ERK1/2-Signalweg basierenden Krankheiten dar.}, subject = {ERK2d4}, language = {de} } @phdthesis{Arnaudov2017, author = {Arnaudov, Theresa Irina}, title = {Anthocyane - Modulation oxidativen Stresses in vivo und in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152593}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die menschliche Nahrung enth{\"a}lt antioxidative Stoffe, die den Menschen m{\"o}glicherweise vor oxidativem Stress und seinen Konsequenzen sch{\"u}tzen k{\"o}nnen. Im Fokus der vorliegenden Arbeit standen Anthocyane, die als vielversprechende antioxidative Pflanzenstoffe in unterschiedlichen Obst- und Gem{\"u}sesorten zu finden sind. Im ersten Teil der Arbeit wurden in einem HT-29-Zellkulturmodell die zwei wichtigsten Vertreter der Anthocyanidine, Delphinidin und Cyanidin, untersucht. Es galt zu pr{\"u}fen, ob beide Pflanzenstoffe in geringen Konzentrationen in humanen Zellen antioxidativ wirken und oxidativen Genomschaden verhindern k{\"o}nnen. Im Comet-Assay reduzierten sowohl Delphinidin (ab 3,2 µM) als auch Cyanidin (ab 1 µM) signifikant die durch 100 µM Wasserstoffperoxid induzierten DNA-Sch{\"a}den in den HT-29-Zellen. Im Comet-Assays mit FPG-Enzym wurde deutlich, dass eine Pr{\"a}inkubation mit Cyanidin wirksam die Oxidation der DNA-Basen verringert. Die Auswirkungen auf den Glutathionspiegel wurden mit Hilfe des Glutathion-Recycling-Assays nach Tietze untersucht. Die Pr{\"a}inkubation mit Cyanidin f{\"u}hrte hierbei zu keinen signifikanten Ver{\"a}nderungen. Um die Auswirkungen der Anthocyanidine auf die intrazellul{\"a}re ROS-Produktion zu beobachten, wurde der fluoreszierenden Farbstoffs DHE verwendet. Sowohl Delphinidin (10 und 15 µM) als auch Cyanidin (10 und 20 µM) senkten signifikant die durch 25 µM Antimycin A angeregte ROS-Produktion. Im zweiten Teil der Arbeit wurde ein anthocyanreicher roter Fruchtsaft in einer 10-w{\"o}chigen Interventionsstudie am Menschen getestet. Hieran nahmen sowohl 19 Fibromyalgiepatienten als auch 10 gesunde Probanden teil. Es sollte die Hypothese gepr{\"u}ft werden, dass die konzentrierte und andauernde Einnahme des Saftes messbar oxidative Stressparameter im Blut ver{\"a}ndert. Außerdem sollten m{\"o}gliche Unterschiede im oxidativen Stresslevel zwischen Patienten und gesunden Probanden aufgedeckt werden. Nach jeder Studienphase erfolgte eine Befragung nach klinischen Symptomen und die Abgabe einer Urin- und Blutprobe in der Schmerzambulanz der Uniklinik W{\"u}rzburg (2 Wochen Einwaschphase, 4 Wochen Fruchtsaftphase mit je 750 ml Saft t{\"a}glich, 4 Wochen Auswaschphase). Das ROS-Level wurde mit 2 Methoden in den mononukle{\"a}ren Blutzellen untersucht: In der photometrischen NBT-Messung konnten keine signifikanten Unterschiede zwischen den Gruppen oder Zeitpunkten beobachtet werden. Bei der durchflusszytometrischen Messung mit Hilfe des fluoreszierenden DCF-Farbstoffes lag das ROS-Level der Patientengruppe vor Fruchtsafteinnahme signifikant h{\"o}her als das der Kontrollgruppe. Zur Messung der antioxidativen Kapazit{\"a}t wurde die Eisen-Reduktionsf{\"a}higkeit (FRAP) im Plasma untersucht. In der Patientengruppe zeigte sich eine Steigerung der antioxidativen Kapazit{\"a}t nach Einnahme des Fruchtsaftes. Die Unterschiede zwischen den beiden Gruppen waren gering. Sowohl das Gesamtglutathion als auch die oxidierte und reduzierte Form wurden in den Erythrozyten der Probanden mit dem Glutathion-Recycling-Assay gemessen. Nach der Fruchtsafteinnahme stieg die Konzentration des Gesamtglutathions in der Patientengruppe an. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass Delphinidin und Cyanidin auch in geringen Konzentrationen (1µM - 20µM) einen antioxidativer Effekt in HT-29-Zellen haben und vor oxidativem DNA-Schaden sch{\"u}tzen k{\"o}nnen. Die Ergebnisse der Interventionsstudie unterschieden sich teilweise in den einzelnen Endpunkten. Es war nicht m{\"o}glich, den Fibromyalgiepatienten ein h{\"o}heres oxidatives Stresslevel nachzuweisen. Ein Grund f{\"u}r die geringeren Effekte des Fruchtsaftes k{\"o}nnte in der eher geringen Bioverf{\"u}gbarkeit der Anthocyane liegen. Außerdem k{\"o}nnte die Heterogenit{\"a}t der Fibromyalgieerkrankung genauso wie andere endogene oder exogene Faktoren wie etwa Alter oder Medikamenteneinnahme die teilweise großen interindividuellen Schwankungen der Messergebnisse hinsichtlich der oxidativen Stressparameter bedingen. Klinisch profitierten einige der Fibromyalgiepatienten von der Fruchtsafteinnahme insbesondere hinsichtlich der Reizdarmsymptomatik. Dieses Volksleiden k{\"o}nnte ein interessanter Ansatzpunkt f{\"u}r Folgeuntersuchungen mit einem anthocyanreichen Produkt sein.}, subject = {Oxidativer Stress}, language = {de} } @phdthesis{Basali2017, author = {Basali, Timo}, title = {Untersuchung der Nierensch{\"a}digung durch Aldosteron am Rattenmodell {\"u}ber die Quantifizierung von Sch{\"a}digungsmarkern mittels Real-Time PCR-Technik}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151311}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die Breite der Wirkungen von Aldosteron auf Nierenzellen wurde lange Zeit untersch{\"a}tzt. Inzwischen zeigte sich ein nicht unerheblicher Anteil des Hyperaldosteronismus an arterieller Hypertonie und ebenso mehren sich die Hinweise auf damit assoziierter erh{\"o}hter Inzidenz f{\"u}r maligne Entartung von Nierengewebe. In dieser Arbeit wurde der Effekt von Hyperaldosteronismus auf Nierenzellen von Ratten in vivo untersucht. Mittels real time quantitative PCR wurden die relative Expressionsver{\"a}nderungen der mRNA von validierten Nierensch{\"a}digungsmarkern im Hyperaldosteronismusmodell kontrolliert beobachtet und statistisch ausgewertet. Anders als im analog durchgef{\"u}hrten Vorversuch mit DOCA an der Stelle von Aldosteron, ließ sich gr{\"o}ßtenteils kein {\"u}ber der nat{\"u}rlichen Streuung der Daten liegender, signifikanter Effekt der Nierensch{\"a}digung durch {\"u}berh{\"o}hte Aldosteronspiegel nachweisen. Hierf{\"u}r kommen vielf{\"a}ltige Gr{\"u}nde in Frage. Neben der technischen Variabilit{\"a}t, der Beschaffenheit der internen Kontrolle, potentiell vorhandenen Inhibitoren und der Qualit{\"a}t der mRNA, konnten eine Reihe von weiteren Gr{\"u}nden als Ursache f{\"u}r die Diskrepanz zu den Ergebnissen der mit DOCA behandelten Tiere ausgeschlossen werden. Neben der theoretischen M{\"o}glichkeit inter-methodischer Differenzen und sich daraus ergebender Variationen, sowie der noch weiter zu untersuchenden Rolle des Glukokortikoidrezeptors durch dessen variable gleichzeitige Aktivierung, ist die Interpretation im Sinne eines zu gering ausgepr{\"a}gten Sch{\"a}digungseffektes durch den Hyperaldosteronismus f{\"u}r den gew{\"a}hlten Stichprobenumfang naheliegend. Hiermit stimmt auch die Tatsache {\"u}berein, dass der Effekt der Behandlung mit Aldosteron im Vergleich zur Behandlung mit DOCA von vorne herein deutlich geringer ausfallend erwartet wurde.}, subject = {Aldosteron}, language = {de} } @phdthesis{Gruse2020, author = {Gruse, Tamara}, title = {Untersuchung der Rolle der ERK-Dimerisierung bei der ERK1/2- vermittelten Proliferation von Tumorzellen}, doi = {10.25972/OPUS-15984}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159847}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Bei vielen Erkrankungen wie z.B. Herzhypertrophie, Diabetes und Entz{\"u}ndungen spielt die Raf-MEK-ERK-Signalkaskade eine wichtige Rolle. ERK1/2 ist in vielen zellul{\"a}ren Prozessen, u.a. Proliferation, Differenzierung, Wachstum, Hypertrophie und Apoptose involviert. Auch in der Tumorentstehung besitzt dieser MAPK-Signalweg eine signifikante Funktion, da er bei ca. 50\% aller Krebsarten deutlich aktiviert ist. Ziel dieser Arbeit war es, die Rolle einer neu entdeckten Phosphorylierungsstelle an Threonin188 an ERK2 bei der Entstehung und der m{\"o}glichen Therapie von Tumoren zu erarbeiten. Daf{\"u}r wurde ein Myc-ERK2309-357-Peptid verwendet, das 2013 in der Arbeitsgruppe Lorenz entwickelt wurde. Myc-ERK2309-357 zeigte in bisher unver{\"o}ffentlichten Versuchen, dass es direkt an ERK2 bindet, eine Dimerisierung von ERK1/2 hemmen und eine vermehrte Lokalisation von ERK2 im Zellkern verhindern kann. Im Rahmen dieses Projekts konnten wir belegen, dass mit Hilfe des Myc-ERK2309-357-Peptids die Tumorzellproliferation von verschiedenen Krebszelllinien (Caco-2, SCC68, PC/1-1 und PC/13-1) um 60-80\% vermindert werden konnte. Des Weiteren konnten wir zeigen, dass Myc-ERK2309-357 keinen Einfluss auf die Phosphorylierung von ERK1/2 am TEY-Motiv besitzt. Die Aktivierung von ERK1/2 durch die Kinasen MEK1/2 wird somit nicht beeinflusst und die zytosolischen ERK-Funktionen, wie z.B. der anti-apoptotische Effekt, w{\"u}rden somit bestehen bleiben. Außerdem fanden wir heraus, dass Myc-ERK2309-357 im Vergleich zu den MEK-Inhibitoren U0126 und PD98059 und verglichen mit dem EGF-Rezeptor-Antik{\"o}rper Cetuximab die Proliferation signifikant besser hemmt.}, subject = {Dimerisierung}, language = {de} } @phdthesis{MontaggebKukielka2018, author = {Montag [geb. Kukielka], Gracia Anna}, title = {Rolle des Differenzierungszustandes f{\"u}r die Empfindlichkeit von S{\"a}ugerzellen gegen{\"u}ber genotoxischen Agenzien}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164670}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {In der vorliegenden Arbeit wurden die Einfl{\"u}sse verschiedener genotoxischer Substanzen auf S{\"a}ugertierzellen untersucht. Da ein Organismus der Ontogenese unterliegt und sich Zellen aus Stamm- und Vorl{\"a}uferzellen entwickelt, gilt es diese urspr{\"u}nglichen Zellen vor {\"a}ußeren Einfl{\"u}ssen zu sch{\"u}tzen. Da bisher kaum Untersuchungen von Zellen in verschiedenen Differenzierungsstadien durchgef{\"u}hrt wurden, wurden unter Verwendung vieler unterschiedlicher biologischer Endpunkte Effekte auf die Vitalit{\"a}t, Proliferation, Mitose und Apoptose dieser Zellen untersucht. Zudem erfolgte eine Interpretation der Ausbildung von Mikrokernen, Entstehung von DNS-Sch{\"a}den und der zugrundeliegenden Reparaturmechanismen. So konnte mit Hilfe der Untersuchungen der h{\"a}matopoetischen Stammzellen und der TK6-Zellen postuliert werden, dass h{\"a}matopoetische Stammzellen weitestgehend weniger empfindlich gegen{\"u}ber Zytostatika (Doxorubicin, Vinblastin, Methylmethansulfonat und Mitomycin C) sind als die lymphoblastoide Zelllinie TK6, welche in der Entwicklungshierarchie den Stammzellen folgt. Die Bef{\"u}rchtung, dass der Mikrokerntest in immortalisierten TK6-Zellen als Grundlage f{\"u}r Genotoxizit{\"a}tsuntersuchungen nicht gen{\"u}gen w{\"u}rden, konnte mit Hilfe der Versuchsergebnisse dieser Arbeit widerlegt werden. Die Ergebnisse belegen, dass der Mikrokerntest in TK6-Zellen relevant ist, da TK6-Zellen empfindlicher auf genotoxische Agentien im Vergleich zu h{\"a}matopoetischen Stammzellen reagieren. Bei der Untersuchung der Leuk{\"a}miezelllinie HL-60 wurden die Effekte klassischer (Vinblastin, Vincristin, Vinflunin und Vinorelbin) mit neu synthetisierten Vinca-Alkaloiden (4-Chlorochablastin, 4-Chlorochacristin, 16a, 17b und 18a) verglichen. Vinca-Alkaloide werden sehr h{\"a}ufig mit Nebenwirkungen, wie Neuropathien assoziiert, welche w{\"a}hrend einer Chemotherapie oftmals zu Therapieabbr{\"u}chen durch die Patienten f{\"u}hren. Aus diesem Grund war es erstrebenswert, neuartige Vinca-Alkaloide zu entwickeln, welche weniger Nebenwirkungen aber zugleich eine {\"a}hnliche Wirksamkeit aufweisen. Obwohl die Potenz der neuen Substanzen niedriger war als bei Vinblastin, Vincristin und Vinorelbin, zeigte ein Teil eine {\"a}hnliche Wirkung wie das Vinca-Alkaloid Vinflunin auf die Krebszelllinie HL-60 auf. Die Ergebnisse diese Arbeit k{\"o}nnen als erste Indikation in vitro genommen werden, dass sich diese Substanzen in der Krebstherapie als wirksam erweisen k{\"o}nnten und nach weiteren Ergebnissen in vivo als therapeutische Alternativen in Betracht gezogen werden. Auch bei der vergleichenden Untersuchung von exponentiell wachsenden mit differenzierten Zelllinien konnten Unterschiede detektiert werden. Die Zelllinie HT-22, welche selbst keine Krebszelllinie ist, zeigte nach Differenzierung zu nicht exponentiell wachsenden Zellen eine erh{\"o}hte Empfindlichkeit gegen{\"u}ber dem Alkylanz Methylmethansulfonat, was auf einer verminderten Basenexzisionsreparatur beruhen k{\"o}nnte. Auch die differenzierte Form der Adenokarzinom-Zelllinie CaCo2 zeigte eine gesteigerte Sensitivit{\"a}t gegen{\"u}ber dem Topoisomerase II-Inhibitor Etoposid auf, wohingegen der unselektive Topoisomerase II-Hemmer Doxorubicin keinen Effekt aufwies. Um den Sachverhalt zu kl{\"a}ren ob die festgestellten Unterschiede auf das Enzym Topoisomerase II zur{\"u}ckzuf{\"u}hren oder zellartspezifisch waren, wurden weitere Analysen der Zelllinien HL-60 und deren differenzierten Zellart durchgef{\"u}hrt. Auch hier konnten signifikante Unterschiede bei der Einzelzellgelelektrophorese nach Behandlung mit Doxorubicin und Etoposid festgestellt werden. Neben den in dieser Arbeit nachgewiesenen Unterschieden bei der Reparatur zwischen den Zelltypen, k{\"o}nnten aber auch weitere Faktoren zu Varianzen f{\"u}hren und die Mutagenit{\"a}tsforschung beeinflussen. Folglich ist davon auszugehen, dass zuk{\"u}nftige Testungen bei der pharmakologischen Substanzentwicklung in verschiedenen Zellsystemen von N{\"o}ten sind, bevor neue Substanzen zugelassen werden. Alles in allem konnte die Komplexit{\"a}t der Ergebnisse zwischen Zellen der verschiedenen Differenzierungsstadien in dieser Arbeit aufgezeigt werden. Deswegen sollte auch bei weiteren Forschungsvorhaben insbesondere ein Augenmerk auf den Differenzierungszustand der zu untersuchenden Zellpopulation geworfen werden.}, subject = {S{\"a}ugetiere}, language = {de} } @article{ArimanyNardiMinuesaPastorAngladaetal.2016, author = {Arimany-Nardi, Cristina and Minuesa, Gerard and Pastor-Anglada, Mar{\c{c}}al and Keller, Thorsten and Erkizia, Itziar and Koepsell, Hermann and Martinez-Picado, Javier}, title = {Role of Human Organic Cation Transporter 1 (hOCT1) Polymorphisms in Lamivudine (3TC) Uptake and Drug-Drug Interactions}, series = {Frontiers in Pharmacology}, volume = {7}, journal = {Frontiers in Pharmacology}, number = {175}, doi = {10.3389/fphar.2016.00175}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165236}, year = {2016}, abstract = {Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level.}, language = {en} } @article{ProppingLorenzMicheletal.2016, author = {Propping, Stefan and Lorenz, Kristina and Michel, Martin C. and Wirth, Manfred P. and Ravens, Ursula}, title = {beta-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in beta 2-Adrenoceptor Knockout Mice}, series = {Frontiers in Pharmacology}, volume = {7}, journal = {Frontiers in Pharmacology}, number = {118}, doi = {10.3389/fphar.2016.00118}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165245}, year = {2016}, abstract = {Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug's affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation.}, language = {en} } @inproceedings{WernerWakabayashiJahnsetal.2017, author = {Werner, Rudolf and Wakabayashi, Hiroshi and Jahns, Roland and Erg{\"u}n, S{\"u}leyman and Jahns, Valerie and Higuchi, Takahiro}, title = {PET-Guided Histological Characterization of Myocardial Infiltrating Cells in a Rat Model of Myocarditis}, series = {European Heart Journal - Cardiovascular Imaging}, volume = {18}, booktitle = {European Heart Journal - Cardiovascular Imaging}, number = {Supplement}, publisher = {Oxford University Press}, issn = {2047-2404}, doi = {10.1093/ehjci/jex071}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161127}, pages = {i1-i3}, year = {2017}, abstract = {No abstract available.}, subject = {Myokarditis}, language = {en} } @phdthesis{Curtaz2019, author = {Curtaz, Carolin Julia}, title = {\(In\) \(vitro\) Analysen der Wechselwirkung erh{\"o}hter Temperatur mit Zytostatika am Beispiel von Cisplatin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174543}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Neben der Chemotherapie ist heutzutage auch die Hyperthermie-Behandlung eine wichtige S{\"a}ule der antitumor{\"o}sen Therapie. W{\"a}hrend der sogenannten HIPEC Therapie (Hypertherme intraperitoneale Chemoperfusion) werden die beiden Arten der Therapieformen kombiniert und in der klinischen Praxis erfolgreich angewendet. Genauere Kenntnisse {\"u}ber die zu Grunde liegenden toxikologischen in-vitro Mechanismen k{\"o}nnten zu neuen M{\"o}glichkeiten in der klinischen Anwendung f{\"u}hren. In unserer Arbeit untersuchten wir verschiedenen Tumorzelllinien (HT29,CaCo-2,HCT116,HaCaT) in Kombination mit Cisplatin und Hyperthermie mit verschiedenen Methoden, wie zum Beispiel Mikrokerntest, Comet-Assay, Durchflusszytometrie, Vitalit{\"a}tstest und mikroskopischen Analysen. Unsere Ergebnisse f{\"u}hrten uns zu der Hypothese, dass Hyperthermie alleine zu einer sogenannte mitotic catastrophe f{\"u}hrt und zum Absterben der Tumorzellen. Im Gegensatz dazu zeigten Tumorzellen, welche mit Cisplatin alleine oder auch in Kombination mit Hyperthermie nicht in die Mitose eintreten und daher nicht durch Apoptose in den Zelltod gehen.}, subject = {Hyperthermie}, language = {de} } @article{BankogluTschoppSchmittetal.2016, author = {Bankoglu, Ezgi Eyluel and Tschopp, Oliver and Schmitt, Johannes and Burkard, Philipp and Jahn, Daniel and Geier, Andreas and Stopper, Helga}, title = {Role of PTEN in Oxidative Stress and DNA Damage in the Liver of Whole-Body Pten Haplodeficient Mice}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {11}, doi = {10.1371/journal.pone.0166956}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146970}, pages = {e0166956}, year = {2016}, abstract = {Type 2 diabetes (T2DM) and obesity are frequently associated with non-alcoholic fatty liver disease (NAFLD) and with an elevated cancer incidence. The molecular mechanisms of carcinogenesis in this context are only partially understood. High blood insulin levels are typical in early T2DM and excessive insulin can cause elevated reactive oxygen species (ROS) production and genomic instability. ROS are important for various cellular functions in signaling and host defense. However, elevated ROS formation is thought to be involved in cancer induction. In the molecular events from insulin receptor binding to genomic damage, some signaling steps have been identified, pointing at the PI3K/AKT pathway. For further elucidation Phosphatase and Tensin homolog (Pten), a tumour suppressor phosphatase that plays a role in insulin signaling by negative regulation of PI3K/AKT and its downstream targets, was investigated here. Dihydroethidium (DHE) staining was used to detect ROS formation in immortalized human hepatocytes. Comet assay and micronucleus test were performed to investigate genomic damage in vitro. In liver samples, DHE staining and western blot detection of HSP70 and HO-1 were performed to evaluate oxidative stress response. DNA double strand breaks (DSBs) were detected by immunohistostaining. Inhibition of PTEN with the pharmacologic inhibitor VO-OHpic resulted in increased ROS production and genomic damage in a liver cell line. Knockdown of Pten in a mouse model yielded increased oxidative stress levels, detected by ROS levels and expression of the two stress-proteins HSP70 and HO-1 and elevated genomic damage in the liver, which was significant in mice fed with a high fat diet. We conclude that PTEN is involved in oxidative stress and genomic damage induction in vitro and that this may also explain the in vivo observations. This further supports the hypothesis that the PI3K/AKT pathway is responsible for damaging effects of high levels of insulin.}, language = {en} } @phdthesis{Schmid2016, author = {Schmid, Evelyn}, title = {Effekte des Raf Kinase Inhibitor Proteins (RKIP) auf β-adrenerge Signalwege, Herzfunktion und die Entwicklung der Herzinsuffizienz}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142486}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Das Raf kinase inhibitor protein (RKIP) ist ein Kinaseregulator, der im Herzen eine Pr{\"a}ferenz f{\"u}r die G-Protein-gekoppelte Rezeptorkinase 2 (GRK2) zeigt. Die Regulation erfolgt durch direkte Interaktion beider Proteine, wird durch eine PKC-Phosphorylierung an Serin 153 des RKIP induziert und inhibiert die GRK2-vermittelte Phosphorylierung von G-Protein-gekoppelten Rezeptoren (GPCR). Die GRK2 desensitiviert GPCR und eine Hemmung der GRK2-Aktivit{\"a}t wirkt sich so positiv auf die Ansprechbarkeit von GPCR aus. Die \textbeta-adrenergen Rezeptoren (\textbeta AR) sind im Herzen maßgeblich an der Regulation der kardialen Kontraktilit{\"a}t beteiligt. Erste Zusammenh{\"a}nge zwischen der RKIP-Expression und der kontraktilen Antwort von Kardiomyozyten wurden bereits in einer fr{\"u}heren Arbeit untersucht und best{\"a}tigt. Sie begr{\"u}nden die Fragestellung nach Effekten einer verst{\"a}rkten RKIP-Expression auf \textbeta-adrenerge Rezeptorsignale, Herzfunktion und die Entwicklung der Herzinsuffizienz. Im Rahmen dieses Projektes konnten die Effekte des RKIP auf \textbeta-adrenerge Signalwege detaillierter beschrieben werden. Dabei erwies sich die inhibitorische Funktion auf die GRK2 als rezeptorspezifisch ohne Einfluss auf zytosolische Angriffspunkte der GRK2 zu nehmen. Verst{\"a}rkte \textbeta-adrenerge Signale zeigten sich in neonatalen Kardiomyozyten an Hand der erh{\"o}hten cAMP-Level, PKA-Aktivit{\"a}t, sowie Kontraktionsrate und Relaxationsgeschwindigkeit nach \textbeta-adrenerger Stimulation. Im Einklang damit konnte eine erh{\"o}hte PKA- und CaMKII-Aktivit{\"a}t und eine positive Inotropie in transgenen Tieren, mit herzspezifischer {\"U}berexpression von RKIP, beobachtet werden. Durch Messung des Calcium-\textit{Cyclings} in Kardiomyozyten konnte der Ph{\"a}notyp auf eine verbesserte R{\"u}ckf{\"u}hrung des Calciums, einer daraus resultierenden erh{\"o}hten Calciumbeladung des sarkoplasmatischen Retikulums und einem gesteigerten systolischen Calciumspiegel, zur{\"u}ckgef{\"u}hrt werden. Die Untersuchung der Phosphorylierung von Calciumkan{\"a}len, L-Typ-Calciumkanal und Ryanodin-Rezeptor 2, die den einw{\"a}rtsgerichteten Calciumstrom vermitteln konnte ihre Beteiligung an der positiv inotropen Wirkung ausschließen. Neben dem kontraktilen Ph{\"a}notyp konnten zus{\"a}tzliche protektive Effekte beobachtet werden. In Modellen, die eine chronische \textbeta-adrenerge Stimulation imitieren, bzw. eine Nachlasterh{\"o}hung induzieren konnte eine Verringerung der interstitiellen Fibrose und der damit assoziierten Marker, gezeigt werden. Mit Hilfe von \textit{in vivo} EKG-Messungen konnte die Neigung zur Ausbildung von Arrhythmien untersucht werden. Auch im Hinblick auf die Anzahl der Extrasystolen waren RKIP-transgene Tiere gesch{\"u}tzt. Infolge der Untersuchung der Ph{\"a}notypen in Deletionshintergr{\"u}nden der einzelnen \textbeta AR-Subtypen (\textbeta\textsubscript{1}AR, \textbeta\textsubscript{2}AR) konnte die positive Inotropie mit den spezifischen Signalwegen des \textbeta\textsubscript{1}AR assoziiert und die protektiven Effekte gegen{\"u}ber den Umbauprozessen und der Arrhythmieneigung dem \textbeta\textsubscript{2}-adrenergen Signalen zugeschrieben werden. Zus{\"a}tzlich best{\"a}tigt sich eine besondere Rolle der G\textalpha\textsubscript{i}-Kopplung des \textbeta\textsubscript{2}AR, durch die er einen hemmenden Einfluss auf die \textbeta\textsubscript{1}AR-Singale nehmen kann. Die Untersuchung einiger Marker, die eine physiologische von einer pathologischen Hypertrophie unterscheiden, konnte das in den RKIP-transgenen M{\"a}usen auftretende Wachstum der Kardiomyozyten als kompensatorische und physiologische Hypertrophie charakterisieren. Zusammengenommen weisen diese Ergebnisse auf eine ausgeglichene Aktivierung der beiden Rezeptoren hin, die sich gegenseitig regulieren und durch die Inhibition der GRK2 in ihrer Anregbarkeit erhalten bleiben. Mittels einer AAV9-vermittelten Gentherapie konnte das therapeutische Potential dieses Prinzips weiter best{\"a}tigt werden, da es die prominentesten Ver{\"a}nderungen w{\"a}hrend der Herzinsuffizienzentwicklung, wie die Verschlechterung der linksventrikul{\"a}ren Funktion, die Dilatation des linken Ventrikels, die Ausbildung von Lungen{\"o}demen und interstitieller Fibrose sowie die Expression von Herzinsuffizienz-assoziierten Genen, verhindern konnte. Auch konnten die Auswirkungen der Deletion des RKIP, die sich durch eine beschleunigte und gravierendere Herzinsuffizienzentwicklung auszeichnet, durch Reexpression von RKIP verhindert werden. Diese Arbeit kann somit zeigen, dass das RKIP eine ausgeglichene Verst{\"a}rkung von \textbeta-adrenergen Signalwegen verursacht, die positiv inotrop und gleichzeitig protektiv wirkt. Dieses Wirkprinzip k{\"o}nnte ferner eine Strategie zur Erh{\"o}hung der Kontraktilit{\"a}t in der Herzinsuffizienz darstellen, die entgegen etablierter Theorien auf der Stimulation beider \textbeta AR basiert.}, subject = {Herzinsuffizienz}, language = {de} } @phdthesis{Kestler2017, author = {Kestler, Christian}, title = {Untersuchungen {\"u}ber die Dimerisierung der HAD-Phosphatase Chronophin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149777}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Phosphatasen der HAD (haloacid dehalogenase)-Familie sind weit verbreitet in allen Dom{\"a}nen des Lebens und erf{\"u}llen die verschiedensten zellul{\"a}ren Aufgaben, beispielsweise in Metabolismus und Zellregulation. Die HAD-Phosphatase Chronophin zeigt Phosphataseaktivit{\"a}t unter anderem gegen{\"u}ber Pyridoxal-5'-Phosphat (PLP), einem essentiellen Kofaktor vieler biochemischer Prozesse, und Phosphocofilin, einem Regulator des Aktinzytoskeletts. Chronophin dimerisiert {\"u}ber die Interaktion zweier identischer Untereinheiten zu einem Homodimer. Ziel dieser Arbeit war, die Rolle dieser Dimerisierung, eines bei HAD-Phosphatasen weit verbreiteten Oligomerisierungszustandes, n{\"a}her zu untersuchen. Hierzu wurde die Dimerisierung erfolgreich durch den Austausch der Aminos{\"a}uren Alanin 194 und 195 zu Lysinen (Mutation A194K/A195K) gest{\"o}rt. Der Nachweis einer konstitutiv monomeren Chronophin-Mutante mittels Gr{\"o}ßenausschlusschromatographie, Rasterkraftmikroskopie, analytischer Ultra¬zentrifugation und Zellexperimenten wurde schließlich {\"u}ber die Struktur¬aufl{\"o}sung mittels R{\"o}ntgenstrukturanalyse best{\"a}tigt. Aktivit{\"a}tsmessungen der monomeren Mutante gegen{\"u}ber dem Substrat PLP zeigten eine deutliche Verminderung der Phosphataseaktivit{\"a}t. Die R{\"o}ntgenstrukturanalyse von Chronophin A194K/A195K im Vergleich mit Wildtyp-Chronophin enth{\"u}llte einen Mechanismus, wie die sogenannte Substratspezifit{\"a}tsschleife, die f{\"u}r die korrekte Positionierung des PLP sorgt, im Homodimer des Wildtyps durch Interaktionen mit dem zweiten Protomer stabilisiert wird. Diese Stabilisierung fehlt bei der monomeren Mutante und {\"a}ußert sich in einer ver{\"a}nderten Stellung der Substratspezifit{\"a}tsschliefe. Der Strukturvergleich von Chronophin mit weiteren HAD-Phosphatasen der selben strukturellen Untergruppe vom C2a-Typ l{\"a}sst eine allgemeine G{\"u}ltigkeit der hier beschriebenen allosterischen Kontrolle von Substratspezifit{\"a}t {\"u}ber Homodimerisierung bei HAD-Phosphatasen vermuten und k{\"o}nnte so neue Ansatzpunkte f{\"u}r m{\"o}glicherweise auch therapeutisch nutzbare Aktivit{\"a}tshemmungen liefern.}, subject = {Dimerisierung}, language = {de} } @phdthesis{Messerer2017, author = {Messerer, Regina}, title = {Synthesis of Dualsteric Ligands for Muscarinic Acetylcholine Receptors and Cholinesterase Inhibitors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149007}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {The study is dealing with the synthesis and pharmacological investigation of newly designed dualsteric ligands of muscarinic acetylcholine receptors belonging to the superfamily of G protein-coupled receptors. Such bipharmacophoric ligands combine the advantages of the orthosteric binding site (high-affinity) and of the topographically distinct allosteric binding site (subtype-selectivity) resulting in compounds with reduced side effects. This opens the way to a new therapeutic approach in the treatment of e.g. chronic pain, drug withdrawal, Parkinson`s and Alzheimer`s disease. Furthermore, the newly synthesized dualsteric compounds were pharmacologically investigated in order to get a better understanding of the activation and signaling processes in muscarinic acetylcholine receptors, especially with regard to partial agonism. The development of the "dynamic ligand binding" concept offers new perspectives for ligand binding and signaling at G protein-coupled receptors. GPCRs are no longer considered as simple on/off switches. Dualsteric ligands can bind in a dualsteric pose, reflecting an active receptor state as well as in a purely allosteric binding pose, characterized by an inactive receptor state resulting in partial agonism. The degree of partial agonism depends on the ratio of active versus inactive receptor populations. On this basis, orthosteric/orthosteric hybrid ligands consisting of the antagonist atropine and scopolamine, respectively, as well as of the agonist iperoxo and isoxazole, respectively, linked via different alkyl chain length were synthesized in order to investigate partial agonism (Figure 1). Figure 1: Structures of the synthesized iperoxo/isoxazole-atropine/scopolamine-hybrids. Furthermore, different sets of quaternary and tertiary homodimers consisting either of two iperoxo or two acetylcholine units were synthesized in order to study their extent on partial agonism (Figure 2). The two agonists were connected by varying alkyl chain length. Binding studies on CHO-hM2 cells of the quaternary compounds revealed that dimerization of the agonist results in a loss of potency. The iperoxo-dimers reached higher maximum effects on the Gi- as well as on the Gs pathway in comparison to the acetylcholine-dimers. Besides the choice of the orthosteric building block (potency of the agonist), the alkyl chain length is also crucial for the degree of partial agonism. Figure 2: Structures of the synthesized quat./tert. iperoxo/acetylcholine-homodimers. Quinolone-based hybrids connected to the superagonist iperoxo and to the endogenous ligand acetylcholine, respectively, linked through an alkyl chain of different length were synthesized in order to develop further partial agonists (Figure 3). FRET studies confirmed M1 subtype-selectivity as well as linker dependent receptor response. The greatest positive FRET signal was observed with quinolone-C6-iper resulting from a positive cooperativity between the two separated moieties, alloster and orthoster. However, the corresponding hybrids with a longer linker led to an inverse FRET signal indicating a different binding mode, e.g. purely allosteric, in contrast to the shorter linked hybrids. Furthermore, the flexible alkyl spacer was replaced by a rigidified linker resulting in the hybrid quinolone-rigid-iperoxo (Figure 3). FRET studies on the M1 receptor showed reduced FRET kinetics, resulting from interactions between the bulky linker and the aromatic lid, located between the orthosteric and allosteric binding site. A bitopic binding mode of the rigidified hybrid is presumed. For further clarity, mutational studies are necessary. Figure 3: M1-selective hybrid compounds. Another aim of this work was the design and synthesis of new hybrid compounds, acting as agonists at the M1 and M2 receptor and as inhibitors for AChE and BChE in the context of M. Alzheimer. Several sets of hybrid compounds consisting of different pharmacophoric units (catalytic active site: phthalimide, naphthalimide, tacrine; peripheric anionic site: iperoxo, isoxazole) linked through a polymethylene chain of varying length were synthesized. Tac-C10-iper (Figure 4), consisting of tacrine and the superagonist iperoxo linked by a C10 polymethylene spacer, was found to have excellent anticholinesterase activity for both AChE (pIC50 = 9.81) and BChE (pIC50 = 8.75). Docking experiments provided a structural model to rationalize the inhibitory power towards AChE. Additionally, the tacrine related hybrids showed affinity to the M1 and M2 receptor. Such compounds, addressing more than one molecular target are favorable for multifactorial diseases such as Alzheimer. Figure 4: Structure of the most active compound regarding anticholinesterase activity. In summary, the choice of the pharmacophoric units, their connecting point as well as the nature, length, and flexibility of the linker play an important role for the activity of designed bivalent ligands. A shorter linker length cannot bridge both binding sites simultaneously in contrast to longer linker chains. On the other hand, too long linker chains can result in unwanted steric interactions. Further investigations with respect to structural variations of hybrid compounds, with or without quaternary ammonium groups, are necessary in the light of drug development.}, subject = {Cholinesteraseinhibitor}, language = {en} } @article{VoglLutzSchoenfelderetal.2015, author = {Vogl, Silvia and Lutz, Roman W. and Sch{\"o}nfelder, Gilbert and Lutz, Werner K.}, title = {CYP2C9 genotype vs. metabolic phenotype for individual drug dosing - a correlation analysis using flurbiprofen as probe drug}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0120403}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148783}, pages = {e0120403}, year = {2015}, abstract = {Currently, genotyping of patients for polymorphic enzymes responsible for metabolic elimination is considered a possibility to adjust drug dose levels. For a patient to profit from this procedure, the interindividual differences in drug metabolism within one genotype should be smaller than those between different genotypes. We studied a large cohort of healthy young adults (283 subjects), correlating their CYP2C9 genotype to a simple phenotyping metric, using flurbiprofen as probe drug. Genotyping was conducted for CYP2C9*1, *2, *3. The urinary metabolic ratio MR (concentration of CYP2C9-dependent metabolite divided by concentration of flurbiprofen) determined two hours after flurbiprofen (8.75 mg) administration served as phenotyping metric. Linear statistical models correlating genotype and phenotype provided highly significant allele-specific MR estimates of 0.596 for the wild type allele CYP2C9*1, 0.405 for CYP2C9*2 (68 \% of wild type), and 0.113 for CYP2C9*3 (19 \% of wild type). If these estimates were used for flurbiprofen dose adjustment, taking 100 \% for genotype *1/*1, an average reduction to 84 \%, 60 \%, 68 \%, 43 \%, and 19\% would result for genotype *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. Due to the large individual variation within genotypes with coefficients of variation >= 20\% and supposing the normal distribution, one in three individuals would be out of the average optimum dose by more than 20 \%, one in 20 would be 40\% off. Whether this problem also applies to other CYPs and other drugs has to be investigated case by case. Our data for the given example, however, puts the benefit of individual drug dosing to question, if it is exclusively based on genotype.}, language = {en} } @phdthesis{PerpinaViciano2020, author = {Perpi{\~n}{\´a} Viciano, Cristina}, title = {Study of the activation mechanisms of the CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3)}, doi = {10.25972/OPUS-19237}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192371}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β-arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β-arrestin-dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different F{\"o}rster resonance energy transfer (FRET)-based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild-type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET-based receptor sensors in the field of chemokine receptors. Additional FRET-based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand-induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12-induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands.}, subject = {G protein-coupled receptors}, language = {en} } @phdthesis{Link2019, author = {Link, Samuel}, title = {Aldosteron-vermittelte oxidative Nierensch{\"a}digung - Einfluss der antioxidativen Abwehr}, doi = {10.25972/OPUS-18603}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186037}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Patienten mit erh{\"o}hten Aldosteronspiegeln zeigen eine gesteigerte Inzidenz f{\"u}r Malignome, insbesondere von Nierenzellkarzinomen. Das Ziel dieser Arbeit war es, die Aldosteron-vermittelte oxidative Nierensch{\"a}digung n{\"a}her zu analysieren sowie die auf Zellebene gezeigte Beeinflussung der antioxidativen Schutzmechanismen im lebenden Organismus nachzuweisen und m{\"o}gliche therapeutische Ansatzpunkte zu identifizieren. Dazu wurde ein Interventions-versuch {\"u}ber 28 Tage durchgef{\"u}hrt. Neben einer Aldosterongabe wurden folgende Interventionen verwendet: Spironolacton zur Blockade des Mineralkortikoid-Rezeptors (MR), Apocynin als Hemmstoff der NADPH-Oxidasen (Nox), L-NAME zur Blockade der NO-Synthasen (NOS), PDTC, einen Hemmstoff des Transkriptionsfaktors NF-kB sowie Sulforaphan, ein nat{\"u}rlicher Nrf2-Induktor. Eine weitere Gruppe erhielt Sulforaphan ohne additive Aldosterongabe. Die Nierensch{\"a}den wurden mittels histopathologischer Sch{\"a}digungsscores und der Anzahl an DNA-Doppelstrangbr{\"u}che analysiert. Die Beeinflussung der antioxidativen Abwehr wurde durch die Aktivierung des Transkriptionsfaktors Nrf2 und durch die Quantifizierung antioxidativer Enzyme bestimmt. Im Nierengewebe f{\"u}hrte Aldosteron zu einer Zunahme von oxidativem Stress. Histologisch zeigte sich ein Anstieg von glomerul{\"a}ren Sch{\"a}den. Auch kam es zu einer deutlichen Zunahme von Doppelstrangbr{\"u}chen der DNA. Des Weiteren konnten wir zeigen, dass Aldosteron auch in vivo zu einer Zunahme der Nrf2-Aktivit{\"a}t f{\"u}hrte, wobei sich dies auf Proteinebene nicht in einer (dauerhaften) Synthesesteigerung von antioxidativen Enzymen wiederspiegelte und keinen ausreichenden Schutz des Nierengewebes bot. F{\"u}r die Interventionsgruppen konnte keine signifikante Auswirkung auf das Vorliegen von oxidativem Stress gezeigt werden. Dies k{\"o}nnte an der Versuchsdauer bzw. an der gew{\"a}hlten Nachweismethode gelegen haben. Nichtsdestotrotz zeigte die Blockade der Nox durch Apocynin bzw. der NOS durch L-NAME eine effektive Reduktion der histologischen und genomischen Sch{\"a}den. Die L-NAME-Gruppe wies dabei die h{\"o}chsten Blutdruckwerte auf, diese waren auch zur Aldosterongruppe signifikant gesteigert. Die beobachteten Effekte waren folglich nicht durch den in der Aldosterongruppe erfolgten Blutdruckanstieg, sondern vielmehr durch den Anstieg von oxidativem Stress zu erkl{\"a}ren. Ebenfalls blieb die Nrf2-Aktivit{\"a}t bei der Gabe von Apocynin und L-NAME weitgehend auf Kontrollniveau, was daf{\"u}rspricht, dass der in der Aldosterongruppe messbare Nrf2-Anstieg am ehesten als Reaktion auf chronisch erh{\"o}hten oxidativen Stress erfolgte, welcher durch die Interventionen ausblieb. Die Blockade von NF-κB mittels PDTC f{\"u}hrte zu vergleichbaren Effekten wie Apocynin und L-NAME. Das deutet darauf hin, dass Aldosteron {\"u}ber die Aktivierung von NF-κB die vermehrte Synthese von pro-oxidativen Enzymen wie Nox und NOS anregt. Die Gabe von Spironolacton hatte den st{\"a}rksten protektiven Effekt, sowohl auf histologische Ver{\"a}nderungen als auch auf das Entstehen von DNA-Doppelstrangbr{\"u}chen, wobei die Nrf2-Aktivit{\"a}t in dieser Gruppe ebenfalls auf Kontrollniveau blieb. Die Aldosteroneffekte wurden folglich {\"u}ber den MR vermittelt. Eine additive Nrf2-Induktion mittels Sulforaphan konnte auch keinen (dauerhaften) Effekt auf die Synthese antioxidativer Enzyme zeigen. Dennoch zeigte diese Gruppe einen {\"a}hnlich effektiven Schutz vor den oxidativen Nierensch{\"a}den wie die Gabe von Spironolacton. Vieles spricht daf{\"u}r, dass die Wirkung von Sulforaphan dabei {\"u}ber seine Wirkung als direktes Antioxidans bzw. Radikalf{\"a}nger und nicht {\"u}ber den Nrf2-Weg zu erkl{\"a}ren ist. Aldosteron f{\"u}hrt in der Niere {\"u}ber oxidativen Stress zu glomerul{\"a}rer Fibrose und DNA-Sch{\"a}den. Das k{\"o}nnte eine Erkl{\"a}rung f{\"u}r die gesteigerte Inzidenz von Nierenzellkarzinomen in Patienten mit erh{\"o}hten Aldosteronspiegeln darstellen. Unsere Ergebnisse sprechen daf{\"u}r, dass Aldosteron {\"u}ber eine Signalkaskade {\"u}ber den MR zu einer Aktivierung von Nox und NOS f{\"u}hrt. Der Aktivierung des Transkriptionsfaktors NF-κB scheint dabei durch die Synthese pro-oxidativer Enzyme eine Art Verst{\"a}rker-Effekt zuzukommen. Als Reaktion auf den durch Aldosteron gesteigerten oxidativen Stress kommt es zu einer Aktivierung des antioxidativen Transkriptionsfaktors Nrf2, jedoch ohne dass dies zu einem ausreichenden Schutz des Nierengewebes f{\"u}hrt. M{\"o}gliche therapeutische Ansatzpunkte f{\"u}r einen Schutz vor den durch Aldosteron vermittelten oxidativen Nierensch{\"a}den scheinen eher innerhalb der Aldosteronsignalkaskade, insbesondere in der Blockade des MR, als in der antioxidativen Abwehr zu liegen.}, subject = {Aldosteron}, language = {de} } @phdthesis{Zundler2019, author = {Zundler, Matthias}, title = {Einfluss der Phosphoglykolat-Phosphatase auf den Metabolismus von Signal-, Membran- und Speicherlipiden in murinen Embryonen und Lymphozyten}, doi = {10.25972/OPUS-16844}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168442}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Die Phosphoglykolat-Phosphatase PGP (fr{\"u}her auch als AUM bezeichnet) wurde in unserem Labor als Mitglied der HAD-Typ-Phosphatasen identifiziert. Die genetische Inaktivierung des Enzyms im gesamten Mausorganismus f{\"u}hrt ab E8.5 zu einer Wachstumsverz{\"o}gerung muriner Embryonen und bis E12.5 schließlich zu deren Tod. Im Gegensatz dazu sind M{\"a}use mit einer PGP-Inaktivierung in h{\"a}matopoetischen Zellen und im Endothel lebensf{\"a}hig und ph{\"a}notypisch unauff{\"a}llig. Neue Erkenntnisse schreiben dem Enzym neben einer Aktivit{\"a}t gegen{\"u}ber Phosphoglykolat auch Aktivit{\"a}ten gegen{\"u}ber Glycerin-3-phosphat (G3P), P-Erythronat und P-Lactat zu. Da diese Phosphatase-Aktivit{\"a}ten Auswirkungen auf den Lipidstoffwechsel nahelegen, wurde in der vorliegenden Arbeit mittels massenspektrometrischer Methoden der Einfluss der Phosphoglykolat-Phosphatase auf den Metabolismus von Signal-, Membran- und Speicherlipiden in murinen Embryonen und Lymphozyten untersucht. Nach Inaktivierung der PGP im gesamten Organismus wurden in E8.5-Embryonen erh{\"o}hte Diacylglycerin (DG)-, Triacylglycerin (TG)- und Sphingomyelin (SM)-Spiegel gemessen, w{\"a}hrend niedrigere Phosphatidylcholin (PC)-Level vorlagen. In PGP-inaktivierten Lymphozyten waren G3P-, DG-, TG-, PC- und SM-Level nicht ver{\"a}ndert. Daf{\"u}r kam es zu signifikanten Erh{\"o}hungen der Phosphatidylglycerol (PG*)- und Cardiolipin (CL)-Spiegel. Zusammenfassend konnte gezeigt werden, dass die PGP in unterschiedlichen Geweben differenzielle Effekte auf die Spiegel verschiedener Lipide hat. Dies deckt neue Funktionen der PGP f{\"u}r die Regulation des Lipidmetabolismus auf. Die vorliegende Arbeit stellt somit die Grundlage f{\"u}r weitere Untersuchungen {\"u}ber die genauen Ursachen und Folgen dieser Regulation dar und l{\"a}sst auf eine wichtige Rolle der PGP als metabolische Phosphatase im Organismus schließen.}, subject = {Phosphoglykolatphosphatase}, language = {de} } @article{MambrettiKistnerMayeretal.2016, author = {Mambretti, Egle M. and Kistner, Katrin and Mayer, Stefanie and Massotte, Dominique and Kieffer, Brigitte L. and Hoffmann, Carsten and Reeh, Peter W. and Brack, Alexander and Asan, Esther and Rittner, Heike L.}, title = {Functional and structural characterization of axonal opioid receptors as targets for analgesia}, series = {Molecular Pain}, journal = {Molecular Pain}, number = {12}, doi = {10.1177/1744806916628734}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145917}, pages = {1-17}, year = {2016}, abstract = {Background Opioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. µ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in na{\"i}ve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function. Results Comparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOP-mcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [D-Ala\(^2\), N-MePhe\(^4\), Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in na{\"i}ve Wistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using β-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl lead to a recruitment of β-arrestin-2 to the membrane followed by a β-arrestin-2 reappearance in the cytosol and MOP internalization. Pretreatment with hypertonic saline prevented MOP internalization. Conclusion MOPs are present and functional in the axonal membrane from na{\"i}ve animals. Hypertonic saline acutely decreases ligand-induced internalization of MOP and thereby might improve MOP function. Further studies should explore potential clinical applications of opioids together with enhancers for regional analgesia.}, language = {en} } @article{OthmanNaseemAwadetal.2016, author = {Othman, Eman M. and Naseem, Muhammed and Awad, Eman and Dandekar, Thomas and Stopper, Helga}, title = {The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0168386}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147983}, pages = {e0168386}, year = {2016}, abstract = {Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.}, language = {en} } @phdthesis{Berisha2019, author = {Berisha, Filip}, title = {Molekulare Wirkmechanismen von Sulfonylharnstoffen: Direkte Epac-Aktivierung oder Hemmung der Phosphodiesterasen}, doi = {10.25972/OPUS-17653}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176535}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Diabetes mellitus ist die h{\"a}ufigste Stoffwechselerkrankung in Deutschland. Sulfonylharnstoffe (SH) stellen die {\"a}lteste und eine sehr prominente Gruppe in der oralen Therapie des Diabetes mellitus Typ II dar, die eine verst{\"a}rkte Insulinfreisetzung vorrangig durch die Hemmung eines ATP-sensitiven Kaliumkanals (K+ATPKanal) erreichen. Daneben konnten weitere Proteine identifiziert werden, die mit SH interagieren und zu deren Effekten beitragen. W{\"a}hrend bereits in fr{\"u}hen Arbeiten gezeigt werden konnte, dass SH Vertreter der Phosphodiesterasen (PDE)Familie in ihrer Funktion behindern k{\"o}nnen, wurde k{\"u}rzlich Epac2 (exchange protein directly activated by cAMP 2) als weiteres Zielprotein f{\"u}r SH angef{\"u}hrt. Insbesondere die F{\"a}higkeit von SH, direkt an Epac2 zu binden, wird in der Literatur kontrovers diskutiert und eine indirekte Aktivierung durch eine PDE-Hemmung und einen erh{\"o}hten cAMP-Spiegel als Mechanismus vermutet. Zur weiteren Untersuchung wurden in dieser Arbeit FRET-basierte Biosensoren verwendet, um die Wirkung von SH auf Epac und PDEs n{\"a}her zu untersuchen. Dabei konnte sowohl in einem photometrischen Ansatz als auch in lebenden Zellen, die einen Epac2-basierten Sensor enthalten, gezeigt werden, dass eine Aktivierung durch SH stattfindet. Da sowohl Epac2-camps, der von allen hier verwendeten Sensoren mit der h{\"o}chsten Sensitivit{\"a}t f{\"u}r cAMP, als auch CFP-Epac1δDEPYFP nicht auf SH reagieren, ist diese Aktivierung selektiv f{\"u}r die Isoform Epac2 und wird vorrangig nicht durch eine PDE-Hemmung verursacht. Die Verwendung weiterer Sensoren mit verschiedenen Varianten von Epac2 (verl{\"a}ngerte Version von Epac2-camps) zeigen mit zunehmender L{\"a}nge {\"u}ber die cAMP-Bindedom{\"a}ne hinaus eine beginnende Reaktion im Sinne einer instabilen FRET-Kurve (Epac2camps long) bzw. eine deutliche Aktivierung durch den SH (Epac2-camps superlong), wodurch eine direkte Aktivierung best{\"a}tigt wird, und suggerieren eine Bindestelle f{\"u}r SH, die sich von denen von cAMP unterschiedet und weiter eingeengt werden konnte (im n{\"a}heren Bereich von Q454 bzw. E460). Obwohl hierdurch eine direkte Aktivierung gezeigt werden konnte, ist die grunds{\"a}tzliche F{\"a}higkeit der SH, PDE zu beeinflussen, keineswegs gekl{\"a}rt. Daher wurden weitere Sensoren konstruiert bzw. verwendet, die basierend auf Epac1-camps und Epac2-camps verschiedene PDEs enthalten. Dabei konnte durch die Zugabe von SH eine deutliche Aktivierung des jeweiligen Sensors und somit eine PDEHemmung nachgewiesen werden. Dies konnte sowohl f{\"u}r PDE4A als auch f{\"u}r die in Inselzellen {\"u}berwiegend vorkommende PDE3B gezeigt werden. Dadurch ergeben sich einige (klinisch relevante) Implikationen. Zum einen stellt neben der direkten Epac-Aktivierung auch die direkte Hemmung der PDE einen wichtigen Mechanismus f{\"u}r die Sekretion von Insulin dar. Außerdem sind bei PDEHemmung und direkter Epac-Aktivierung außerhalb der Inselzellen auch Nebenwirkungen in anderen Organen zu erwarten wie z.B. die Entstehung lebensgef{\"a}hrlicher Rhythmusst{\"o}rungen in Herzmuskelzellen.}, subject = {Sulfonylharnstoffe}, language = {de} } @phdthesis{Glaeser2017, author = {Gl{\"a}ser, Katharina}, title = {Einfluss hochfrequenter Felder des Mobilfunks auf das blutbildende System in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145733}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Elektromagnetische Felder (EMF) sind in der Umwelt des Menschen allgegenw{\"a}rtig. Unter Verwendung unterschiedlicher Frequenzen bilden sie die Grundlage zahlreicher Technologien und begegnen uns im Alltag in einer Vielzahl von Anwendungen. Eine sehr wichtige Anwendung von EMF ist die mobile Kommunikation. Die hierf{\"u}r verwendeten Frequenzen liegen im hochfrequenten Bereich und variieren mit dem Mobilfunkstandard. Weit verbreitet ist die GSM- und UMTS-Modulation der zweiten (2G) und dritten Generation (3G). Zum neuesten Mobilfunkstandard z{\"a}hlt LTE (4G). Aus statistischen Daten geht hervor, dass derzeit weltweit mehr als sieben Milliarden Mobilfunk-Endger{\"a}te existieren. Die weitverbreitete und stetig ansteigende Verwendung dieser Technologien verdeutlicht, dass viele Menschen, darunter auch zunehmend Kinder und Jugendliche, regelm{\"a}ßig einer Exposition gegen{\"u}ber EMF ausgesetzt sind. Die wichtigste Expositionsquelle stellt dabei das Mobiltelefon dar, da sich in diesem Szenario die Quelle sehr nah am menschlichen K{\"o}rper befindet. In der Vergangenheit wurden zahlreiche in-vitro- und in-vivo-Untersuchungen sowie epidemiologische Studien durchgef{\"u}hrt, um potentielle, nicht-thermische Effekte von Mobilfunkstrahlung auf biologische Systeme beurteilen zu k{\"o}nnen. Ein vollst{\"a}ndiger Konsens konnte auf der Basis der erhaltenen Ergebnisse jedoch nicht erzielt werden, sodass weiterhin Bedenken zum sch{\"a}dlichen Potential dieser nichtionisierenden Strahlung bestehen. Insbesondere wurden Fragestellungen zu Langzeiteffekten sowie zu Effekten, die speziell bei Kindern eine besondere Rolle spielen, bisher nicht ausreichend adressiert. Kinder k{\"o}nnen empfindlicher auf Umwelteinfl{\"u}sse reagieren und sind im Vergleich zu Erwachsenen teilweise h{\"o}her gegen{\"u}ber EMF exponiert. Dies gilt vor allem f{\"u}r Kopfregionen, in denen sich das aktive, f{\"u}r die H{\"a}matopoese verantwortliche Knochenmark befindet. Vor diesem Hintergrund war es das Ziel der vorliegenden Arbeit, den Einfluss von Mobilfunkstrahlung auf das humane blutbildende System zu untersuchen. Im Fokus standen dabei humane h{\"a}matopoetische Stammzellen, die mit Frequenzen der Mobilfunkstandards GSM (900 MHz), UMTS (1.950 MHz) und LTE (2.535 MHz) jeweils {\"u}ber einen kurzen (4 h) und einen langen (20 h) Zeitraum und mit unterschiedlichen Intensit{\"a}ten (0 W/kg, 0,5 W/kg, 1 W/kg, 2 W/kg und 4 W/kg) exponiert wurden. Vergleichende Experimente erfolgten mit Zellen der Promyelozyten-Zelllinie HL-60. M{\"o}gliche Effekte wurden mit den Endpunkten Apoptose, oxidativer Stress, Zellzyklus, DNA-Schaden und -Reparatur sowie Differenzierung und Epigenetik in Form von Histonacetylierung bewertet. In keinem der genannten Endpunkte konnten klare Effekte durch Mobilfunkstrahlung ausgemacht werden, weder f{\"u}r die h{\"a}matopoetischen Stammzellen, noch f{\"u}r die Zelllinie HL-60. Die einzige Ver{\"a}nderung wurde bei der Quantifizierung von DNA-Sch{\"a}den beobachtet. Hier zeigte sich nach der Kurzzeitexposition der Stammzellen mit der Modulation GSM eine kleine, aber statistisch signifikante Abnahme der DNA-Sch{\"a}den verglichen mit der Scheinexposition. Diese Beobachtung ließ sich in weiteren Replikaten jedoch nicht reproduzieren und wurde daher als nicht biologisch relevant eingestuft. Insgesamt konnte mit dieser Arbeit gezeigt werden, dass durch Mobilfunkstrahlung mit Frequenzen der verbreiteten Modulationen GSM, UMTS und LTE sowie SAR-Werten, die unterhalb und oberhalb des empfohlenen Sicherheitsstandards liegen und typischerweise bei Handytelefonaten auftreten, keine Effekte in Zellen des blutbildenden Systems unter den gegebenen Versuchsbedingungen induziert wurden. Ein besonderer Fokus lag hierbei auf der Reproduzierbarkeit der Ergebnisse. Weiterhin wurden zum ersten Mal humane h{\"a}matopoetische Stammzellen f{\"u}r derartige Untersuchungen eingesetzt. Dies hat insofern eine besondere Bedeutung, als h{\"a}matopoetische Stammzellen aufgrund ihrer multipotenten Eigenschaften eine breitere Analyse mit Hinblick auf die Kanzerogenese und auf das Immunsystem erm{\"o}glichen. Um {\"u}ber die Mobilfunk-Untersuchungen hinaus die h{\"a}matopoetischen Stammzellen besser charakterisieren zu k{\"o}nnen, sowie die Sensitivit{\"a}t von Blutzellen mit unterschiedlichem Differenzierungsstatus zu analysieren, wurden sie anderen Zellen des blutbildenden Systems (undifferenzierte und differenzierte HL-60-Zellen und TK6-Zellen) gegen{\"u}bergestellt. Eine Behandlung der verschiedenen Zelltypen mit mutagenen Substanzen zeigte, dass sich die h{\"a}matopoetischen Stammzellen in den meisten der untersuchten Endpunkte von den Zelllinien unterschieden. Deutliche Abweichungen zeigten sich beim oxidativen Stress, der DNA-Reparatur und der Histonacetylierung; kein Unterschied konnte dagegen bei den DNA-Sch{\"a}den beobachtet werden. Eine erste Interpretation der erhaltenen Ergebnisse ist auf der Grundlage der unterschiedlichen Eigenschaften von Zellen mit abweichendem Differenzierungsstatus m{\"o}glich. Um jedoch eine eindeutige Aussage treffen zu k{\"o}nnen, m{\"u}ssten noch weitere Untersuchungen durchgef{\"u}hrt werden.}, subject = {Mobilfunk}, language = {de} } @phdthesis{Ramm2012, author = {Ramm, Susanne}, title = {Mechanismen idiosynkratischer Lebertoxizit{\"a}t - Einfluss von Arzneistoff-unabh{\"a}ngigen Stressfaktoren auf die Bildung reaktiver Metaboliten und zellul{\"a}ren Stress}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74342}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Idiosynkratische Lebersch{\"a}digung durch Arzneimittel (z.B. Diclofenac) stellt trotz ihres seltenen Auftretens eine erhebliche Komplikation in der Arzneimittelentwicklung und -therapie dar. Die zu idiosynkratischen Reaktionen f{\"u}hrenden, komplexen chemischen und biologischen Abl{\"a}ufe sind noch weitgehend unklar. Inzwischen wird jedoch vermutet, dass die Toxizit{\"a}t eines Arzneimittels durch Arzneistoff-unabh{\"a}ngige Risikofaktoren, wie Krankheiten, Entz{\"u}ndungsreaktionen, Co-Medikation oder Alkohol, erh{\"o}ht werden kann. M{\"o}gliche Mechanismen k{\"o}nnten hierbei eine vermehrte Bildung reaktiver Metaboliten bzw. eine ver{\"a}nderte zellul{\"a}re Stress- und Immunantwort sein. Um tiefere Einblicke in die Bedeutung m{\"o}glicher Arzneistoff-unabh{\"a}ngiger Risikofaktoren zu erhalten, wurde in der vorliegenden Arbeit der Einfluss drei verschiedener Stressfaktoren auf die Toxizit{\"a}t von Diclofenac (Dcl) untersucht. Bei diesen Stressfaktoren handelte es sich um Lipopolysaccharid (LPS) und Poly I:C (PIC) zur Simulation einer bakteriellen bzw. viralen Entz{\"u}ndung sowie um Buthionin-Sulfoximin (BSO) zur Depletion zellul{\"a}ren Glutathions. Zus{\"a}tzlich wurde getestet, ob eine durch Stressfaktoren ausgel{\"o}ste Erh{\"o}hung der Toxizit{\"a}t von Dcl in Ratten mit Ver{\"a}nderungen in der Biotransformation bzw. mit einer Hochregulation co-stimulatorischer Faktoren (z.B. Zytokine oder Alarmsignale) einhergeht. Die Kombination einer einw{\"o}chigen therapeutisch dosierten Dcl-Behandlung mit einer einmaligen LPS-Dosis erzeugte in den Tieren eine ausgepr{\"a}gte Hepatotoxizit{\"a}t, die mit erh{\"o}hten Aktivit{\"a}ten der Aminotransferasen im Serum einherging. Diese adversen Effekte konnten jedoch nicht durch LPS oder Dcl alleine, bzw. in Kombination mit PIC oder BSO erzeugt werden. Es besteht die Annahme, dass die Bioaktivierung von Diclofenac zu 5-OH-Dcl oder Dcl-Acylglucuronid (AG) sowie die folgende Bildung kovalenter Proteinaddukte zur Entwicklung von Lebertoxizit{\"a}t beitr{\"a}gt. Mittels LC-MS/MS-Messungen konnten wir jedoch nachweisen, dass die Gabe von LPS + Dcl keine erh{\"o}hte Bildung reaktiver Metaboliten oder Dcl-AG-abh{\"a}ngiger Proteinaddukte ausl{\"o}st. Im Einklang damit wurden Enzyme, die f{\"u}r die Bio-aktivierung von Dcl zu reaktiven Metaboliten verantwortlich sind (z.B. Cyp2C11, Cyp2C7 und UGT2B1), sowie die MRP-Effluxtransporter der Leber durch die Co-Behandlung mit LPS in ihrer Genexpression gehemmt. Zus{\"a}tzliche qRT-PCR-Analysen Nrf2-abh{\"a}ngiger Gene, als Sensor f{\"u}r elektrophilen oder oxidativen Stress, zeigten keine Hochregulation zytoprotektiver Faktoren und unterst{\"u}tzen die Schlussfolgerung, dass Arzneistoff-unabh{\"a}ngige Stress-faktoren keine erh{\"o}hte Bildung toxischer Dcl-Metaboliten ausl{\"o}sen. Schließlich ergaben unsere Analysen, dass eine Aktivierung co-stimulatorischer NFκB- und MAPK-Signalwege mit Hochregulation co-stimulatorischer Faktoren (z.B. IL-1β, TNF-α, CINC-1, iNOS) und Akkumulation neutrophiler Granulozyten in der Leber sowohl durch Behandlung mit LPS + Dcl als auch mit PIC + Dcl induziert wurde. Nur die Kombination von LPS und Diclofenac bewirkte jedoch dar{\"u}ber hinaus eine massive Freisetzung pro-inflammatorischer Zytokine, Chemokine sowie toxizit{\"a}tsf{\"o}rdernder Alarmsignale (z.B. IL-1β, TNF-α, CINC-1, HMGB1, LTB4) ins Plasma. Zus{\"a}tzlich waren sch{\"u}tzende negative Feed-back-Mechanismen, wie die Hitzeschockreaktion, in den mit LPS und Dcl behandelten Tieren gehemmt. Zusammenfassend zeigen unsere Ergebnisse, dass eine metabolische Aktivierung von Dcl bzw. eine Akkumulation reaktiver Dcl-Metaboliten an der Entwicklung idiosynkratischer Lebersch{\"a}digung nicht ausschlaggebend beteiligt ist. Im Gegensatz zu PIC oder BSO f{\"u}hrte in den verabreichten Dosen nur die Gabe von LPS als Stressfaktor zu einer Aktivierung co-stimulatorischer Signalwege sowie zu einer Hemmung protektiver Systeme, wodurch die lebersch{\"a}digende Wirkung von Dcl potenziert wurde.}, subject = {Leber}, language = {de} } @article{FoertschHuppMaetal.2011, author = {F{\"o}rtsch, Christina and Hupp, Sabrina and Ma, Jiangtao and Mitchell, Timothy J. and Maier, Elke and Benz, Roland and Iliev, Asparouh I.}, title = {Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69084}, year = {2011}, abstract = {Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin's pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20-40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin's lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.}, subject = {Toxikologie}, language = {en} } @phdthesis{Mueller2012, author = {M{\"u}ller, Stephanie}, title = {Identification of early molecular changes associated with Fumonisin B1-induced carcinogenesis in vivo and in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71336}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Fumonisin B1 (FB1) is a mycotoxin produced by various Fusarium species and constitutes a major contaminant of maize worldwide. A 2-year carcinogenicity study of the National Toxicology Program (NTP) in Fischer N344 rats showed that male rats were most susceptible to FB1-induced tumor formation in the kidney. Histopathologically, a rare and highly malignant tumor type originating from the proximal tubules of rat kidney with increased potential for invasion and metastasis was identified. However, mechanisms underlying the FB1-induced carcinogenesis in kidneys of male rats are still not clear. Previous studies have shown that FB1-mediated disruption of sphingolipid metabolism via inhibition of ceramide synthase is a primary key event in FB1 toxicity. The disruption of sphingolipid metabolism may cause time- and dose-related changes in the relative balance of various bioactive intermediates. Furthermore, the ability of FB1 to induce renal cell death and subsequent compensatory cell proliferation is well known, but it does not completely explain the invasive growth characteristics and exceptionally high metastatic potential of FB1-induced tumors. Considering the complexity of sphingolipid metabolism and the fact that various sphingolipids (e.g. ceramide, sphingoid bases and their respective 1-phosphates) act on opposing signaling pathways, it is hypothesized that the balance between individual sphingolipids and thus the overall cellular response to FB1 may shift with time and by continuing FB1 exposure, resulting in the disruption of specific cell signaling pathways, which may promote tumor formation in kidney. To identify early FB1-induced gene expression patterns in the kidney, which may be associated with sphingolipid-mediated signaling pathways in cancer, a short-term i.p. study on FB1 in male Sprague Dawley rats was performed and changes in gene expression were analyzed using a qRT-PCR array that comprises 84 relevant genes of 6 pathways pivotally involved in the formation of cancer. Furthermore, apoptosis and cell proliferation as well as changes in specific sphingolipids were investigated in FB1-treated kidneys. As shown by classical histopathology (H\&E) and (immuno)-histochemical staining (TUNEL and BrdU), FB1 caused a time- and dose-dependent increase in tubular apoptosis in the cortex and OSOM of the kidney, which was compensated by the induction of proliferation in the affected areas. HPLC-MS/MS analysis of bioactive sphingolipids demonstrated that FB1 induced a marked elevation of the pro-apoptotic sphingoid bases sphinganine and sphingosine, which paralleled the time- and dose-dependent increase in renal tubular apoptosis. With prolonged exposure to FB1, increased metabolic conversion of the accumulated sphinganine to the sphinganine-1-phosphate, a second messenger with anti-apoptotic and proliferative properties, was observed in kidney. This finding was compliant with the increased regenerative cell proliferation in the cortex and OSOM. In addition to effects on sphingoid bases and their 1-phosphate metabolites, this study, for the first time, demonstrated reduced levels of specific ceramides in rat kidney after FB1 exposure. In particular, C16-ceramide, which is a widespread constituent of membrane-bound complex sphingolipids involved in cell adhesion, was time- and dose-dependently decreased after treatment with FB1. Besides its role as component of the cell membrane, C16-ceramide functions as a signaling molecule for the initiation of apoptosis in response to various stress stimuli. Under conditions of chronic FB1 exposure, a significant reduction in pro-apoptotic C16-ceramide together with markedly increased levels of anti-apoptotic and proliferation-promoting sphingoid base 1-phosphates may thus favor resistance to stress-induced apoptosis and facilitate the survival of abnormal cells with potential to initiate tumor formation. Our study also revealed that early exposure to FB1 resulted in increased expression of a plethora of genes involved in tumor initiation as well as tumor progression. While single FB1 exposure was demonstrated to predominately induce gene expression of proto-oncogenic transcription factors (e.g. Fos, Jun, Myc) and apoptotis-related genes (e.g. members of the tumor-necrosis factor family), repeated exposure resulted in marked upregulation of genes mediating cell survival and cell proliferation (e.g. Bcl-XL, Bcl-2, Nfκb1 and Egfr). Moreover, continued exposure to FB1 initiated increased expression of genes critically involved in tumor migration, adhesion, invasion and metastasis. A close correlation was established between gene expression changes in response to FB1 and known signaling pathways mediated by extracellular or intracellular action of sphingoid base 1-phosphates - bioactive lipids that were markedly increased after FB1 treatment. In particular, genes encoding components of the plasminogen activator system were abundantly upregulated. These mediate invasion and metastasis in response to So1P, and may hence particularly promote the formation of highly aggressive and invasive tumors in kidney as observed after chronic exposure to FB1. Thus, it is conceivable that upregulation of a majority of genes in response to FB1 may be a direct or indirect consequence of increased So1P signaling. Another aim of this study was to identify differences in the organ-specific susceptibility for tumor formation by comparing FB1-mediated effects on apoptosis, cell proliferation, sphingolipids, and selected cancer-related genes in kidney and liver. Collectively, the present results revealed that kidney and liver showed marked differences in several endpoints of FB1 toxicity, which seemed to be primarily associated with their different susceptibility to FB1-mediated alterations in sphingolipid metabolism. The strong correlation between histopathological lesions and alterations in sphingolipid metabolism as well as sphingoid base 1-phosphate accumulation and concomitant S1P receptor expression suggested that tumor formation and progression to highly malignant carcinomas seems to be rather favored in kidney compared to liver. However, genes mostly deregulated by FB1 treatment in kidney (PAI-1, Thbs1 and Itga2) were also found to be induced in liver. To verify FB1-induced gene expression in kidney, normal rat tubular epithelial (NRK-52E) cells were analyzed for FB1-induced expression changes of the same cancer-related genes as in vivo. The results of qRT-PCR analysis revealed that gene expression changes in NRK-52E cells after FB1 treatment strongly correlated with those found in rat kidney and paralleled the marked alterations in sphingolipid metabolism. Furthermore, a good correlation between FB1-induced expression changes of cancer-related genes obtained in vivo and in vitro and those known to be mediated by bioactive sphingoid base 1-phosphates in cancer was established. Moreover, experiments modeling the invasive behavior of NRK-52E cells showed that FB1 may enhance cell invasion, which also correlated with both the increase in invasion- and metastasis-associated genes and bioactive sphingoid base 1-phophates. Importantly, NRK-52E cells basally expressed the S1P receptors S1P2 and S1P3, which are known to be involved in tumor migration and invasion. Since these receptors were also identified as most abundant S1PRs in kidneys of male Sprague Dawley rats, they may present important mediators of gene expression and invasion in response to FB1 in vivo. In summary, FB1-mediated disruption of sphingolipid metabolism and subsequent time- and dose-related increase in intermediates, such as bioactive sphingoid base 1-phosphates, correlate with early changes in genes and signaling pathways that may mediate loss of growth control, replication, evasion of apoptosis, cell motility and invasion, and thus favor renal tumor formation in response to FB1. However, to clarify whether the obtained gene expression changes in cancer-related genes in kidney are specific to the biological action of sphingoid base 1-phosphates and their respective receptors, further mechanistic studies are necessary.}, subject = {Nephrotoxizit{\"a}t}, language = {en} } @article{StopperJonesZimmermann1987, author = {Stopper, Helga and Jones, H. and Zimmermann, U.}, title = {Large scale transfection of mouse L-cells by electropermeabilization}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63497}, year = {1987}, abstract = {Mouse L-cells were transfected by electropenneabilization using the selectable plasmid pSV2-neo which confers resistance to G-418 (Geneticin). 1be DNA concentration used was 1 l'gfml, the field strength was 10 kV fcm, the duration of the pulse was S ~s. Transfeetion yield was optimal at a temperature of 4°C when using a time in between consecutive pulses of 1 minute compared to shorter (of the order of seoonds) or Ionger (3 minutes) time intervals. A more detailed study of the relationship between the number of pulses applied (up to 10) and transfection yield showed it to be almost linear in this range at 4 o C. The yield of transfectants in response to 10 pulses was up to 1000 per 106 cells (using 3.3 pg DNA per cell). The inßuence of the growth phase of the cells on the transfection yield and I or the subpopulation of the mouse L--ceU line used was shown. Furthennore the clone yield depended on the DNA per ceU ratio within a very small range.}, subject = {Toxikologie}, language = {en} } @article{HollerFischerWeberetal.1987, author = {Holler, E. and Fischer, H. and Weber, C. and Stopper, Helga and Steger, H. and Simek, H.}, title = {A DNA polymerase with unusual properties from the slime mold Physarum polycephalum}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63501}, year = {1987}, abstract = {Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase cx on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzymewas 30-40\% pure and form HS2 enzyme 60\% with regard toprotein contents of the preparations. Form HS2 enzymewas generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptidein a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as weil as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase ß by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.}, subject = {Toxikologie}, language = {en} } @article{ZimmermannStopper1987, author = {Zimmermann, U. and Stopper, Helga}, title = {Elektrofusion und Elektropermeabilisierung von Zellen : Eine neuartige Methode der Biotechnologie zur gezieltenVer{\"a}nderung der genetischen Eigenschaften von Zellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63514}, year = {1987}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @phdthesis{Baetz2012, author = {B{\"a}tz, Julia}, title = {FRET-basierte Untersuchungen zur ligandenselektiven Beeinflussung der Rezeptorkonformation durch orthosterische und allosterische Liganden am Beispiel des muskarinischen M2 Acetylcholinrezeptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72836}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Zahlreiche experimentelle Befunde lassen vermuten, dass G-Protein gekoppelte Rezeptoren (GPCR) nach ihrer Aktivierung einer ligandenselektiven {\"A}nderung der Rezeptorkonformation unterliegen. Ziel der vorliegenden Arbeit war es dieses Ph{\"a}nomen am Subtyp 2 der muskarinischen Acetylcholinrezeptoren (M2 AChR) zu untersuchen. Muskarinische Acetylcholinrezeptoren (mAChR) k{\"o}nnen in f{\"u}nf Subtypen (M1-M5) unterschieden werden. Durch die Beteiligung der mAChR an zahlreichen physiologischen Prozessen stellen sie wichtige Zielstrukturen pharmakologischer Therapien dar. Da die orthosterische Ligandenbindestelle (= Bindestelle des endogenen Liganden) in allen f{\"u}nf Subtypen hoch konserviert ist, wird ihr pharmakologischer Nutzen derzeit allerdings durch die unselektive Rezeptormodulation und dem damit verbundenen Auftreten unerw{\"u}nschter Arzneimittelwirkungen stark limitiert. Ein Ansatz zur Erzielung subtypselektiver Effekte besteht in der Verwendung allosterischer Modulatoren. Da die allosterische Bindestelle der mAChR eine geringere Sequenzhomologie aufweist, k{\"o}nnen so gezielt einzelne Subtypen der mAChR reguliert werden. Der M2 AChR stellt hinsichtlich allosterischer Modulation ein gut charakterisiertes Modellsystem dar. F{\"u}r ihn wurde bereits eine Vielzahl allosterischer Liganden entwickelt. Auch bitopische Liganden, die sowohl einen allosterischen als auch einen orthosterischen Anteil enthalten, wurden f{\"u}r den M2 AChR bereits beschrieben. Im ersten Teil der vorliegenden Arbeit wurden verschiedene FRET-Sensoren des M2 AChR generiert und charakterisiert. Als FRET-Paar wurden das cyan fluoreszierende Protein (CFP) und der niedermolekulare fluorescein-basierte Fluorophor FlAsH (fluorescein arsenical hairpin binder) gew{\"a}hlt. CFP wurde in den Sensoren am Ende des C-Terminus angef{\"u}gt. Die zur Markierung mit FlAsH n{\"o}tige Tetracysteinsequenz wurde in verschiedenen Bereichen der dritten intrazellul{\"a}ren Rezeptorschleife (IL) eingebracht. Die auf diese Weise erstellten Re-zeptorsensoren trugen das Tetracysteinmotiv in der N terminalen (M2i3-N) bzw. in der C terminalen Region (M2i3-C) von IL 3. Die Charakterisierung der Rezeptorsensoren bez{\"u}glich Ligandenbindung, Gi-Protein Aktivierung und β-Arrestin2 Translokation ergab keine signifikanten Unterschiede zwischen M2i3-N, M2i3 C und M2CFP oder Wildtyp M2 AChR. Zun{\"a}chst wurden sowohl unterschiedliche orthosterische, als auch allosterische Liganden hinsichtlich ihrer mittleren effektiven Konzentration und ihrer maximalen Wirkst{\"a}rke an den Rezeptorsensoren untersucht. Mit Hilfe von FRET-Messungen konnte ein superago-nistisches Verhalten des orthosterischen Testliganden Iperoxo gezeigt werden. Die Eigenschaften der allosterischen Substanzen wurden durch Messung der Rezeptordeakti-vierungskinetik und des maximalen Hemmeffekts auf einen vorstimulierten Rezeptor charakterisiert. Alle allosterischen Liganden deaktivierten den vorstimulierten M2 AChR mit einer schnelleren Kinetik als Atropin. Die EC50-Werte der unterschiedlichen Substanzen waren unabh{\"a}ngig von der Markierungsposition im verwendeten Rezeptorsensor vergleich-bar. Ausnahmen bildeten die allosterischen Liganden JK 289, JK 338, ½ W84 und EHW 477, die liganden- und sensorabh{\"a}ngig unterschiedliche mittlere effektive Konzentrationen aufwie-sen. Bei der Untersuchung der Konformations{\"a}nderung des M2 AChR konnte kein liganden-selektiver Unterschied zwischen den FRET-Signalen f{\"u}r 19 getestete orthosterische Liganden beobachtet werden. Dies deutet darauf hin, dass alle orthosterischen Testliganden eine dem Acetylcholin (ACh) vergleichbare {\"A}nderung der M2 AChR Konformation induzier-ten. Um zu untersuchen, ob f{\"u}r die orthosterischen Testliganden eine Korrelation zwischen ihrer maximalen Wirkst{\"a}rke hinsichtlich Rezeptoraktivierung in FRET-Experimenten und der Aktivierung nachgeschalteter Signalwege besteht, wurde die orthosterisch-vermittelte Translokation von β-Arrestin2 mit Hilfe der Konfokalmikroskopie bestimmt. Bis auf 5-Methyl-furmethiodid translozierten alle orthosterischen Liganden β-Arrestin2 in einem Ausmaß, das mit der maximalen Rezeptoraktivierung vergleichbar war. Dagegen rief 5 Methylfurmethiodid verglichen mit dem endogenen Liganden ACh zwar eine ca. halbmaximale Rezeptorakti-vierung, aber nur eine {\"a}ußerst geringe β-Arrestin2 Translokation hervor. Im zweiten Teil der Arbeit wurde der Einfluss verschiedener Allostere auf eine ligandenselektive Konformations{\"a}nderung des M2 AChR untersucht. Die allosterischen Liganden JK 337 und Seminaph beeinflussten den M2i3-C Sensor signifikant st{\"a}rker, als das M2i3-N Konstrukt. Dagegen zeigte EHW 477 eine st{\"a}rkere Beeinflussung der Rezeptorkon-formation des M2i3-N-, als des M2i3-C Sensors. Dies erlaubt die Vermutung, dass JK 337 und Seminaph eine st{\"a}rkere Bewegung unterhalb von Transmembrandom{\"a}ne (TM) 6, als unterhalb von TM 5 hervorriefen. Die Ergebnisse f{\"u}r EHW 477 legen nahe, dass TM 5 eine gr{\"o}ßere Bewegung eingeht, als TM 6. FRET-basierte Untersuchungen der Einfl{\"u}sse der allosterischen Testliganden auf nachgeschaltete Signalwege ergaben, dass sowohl die Akti-vierung des Gi Proteins, als auch die β-Arrestin2 Translokation selektiv durch einzelne allosterische Liganden beeinflusst werden. Auch ein Zusammenhang zwischen Rezeptor-aktivierung und der Regulation nachgeschalteter Signalwege war erkennbar. Allerdings waren auf Grund der Versuchsbedingungen keine quantitativen Aussagen m{\"o}glich. Im Folgenden wurden die bitopischen Liganden Hybrid 1 und 2 (H 1, H 2) hinsichtlich ihres Effekts auf die Konformations{\"a}nderung des M2 AChR untersucht. W{\"a}hrend eine Stimulation mit H 1 vergleichbare FRET-Signale an beiden Sensoren ergab, konnte mit H 2 weder am M2i3-N-, noch an M2i3-C Sensor eine FRET-{\"A}nderung detektiert werden. Um den mangeln-den Effekt der Hybridsubstanzen in FRET-mikroskopischen Untersuchungen aufzukl{\"a}ren, wurden verschiedene Ans{\"a}tze gew{\"a}hlt: Mit kettenverl{\"a}ngerten Derivaten der Hybridsubstanzen konnte in FRET-Messungen eine {\"A}nderung des FRET-Signals detektiert werden. Die Entfernung des allosterischen Bausteins f{\"u}hrte in FRET-Experimenten zu einer verglichen mit dem endogenen Liganden ACh etwa halbmaximalen Aktivierung beider Sensoren. Dagegen resultierte die Mutation der alloste-rischen Bindestelle in nachfolgenden FRET-Untersuchungen mit H 1 und H 2 in keiner Signal{\"a}nderung des FRET-Ratio. Diese Beobachtungen f{\"u}hrten zu der Annahme, dass die Linkerkette, die orthosterischen und allosterischen Baustein der Hybride miteinander verbindet, zu kurz war um eine gleichzeitige Bindung an die allosterische und orthosterische Bindestelle zu erm{\"o}glichen. Ein anderer Erkl{\"a}rungsansatz besteht darin, dass nach Bindung des Orthosters der Kanal zwischen orthosterischer und allosterischer Bindestelle durch die Konformations{\"a}nderung des Rezeptors verschlossen wird, weshalb keine dauerhafte, dualsterische Bindung der Hybridsubstanzen an den M2 AChR m{\"o}glich ist. Im Rahmen der vorliegenden Arbeit ist es gelungen mittels FRET-Experimenten die Existenz einer ligandenselektiven Rezeptorkonformation des M2 AChR mit allosterischen Liganden nachzuweisen. Dar{\"u}ber hinaus konnte auch ein Bezug zum Auftreten einer funktionellen Selektivit{\"a}t mit allosterischen Liganden hergestellt werden. Die Untersuchung von 19 orthosterischen Liganden hinsichtlich ihres Einflusses auf die Rezeptorkonformation des M2 AChR ergab keinen Hinweis auf eine ligandenselektive Konformations{\"a}nderung. Die Betrachtung der orthosterisch-vermittelten Translokation von β-Arrestin2 zeigte, dass zwischen der Effizienz der orthosterischen Testliganden, den M2 AChR zu aktivieren und dem Ausmaß, in dem sie eine β Arrestin2 Translokation induzierten eine direkte Korrelation besteht. Lediglich 5-Methylfurmethiodid rief eine ungleich geringere β-Arrestin2 Translokation hervor, verglichen mit dem Ausmaß an Rezeptoraktivierung. Diese Beobachtung deutet auf die Existenz eines signaling-bias f{\"u}r diesen Liganden hin. Die Untersuchung der dualsterischen Liganden H 1 und 2 bez{\"u}glich ihrer F{\"a}higkeit zur Rezeptoraktivierung ergab, dass erst durch eine Verl{\"a}ngerung der Linkerkette, durch die orthosterischer und alloste-rischer Baustein miteinander verbunden sind eine Konformations{\"a}nderung des M2 AChR hin zu einer aktiven Konformation erreicht werden kann. Es kann somit angenommen werden, dass in den urspr{\"u}nglichen Hybridsubstanzen H 1 und H 2 eine zu kurze Linkerkette, durch die keine dualsterische Bindung der Hybride an die allosterische und orthosterische Bindestelle m{\"o}glich ist, urs{\"a}chlich f{\"u}r die mangelnde Rezeptoraktivierung des M2 AChR war.}, subject = {Muscarinrezeptor}, language = {de} } @phdthesis{Glaser2012, author = {Glaser, Nina}, title = {Influence of natural food compounds on DNA stability}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72872}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Cancer is one of the leading causes of death all over the world. Malnutrition and toxic contaminations of food with substances such as mycotoxins have been thought to account for a high percentage of cancers. However, human diet can deliver both mutagens and components that decrease the cancer risk. Genomic damage could be reduced by food components through different mechanisms such as scavenging of reactive oxygen species. In the first part of this study we tried to investigate the effects of patulin and resveratrol on DNA stability in V79 cells. Patulin is a mycotoxin, which is frequently found in spoiled apples and other fruits. The WHO has established a safety level of 50 µg/L, which is indeed not observed by all manufacturers. The acute toxicity of patulin in high concentrations is well known, however its potential carcinogenicity is still a matter of debate. Therefore we wanted to investigate further steps in the mechanism of patulin-induced genotoxicity. Patulin caused the formation of micronuclei and nucleoplasmic bridges in a dose-dependent manner. Further analysis revealed that patulin induced both kinetochore-negative and positive micronuclei. Time course of incubation indicate a new mechanism for patulin-induced nucleoplasmic bridge formation. We hypothized a mechanism via cross-linking of DNA, which was confirmed by a modified version of comet assay. Incubations of cells with patulin led to an increased number of multinucleated cells and multipolar mitoses. Cell cytometry revealed a G2 arrest by patulin, which might explain the amplification of centrosomes and patulin-induced aneuploidy. Patulin cause a dose-dependent DNA damage in comet assay which was influenced by the cellular GSH content. However, an induction of oxidative stress was just seen with higher concentrations of patulin. Levels of cellular glutathione were increased after 24 h incubation indicating an adaptive response to patulin-induced stress. There is growing interest in polyphenols such as resveratrol which have shown many positive effects on human health. The beneficial properties are partially attributed to their ability to scavenge reactive oxygen species. Co-incubation of V79 cells with patulin and 10 µM of the antioxidant resveratrol led to a slight reduction of micronucleus frequency compared to cells which were just treated with patulin. However, in higher concentrations resveratrol themselves caused the formation of micronuclei in V79 cells. Kinetochore analysis indicated only clastogenic properties for resveratrol but no disturbance of mitosis. The antioxidant properties of resveratrol were shown in ferric reducing antioxidant power (FRAP) assay. However, in cellular system resveratrol in higher concentrations revealed also prooxidative properties, as shown in 2,7-dichlordihydrofluorescein (DCF) assay. The increased level of glutathione after resveratrol treatment might reflect an adaptive response to resveratrol-induced oxidative stress. For the second part of this thesis we investigated the effects of an anthocyanin-rich grape extract on hypertensive Ren-2 rats. Ren-2 rats are an accepted genetically modified rat model for the investigation of hypertension and increased oxidative stress. We divided 23 female Ren-2 rats into three groups. One group was fed with an anthocyanin-rich Dacapo grape extract, one group was treated with the angiotensin converting enzyme (ACE) inhibitor ramipril and the third group was kept without medication during the experiment. After one week untreated group showed a clear increase in systolic and diastolic blood pressure compared to the ramipril treated rats. This was in part attenuated in the animals fed with anthocyanin-rich Dacapo grape extract. Effects on blood pressure were also reflected in an increased thirst of untreated and extract fed animals. Comet assay with cells of kidney and liver revealed a slight protective impact of Dacapo extract on DNA damage compared to the other groups. Similar results were obtained after evaluation of ɣ-H2AX-staining of kidney and heart sections. However, in the small intestine oppositional effects were seen, indicating an increased number of double strand breaks probably due to the high local concentration of polyphenols after oral ingestion. Antioxidative properties of the extract were shown in FRAP assay. However, this effect was not reflected in an increased antioxidative capacity in serum or a protective impact in the dihydroethidium (DHE) assay. The extract showed protective effects on DNA damage in comet assay and ɣ-H2AX-staining, but was not able to reduce hypertension back to the control level of ramipril treated animals. High local concentrations could also result in an increased damage of the affected tissue. Therefore, the administration of such concentrated compounds should be handled with care.}, subject = {Patulin}, language = {en} } @phdthesis{Wippel2012, author = {Wippel, Carolin}, title = {Alterations of brain dendrite and synapse structure by the Streptococcus pneumoniae neurotoxin pneumolysin - Insights and pharmacological modulation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72016}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Streptococcus pneumoniae (Pneumococcus) is one of the leading causes of childhood meningitis,pneumonia and sepsis. Despite the availability of childhood vaccination programs and antimicrobial agents, childhood pneumococcal meningitis is still a devastating illness with mortality rates among the highest of any cause of bacterial meningitis. Especially in low-income countries, where medical care is less accessible, mortality rates up to 50 \% have been reported. In surviving patients, neurological sequelae, including hearing loss, focal neurological deficits and cognitive impairment, is reported in 30 to 50 \%. Growing resistance of pneumococci towards conventional antibiotics emphasize the need for effective therapies and development of effective vaccines against Streptococcus pneumoniae. One major virulence factor of Streptococcus pneumoniae is the protein toxin Pneumolysin (PLY). PLY belongs to a family of structurally related toxins, the so-called cholesterol-dependent cytolysins (CDCs). Pneumolysin is produced by almost all clinical isolates of the bacterium. It is expressed during the late log phase of bacterial growth and gets released mainly through spontaneous autolysis of the bacterial cell. After binding to cholesterol in the host cell membranes, oligomerization of up to 50 toxin monomers and rearrangement of the protein structure, PLY forms large pores, leading to cell lysis in higher toxin concentrations. At sub-lytic concentrations, however, PLY mediates several other effects, such as activation of the classic complement pathway and the induction of apoptosis. First experiments with pneumococcal strains, deficient in pneumolysin, showed a reduced virulence of the organism, which emphasizes the contribution of this toxin to the course of bacterial meningitis and the urgent need for the understanding of the multiple mechanisms leading to invasive pneumococcal disease. The aim of this thesis was to shed light on the contribution of pneumolysin to the course of the disease as well as to the mental illness patients are suffering from after recovery from pneumococcal meningitis. Therefore, we firstly investigated the effects of sub-lytic pneumolysin concentrations onto primary mouse neurons, transfected with a GFP construct and imaged with the help of laser scanning confocal microscopy. We discovered two major morphological changes in the dendrites of primary mouse neurons: The formation of focal swellings along the dendrites (so-called varicosities) and the reduction of dendritic spines. To study these effects in a more complex system, closer to the in vivo situation, we established a reproducible method for acute brain slice culturing. With the help of this culturing method, we were able to discover the same morphological changes in dendrites upon challenge with sub-lytic concentrations of pneumolysin. We were able to reverse the seen alterations in dendritic structure with the help of two antagonists of the NMDA receptor, connecting the toxin´s mode of action to a non-physiological stimulation of this subtype of glutamate receptors. The loss of dendritic spines (representing the postsynapse) in our brain slice model could be verified with the help of brain slices from adult mice, suffering from pneumococcal meningitis. By immunohistochemical staining with an antibody against synapsin I, serving as a presynaptic marker, we were able to identify a reduction of synapsin I in the cortex of mice, infected with a pneumococcal strain which is capable of producing pneumolysin. The reduction of synapsin I was higher in these brain slices compared to mice infected with a pneumococcal strain which is not capable of producing pneumolysin, illustrating a clear role for the toxin in the reduction of dendritic spines. The fact that the seen effects weren´t abolished under calcium free conditions clarifies that not only the influx of calcium through the pneumolysin-pore is responsible for the alterations. These findings were further supported by calcium imaging experiments, where an inhibitor of the NMDA receptor was capable of delaying the time point, when the maximum of calcium influx upon PLY challenge was reached. Additionally, we were able to observe the dendritic beadings with the help of immunohistochemistry with an antibody against MAP2, a neuron-specific cytoskeletal protein. These observations also connect pneumolysin´s mode of action to excitotoxicity, as several studies mention the aggregation of MAP2 in dendritic beadings in response to excitotoxic stimuli. All in all, this is the first study connecting pneumolysin to excitotoxic events, which might be a novel chance to tie in other options of treatment for patients suffering from pneumococcal meningitis.}, subject = {Nervenzelle}, language = {en} } @article{StopperMetzler1991, author = {Stopper, Helga and Metzler, M.}, title = {Carcinogenic oestrogens induce respiration deficiency mutation in yeast}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63466}, year = {1991}, abstract = {In addition to hormonal activity, genetic darnage has been proposed as an important factor in oestrogen-mediated carcinogenesis. However, as short-term tests for oestrogens usually fail to show DNA mutations, lesions other than dassie nuclear DNA mutation have to be considered. Oestrogeninduced mitochondrial darnage was studied in the yeast Saccharomyces cerevisiae. Stilbene-type, but not steroidal, oestrogens were found to induce respiration-dcficient petite mutation. The effect was inversely correlated with cytotoxicity and required aromatic hydroxyl groups at the stilbene molecule. It only occurred under growth conditions and apparently was not due to the A TPase inhibitory qualities of stilbene oestrogens. Other studies have shown that petite mutation clones, which can be induced by a variety of substances, contain altered mitochondrial DNA. The mechanism of petite mutation induction might be important in tumorigenesis by also acting on nuclear DNA or facilitating carcinogenesis by disturbance of mitochondrial function.}, subject = {Toxikologie}, language = {en} } @phdthesis{Deiss2012, author = {Deiß, Katharina}, title = {Die Regulation des Kinasemodulators Raf Kinase Inhibitor Protein (RKIP): Einfluss von Phosphorylierung und Dimerisierung auf die Interaktion mit Raf1 und G Protein-gekoppelter Rezeptorkinase 2 (GRK2)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73884}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {RKIP reguliert Proteinkinasen der Signaltransduktionskaskaden von G Protein-gekoppelten Rezeptoren, der Raf/MEK/ERK-MAPK, des Transkriptionsfaktors NFκB und von GSK3β. Unklar war bisher, wie die spezifische Interaktion von RKIP mit seinen mannigfaltigen Interaktionspartnern erm{\"o}glicht und reguliert wird. Raf1 und GRK2 sind die einzigen bekannten direkten Interaktionspartner von RKIP und wurden deshalb gew{\"a}hlt, um die zugrundeliegenden molekularen Mechanismen dieser Interaktion genauer zu untersuchen. In dieser Arbeit wurde gezeigt, dass RKIP nach PKC-vermittelter Phosphorylierung von Serin153 dimerisiert und dass diese Dimerisierung f{\"u}r die RKIP/Raf1-Dissoziation und die RKIP/GRK2-Interaktion essentiell ist. Co-Immunpr{\"a}zipitationsexperimente mit einer phosphorylierungsdefizienten Mutante zeigten, dass f{\"u}r diese Dimerisierung die Phosphorylierung von beiden RKIP-Molek{\"u}len notwendig ist. Als Dimerinteraktionsfl{\"a}che wurden die Aminos{\"a}uren 127-146 von RKIP identifiziert, da das Peptid RKIP127-146 die Dimerisierung von RKIP spezifisch und effizient hemmte. Um die Bedeutung dieser phosphorylierungsinduzierten Dimerisierung von RKIP f{\"u}r seine Interaktion mit Raf1 und GRK2 zu untersuchen, wurden eine phosphomimetische Mutante (RKIPSK153/7EE) und eine Mutante von RKIP generiert, welche bereits unphosphoryliert dimerisiert (RKIP∆143-6). Folgende Ergebnisse legen nahe, dass die Dimerisierung von RKIP f{\"u}r die spezifische Interaktion mit Raf1 bzw. GRK2 entscheidend ist: (i) Die Dimerisierung von phosphoryliertem RKIP ging mit der Dissoziation von RKIP und Raf1 und der Assoziation von RKIP und GRK2 einher; (ii) die Mutanten RKIPSK153/7EE und RKIP∆143-6, die bereits in unstimulierten Zellen eine starke Dimerisierung zeigten, hatten eine h{\"o}here Affinit{\"a}t zu GRK2 als zu Raf1; (iii) die Hemmung der RKIP-Dimerisierung interferierte nur mit der RKIP/GKR2- aber nicht mit der RKIP/Raf1-Interaktion; (iv) in vitro und in Mausherzen konnte ein RKIP- und GRK2-immunreaktiver Komplex nachgewiesen werden; (v) Untersuchungen zur RKIP-vermittelten Hemmung der Kinaseaktivit{\"a}t von GRK2 und Raf implizierten, dass dimerisiertes RKIP nur die Aktivit{\"a}t von GRK2, nicht aber von Raf hemmt. Diese Arbeit zeigt, dass die phosphorylierungsinduzierte Dimerisierung von RKIP die spezifische Interaktion von RKIP mit Raf1 und GRK2 koordiniert. Die Aufkl{\"a}rung dieses Mechanismus erweitert unser Verst{\"a}ndnis der spezifischen Interaktion von Kinasen mit ihren Regulatorproteinen.}, subject = {Signaltransduktion}, language = {de} } @article{SchuppHeidlandStopper2010, author = {Schupp, Nicole and Heidland, August and Stopper, Helga}, title = {Genomic Damage in Endstage Renal Disease - Contribution of Uremic Toxins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68653}, year = {2010}, abstract = {Patients with end-stage renal disease (ESRD), whether on conservative, peritoneal or hemodialysis therapy, have elevated genomic damage in peripheral blood lymphocytes and an increased cancer incidence, especially of the kidney. The damage is possibly due to accumulation of uremic toxins like advanced glycation endproducts or homocysteine. However, other endogenous substances with genotoxic properties, which are increased in ESRD, could be involved, such as the blood pressure regulating hormones angiotensin II and aldosterone or the inflammatory cytokine TNF-. This review provides an overview of genomic damage observed in ESRD patients, focuses on possible underlying causes and shows modulations of the damage by modern dialysis strategies and vitamin upplementation.}, subject = {Toxin}, language = {en} } @phdthesis{Bieber2010, author = {Bieber, Daniela}, title = {Der A2B-Adenosinrezeptor und MAP-Kinase Aktivit{\"a}t in MDA-MB-231 Brustkrebszellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65707}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Sowohl MAPK als auch Adenosin werden mit Tumorproliferation und Angiogenese in Verbindung gebracht. MDA-MB-231 {\"O}strogenrezeptor-negative Brustkrebszellen zeigen eine sehr starke Expression des A2BAR, der außerdem der einzige von dieser Zelllinie exprimierte Adenosinrezeptor ist. Es konnte gezeigt werden, dass MDA-MB-231-Brustkrebszellen eine hohe basale MAPK-Aktivit{\"a}t aufweisen, welche durch Stimulation mit FCS nicht weiter gesteigert werden kann. Diese hohe basale MAPK-Aktivit{\"a}t wird durch die src-Kinase und Her2 verursacht, da eine Inhibition dieser beiden Tyrosinkinasen eine Hemmung der basalen ERK-Phosphorylierung induziert. Interessanterweise f{\"u}hrt die Stimulation des A2BAR der MDA-MB-231-Brustkrebszellen mit dem unselektiven Agonisten NECA zu einer zeitanh{\"a}ngigen Inhibition der ERK-1/2-Phosphorylierung. Eine Behandlung der Brustkrebszelllinie mit 10 µM CGS 21680 zeigten keinen Einfluss auf die ERK-Aktivit{\"a}t, weshalb davon ausgegangen werden kann, dass die zeitabh{\"a}ngige Inhibition der ERK-1/2-Phosphorylierung durch den A2BAR vermittelt wird. Eine Beteiligung von cAMP an der MAPK-Signaltransduktion des A2BAR scheint insofern wahrscheinlich, als sowohl eine Behandlung der Zellen mit Forskolin als auch der Kombination aus cAMP-AM und dem PDE4-Inhibitor Rolipram eine zeitabh{\"a}ngige Hemmung der ERK-1/2-Phosphorylierung induzieren. Jedoch scheint weder die PKA noch die PI3K an dieser Signaltransduktion des A2BAR beteiligt zu sein, da die A2BAR-vermittelte Inhibition der MAPK auch in Anwesenheit von PKA- und PI3K-Inhibitoren bestehen bleibt. Auch scheinen cAMP-GEFs wie beispielsweise Epac in diesem Zusammenhang keine Rolle zu spielen. In Gegenwart des PLC-Inhibitors U-73122 und des Ca2+-Chelators BAPTA verschwand die NECA-induzierte Hemmung der ERK-1/2-Phosphorylierung, was f{\"u}r eine Beteiligung der PLC und des Ca2+ an der A2BAR-vermittelten Hemmung der MAPK-Aktivit{\"a}t spricht. Letzten Endes konnte jedoch kein Mechanismus eruiert werden, welcher diese A2BAR-vermittelte, Ca2+-abh{\"a}ngige MAPK-Hemmung mediiert, da weder eine Inhibition der PKC, der CamKII oder des Calcineurins Einfluss auf die NECA-induzierte MAPK-Hemmung hatten. Was Wachstum und Proliferation der {\"O}strogenrezeptor-negativen Brustkrebszelllinie MDA-MB-231 anbelangt, so konnte gezeigt werden, dass der unselektive Agonist NECA zu einer signifikanten Wachstumshemmung dieser Brustkrebszelllinie f{\"u}hrt. Allerdings kommt es aufgrund einer Desensitisierung der A2BAR in MDA-MB-231-Brustkrebszellen lediglich zu einem transienten proliferationshemmenden Effekt nach Stimulation mit NECA.}, subject = {Adenosinrezeptor}, language = {de} } @article{FischerLutz1994, author = {Fischer, W. H. and Lutz, Werner K.}, title = {Short communication : Mouse skin papilloma formation by chronic dermal application of 7,12-dimethylbenz[a]anthracene is not reduced by diet restriction}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60644}, year = {1994}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{GrunickePyerinEisenbrandetal.1994, author = {Grunicke, H. and Pyerin, W. and Eisenbrand, G. and Havemann, K. and Rabes, H. M. and Molling, K. and Schwab, M. and Lutz, Werner K. and Wahrendorf, J. and Schirrmacher, V.}, title = {7th International Symposium of the Division of Experimental Cancer Research (AEK) of the German Cancer Society : [Meeting report]}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60651}, year = {1994}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{KirchnerStopperPappetal.1993, author = {Kirchner, S. and Stopper, Helga and Papp, T. and Eckert, I. and Yoo, H. J. and Vig, B. K. and Schiffmann, D.}, title = {Cytogenetic changes in primary, immortalized and malignant mammalian cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63439}, year = {1993}, abstract = {Some chromosomes in transformed rat cells and somatic cell hybrids fail to display the presence of kinetochore proteins as detected by antikinetochore antibodies. Suchchromosomes (K- Chromosomes) may constitute a novel mechanism for the genesis of aneuploidy. Wehave analyzed primary~ immortalized and malignant marnmalian cells for the presence of kinetochore proteins and micronuclei. Our resuJts suggest a correlation of the K- chromosome and micronucleus frequency with the variability in chromosome number. Upon in situ hybridization with the minor satellite and alpha satellite sequences some Kchromosomes showed a signal. This indicates that the observed lack of kinetocbores is not necessarily due to a lack of centromeric DNA. We conclude that dislocated K- chromosomes may become incorporated into micronuclei which are prone to loss. Such events would be associated with the generation of aneuploidy.}, subject = {Toxikologie}, language = {en} } @article{StopperPechanSchiffmann1992, author = {Stopper, Helga and Pechan, R. and Schiffmann, D.}, title = {5-azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63443}, year = {1992}, abstract = {lt is known that 5-azacytidine (5-AC) induces tumors in several organs of rats and mice. The mechanisms of these effects are still poorly understood although it is known that 5-AC can be incorporated into DNA. Furthermore, it can inhibit DNA methylation. The known data on its clastogenic andjor gene mutation-inducing potential are still controversial. Therefore, we have investigated the kinds of genotoxic effects caused by 5-AC in Syrian hamster embryo (SHE) fibroblasts. Three different endp6ints (micronucleus formation, unscheduled DNA synthesis (UDS) and cell transforrnation) were assayed under similar conditions of metabolism and dose at target in this cell system. 5-AC induces morphological transformation of SHE cells, but not UDS. Therefore, 5-AC does not seem to cause repairable DNA lesions. Furthermore, our studies revealed that 5-AC is a potent inducer of mkronuclei in the SHE system. Immunocytochemical analysis revealed that a certain percentage of these contain kinetochores indicating that 5-AC may induce both clastogenic events and numerical chromosome changes.}, subject = {Toxikologie}, language = {en} } @article{TasStopperKoscheletal.1991, author = {Tas, P. and Stopper, Helga and Koschel, K. and Schiffmann, D.}, title = {Influence of the carcinogenic oestrogen diethylstilboestrol on the intracellular calcium level in C6 rat glioma cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63459}, year = {1991}, abstract = {The ~fthetic oes~rog~n diethylsti~boestrol (DES) causes a dose-dependent elevation of the cytoplasuuc Ca concentratton m C6 rat ghoma cells. This Ca2+ rise is caused neither by Ca2+ influx nor ~-r release from the ~a2 + stores of the endoplasmic reticulum. Therefore it seems likely that DES mob!hzes Ca2+ from a nutochondrial source. The DES-induced Ca2+ signal is remarkably similar to the one mduced by the. tumou~ promotor ~hapsigargin. As this compound causes leakage of calcium from the endoplasmt~ rettculum tt ~ms posstble that DES induces a similar leakage from mitochondrial Ca2+ stores. It remaans to be estabhshed whether the DES-mediated rise in intracellular calcium is causally related to the tumour-promoting properties of this compound}, subject = {Toxikologie}, language = {en} } @article{EpeHarttigStopperetal.1990, author = {Epe, B. and Harttig, U. and Stopper, Helga and Metzler, M.}, title = {Covalent binding of reactive estrogen metabolites to microtubular protein as a possible mechanism of aneuploidy induction and neoplastic cell transformation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63478}, year = {1990}, abstract = {Neoplastic cell transfonnation induced by estrogens and some other carcinogen\& such as benzene appears to involve the induction of mitotic aneuploidy rather than DNA damage and point mutations. As metabolic activation may also play an important roJe in the mechanism of carcinogenesis of these nongenotoxic compounds, we have studied the Interaction of reactive quinone metabolites of various estrogens and of benzene with the major microtubular protein, tubulin, in a cell-free system. Covalent binding of the radioactively labeled metabolites to the a- and 13-subunit of tubulin was found to depend on the structure of the metabolite. When the adducted tubulins were tested in vitro for their ability to polymerize to microtubules, Inhibition of microtubule assembly was obsened in every case, although to varying extents. It is proposed that the fonnation of covalent tubulin adducts may impair the formation of mitotic spindies and thus contribute to chromosomal nondisjunction and aneuploidy induction.}, subject = {Toxikologie}, language = {en} } @article{StopperZimmermannNeil1988, author = {Stopper, Helga and Zimmermann, U. and Neil, G. A.}, title = {Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63488}, year = {1988}, abstract = {Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 p.s) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV jcm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma celllines for use in subsequent fusion experiments.}, subject = {Toxikologie}, language = {en} } @article{LutzPoetzschSchlatteretal.1991, author = {Lutz, Werner K. and Poetzsch, J. and Schlatter, J. and Schlatter, C.}, title = {The real role of risk assessment in cancer risk management}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60730}, year = {1991}, abstract = {Rtgulatory aclio11s Iaken to reduu tht risk of harmfultffects of exposure to chemieals ofltn arenot commensurDtt with the toxicologicDf risk SJsstS\&ment. A numbtr of factors relating to psychology, sociology, economics Dntl politics rather than science and medicine afftct tht final decision. Wemer Lutz and colleagues illustratt the situation using tht feuktmia-indudng chtmiCJJI benzene as an examplt.}, subject = {Toxikologie}, language = {en} } @phdthesis{Froelich2012, author = {Fr{\"o}lich, Nadine}, title = {Analyse der µ-Opiatrezeptoraktivierung und Signaltransduktion in lebenden Zellen mittels FRET-Mikroskopie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71009}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Der Fluoreszenz-Resonanz-Energie-Transfer ist ein Ph{\"a}nomen, welches erstmals 1948 von Theodor F{\"o}rster beschrieben wurde. Mit der Entwicklung von Fluoreszenzproteinen konnten in Kombination mit Mikroskopietechniken Einblicke in zellbiologische Vorg{\"a}nge gewonnen werden, die durch biochemische oder physiologische Experimente nicht m{\"o}glich sind. Dabei spielt die hohe zeitliche und r{\"a}umliche Aufl{\"o}sung eine wichtige Rolle. Auf dem Forschungsgebiet der GPCR, welche die gr{\"o}ßte Gruppe von Membranproteinen bei den S{\"a}ugetieren darstellen, wurden insbesondere Erkenntnisse {\"u}ber Konformations{\"a}nderungen der Rezeptoren, die Kinetik der Rezeptoraktivierung und die Interaktion mit intrazellul{\"a}ren Signalproteinen gewonnen. Der µ-Opioidrezeptor geh{\"o}rt zur Familie der GPCR und stellt aufgrund seiner analgetischen Wirkungen eine wichtige pharmakologische Zielstruktur dar. Das Ziel dieser Arbeit war sowohl den Rezeptor als auch seine Signalwege mittels FRET-Mikroskopie zu untersuchen. Zun{\"a}chst sollte ein intramolekularer FRET-Sensor des µ-Opioidrezeptors entwickelt werden, dazu wurden basierend auf den Kenntnissen {\"u}ber die Terti{\"a}rstruktur und dem Aufbau bereits bekannter GPCR-Sensoren verschiedene Rezeptorkonstrukte kloniert. Bei den Konstrukten wurden entweder zwei Fluoreszenzproteine oder ein Fluoreszenzprotein und ein Fluorophor-bindendes Tetracysteinmotiv kombiniert. Auch die Positionen der eingef{\"u}gten Sequenzen wurden in den intrazellul{\"a}ren Dom{\"a}nen variiert, da der Rezeptor auf die Modifikationen mit beeintr{\"a}chtigter Membranlokalisation reagierte. Durch die Optimierung wurden Rezeptoren konstruiert, die an der Zellmembran lokalisiert waren. Jedoch zeigte keines der Rezeptorkonstrukte Funktionalit{\"a}t im Hinblick auf die Rezeptoraktivierung. Im zweiten Teil wurden die pharmakologischen Effekte der Metabolite von Morphin am humanen µ-Opioidrezeptor systematisch analysiert. Dazu wurde die F{\"a}higkeit der Metabolite, Gi-Proteine zu aktivieren und β-Arrestin2 zu rekrutieren, mittels FRET-basierter Messungen an lebenden Zellen untersucht. Außerdem wurde die Affinit{\"a}t der Metabolite zum humanen µ Opioidrezeptor anhand der Verdr{\"a}ngung eines radioaktiven Liganden analysiert. Meine Experimente identifizierten eine Gruppe mit stark agonistischen und eine mit schwach agonistischen Eigenschaften. Die starken Partialagonisten aktivieren den Rezeptor bereits bei nanomolaren Konzentrationen, w{\"a}hrend die schwachen Metabolite den Rezeptor erst bei Konzentrationen im mikromolaren Bereich aktivieren. Die Metabolite Normorphin, Morphin-6-Glucuronid und 6-Acetylmorphin zeigen geringere Potenz als Morphin bei der Gi-Aktivierung aber {\"u}berraschenderweise h{\"o}here Potenz und Effizienz f{\"u}r die β-Arrestin-Rekrutierung. Dies deutet auf eine bevorzugte Aktivierung von β-Arrestin2 hin. Die aus diesen Studien gewonnenen Ergebnisse liefern Hinweise darauf, welche Metabolite bei der Signalverarbeitung am µ Opioidrezeptor in vivo beteiligt sind.}, subject = {Opiatrezeptor}, language = {de} } @phdthesis{Blankenburg2010, author = {Blankenburg, Robert}, title = {Longitudinale Untersuchungen der kardialen Morphologie von knockin-M{\"a}usen mit humanen Myosinmutationen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71417}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Longitudinale Untersuchungen der kardialen Morphologie von knockin-M{\"a}usen mit humanen Myosinmutationen}, subject = {Kardiomyopathie}, language = {de} } @phdthesis{Hupp2012, author = {Hupp, Sabrina}, title = {Modulation of Actin Dynamics by the Cholesterol-Dependent Cytolysin Pneumolysin - a novel mechanism beyond pore formation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70889}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Streptococcus pneumoniae is one of the major causes of bacterial meningitis, which mainly affects young infants in the developing countries of Africa, Asia (esp. India) and South America, and which has case fatality rates up to 50\% in those regions. Bacterial meningitis comprises an infection of the meninges and the sub-meningeal cortex tissue of the brain, whereat the presence of pneumolysin (PLY), a major virulence factor of the pneumococcus, is prerequisite for the development of a severe outcome of the infection and associated tissue damage (e. g. apoptosis, brain edema, and ischemia). Pneumolysin belongs to the family of pore forming, cholesterol-dependent cytolysins (CDCs), bacterial protein toxins, which basically use membrane-cholesterol as receptor and oligomerize to big aggregates, which induce cell lysis and cell death by disturbance of membrane integrity. Multiple recent studies, including this work, have revealed a new picture of pneumolysin, whose cell-related properties go far beyond membrane binding, pore formation and the induction of cell death and inflammatory responses. For a long time, it has been known that bacteria harm the tissues of their hosts in order to promote their own survival and proliferation. Many bacterial toxins aim to rather hijack cells than to kill them, by interacting with cellular components, such as the cytoskeleton or other endogenous proteins. This study was able to uncover a novel capacity of pneumolysin to interact with components of the actin machinery and to promote rapid, actin-dependent cell shape changes in primary astrocytes. The toxin was applied in disease-relevant concentrations, which were verified to be sub-lytic. These amounts of toxin induced a rapid actin cortex collapse in horizontal direction towards the cell core, whereat membrane integrity was preserved, indicating an actin severing function of pneumolysin, and being consistent with cell shrinkage, displacement, and blebbing observed in live cell imaging experiments. In contrast to neuroblastoma cells, in which pneumolysin led to cytoskeleton remodeling and simultaneously to activation of Rac1 and RhoA, in primary astrocytes the cell shape changes were seen to be primarily independent of small GTPases. The level of activated Rac1 and RhoA did not increase at the early time points after toxin application, when the initial shape changes have been observed, but at later time points when the actin-dependent displacement of cells was slower and less severe, probably presenting the cell's attempt to re-establish proper cytoskeleton function. A GUV (giant unilamellar vesicle) approach provided insight into the effects of pneumolysin in a biomimetic system, an environment, which is strictly biochemical, but still comprises cellular components, limited to the factors of interest (actin, Arp2/3, ATP, and Mg2+ on one side, and PLY on the other side). This approach was able to show that the wildtype-toxin, but not the Δ6 mutant (mutated in the unfolding domain, and thus non-porous), had the capacity to exhibit its functions through a membrane bilayer, meaning it was able to aggregate actin, which was located on the other side of the membrane, either via direct interaction with actin or in an Arp2/3 activating manner. Taking a closer look at these two factors with the help of several different imaging and biochemical approaches, this work unveiled the capacity of pneumolysin to bind and interact both with actin and Arp2 of the Arp2/3 complex. Pneumolysin was capable to slightly stabilize actin in an actin-pyrene polymerization assay. The same experimental setup was applied to show that the toxin had the capacity to lead to actin polymerization through activation of the Arp2/3 complex. This effect was additionally confirmed with the help of fluorescent microscopy of rhodamine (TRITC)-tagged actin. Strongest Arp2/3 activation, and actin nucleation/polymerization is achieved by the VCA domain of the WASP family proteins. However, addition of PLY to the Arp2/3-VCA system led to an enhanced actin nucleation, suggesting a synergistic activation function of pneumolysin. Hence, two different effects of pneumolysin on the actin cytoskeleton were observed. On the one hand an actin severing property, and on the other hand an actin stabilization property, both of which do not necessarily exclude each other. Actin remodeling is a common feature of bacterial virulence strategies. This is the first time, however, that these properties were assigned to a toxin of the CDC family. Cytoskeletal dysfunction in astrocytes leads to dysfunction and unregulated movement of these cells, which, in context of bacterial meningitis, can favor bacterial penetration and spreading in the brain tissue, and thus comprises an additional role of pneumolysin as a virulence factor of Streptococcus pneumonia in the context of brain infection.}, subject = {Hirnhautentz{\"u}ndung}, language = {en} } @phdthesis{Foertsch2012, author = {F{\"o}rtsch, Christina}, title = {Pneumolysin: the state of pore-formation in context to cell trafficking and inflammatory responses of astrocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70892}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Pneumolysin, a protein toxin, represents one of the major virulence factors of Streptococcus pneumoniae. This pathogen causes bacterial meningitis with especially high disease rates in young children, elderly people and immunosuppressed patients. The protein toxin belongs to the family of cholesterol-dependent cytolysins, which require membrane cholesterol in order to bind and to be activated. Upon activation, monomers assemble in a circle and undergo conformational change. This conformational change leads to the formation of a pore, which eventually leads to cell lysis. This knowledge was obtained by studies that used a higher concentration compared to the concentration of pneumolysin found in the cerebrospinal fluid of meningitis patients. Thus, a much lower concentration of pneumolysin was used in this work in order to investigate effects of this toxin on primary mouse astrocytes. Previously, a small GTPase activation, possibly leading to cytoskeletal changes, was found in a human neuroblastoma cell line. This led to the hypothesis that pneumolysin can lead to similar cytoskeletal changes in primary cells. The aim of this work was to investigate and characterise the effects of pneumolysin on primary mouse astrocytes in terms of a possible pore formation, cellular trafficking and immunological responses. Firstly, the importance of pore-formation on cytoskeletal changes was to be investigated. In order to tackle this question, wild-type pneumolysin and two mutant variants were used. One variant was generated by exchanging one amino acid in the cholesterol recognising region, the second variant was generated by deleting two amino acids in a protein domain that is essential for oligomerisation. These variants should be incapable of forming a pore and were compared to the wild-type in terms of lytic capacities, membrane binding, membrane depolarisation, pore-formation in artificial membranes (planar lipid bilayer) and effects on the cytoskeleton. These investigations resulted in the finding that the pore-formation is required for inducing cell lysis, membrane depolarisation and cytoskeletal changes in astrocytes. The variants were not able to form a pore in planar lipid bilayer and did not cause cell lysis and membrane depolarisation. However, they bound to the cell membrane to the same extent as the wild-type toxin. Thus, the pore-formation, but not the membrane binding was the cause for these changes. Secondly, the effect of pneumolysin on cellular trafficking was investigated. Here, the variants showed no effect, but the wild-type led to an increase in overall endocytotic events and was itself internalised into the cell. In order to characterise a possible mechanism for internalisation, a GFP-tagged version of pneumolysin was used. Several fluorescence-labelled markers for different endocytotic pathways were used in a co-staining approach with pneumolysin. Furthermore, inhibitors for two key-players in classical endocytotic pathways, dynamin and myosin II, were used in order to investigate classical endocytotic pathways and their possible involvement in toxin internalisation. The second finding of this work is that pneumolysin is taken up into the cell via dynamin- and caveolin-independent pinocytosis, which could transfer the toxin to caveosomes. From there, the fate of the toxin remains unknown. Additionally, pneumolysin leads to an overall increase in endocytotic events. This observation led to the third aim of this work. If the toxin increases the overall rate of endocytosis, the question arises whether toxin internalisation favours bacterial tissue penetration of the host or whether it serves as a defence mechanism of the cell in order to degrade the protein. Thus, several proinflammatory cytokines were investigated, as previous studies describe an effect of pneumolysin on cytokine production. Surprisingly, only interleukin 6-production was increased after toxin-treatment and no effect of endocytotic inhibitors on the interleukin 6-production was observed. The conclusion from this finding is that pneumolysin leads to an increase of interleukin 6, which would not depend on the endocytotic uptake of pneumolysin. The production of interleukin 6 would enhance the production of acute phase proteins, T-cell activation, growth and differentiation. On the one hand, this activation could serve pathogen clearance from infected tissue. On the other hand, the production of interleukin 6 could promote a further penetration of pathogen into host tissue. This question should be further investigated.}, subject = {Streptococcus pneumoniae}, language = {en} } @article{StopperKuehnelPodschun1994, author = {Stopper, Helga and K{\"u}hnel, A. and Podschun, B.}, title = {Combination of the chemotherapeutic agent 5-fluorouracil with an inhibitor of its catabolism results in increased micronucleus induction}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63383}, year = {1994}, abstract = {The rate limiting step in 5-fluorouracil catabolism is catalyzed by the enzyme dihydropyrimidine dehydrogenase. Since degradation of 5-fluorouracil decreases its efficacy in chemotherapy, the inhibition of its catabolism is a promising tool. We investigated the formation of micronuclei in vitro in mouse L5178Y cells. 5-fluorouracil induced an increase in micronucleus frequency, which could significantly be enhanced by the concurrent application of 2,6-dihydroxypyridine, an inhibitor of dihydropyrimidine dehydrogenase. The 5-fluorouracil concentration necessary to reach maximal genotoxic effects could be reduced to half in the presence of inhibitor. 2,6-Dihydroxypyridine alone and the naturally occuring enzyme substrate uracil did not induce micronucleus formation. Combined application of the chemotherapeutic agent 5-fluorouracil and an inhibitor of its could reduce side-effects by lowering the effective dose of the active drug. With this study we provide further support for the usefulness of this concept.}, subject = {Toxikologie}, language = {en} } @article{StopperEckertSchiffmannetal.1994, author = {Stopper, Helga and Eckert, I. and Schiffmann, D. and Spencer, D. L. and Caspary, W. J.}, title = {Is micronucleus induction by aneugens an early event leading to mutagenesis?}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63390}, year = {1994}, abstract = {This study was designed to investigate a previously unidentified potential mechanism for mutation induction as well as to clarify a biological comequence of micronucleus formation. We compared the induction of micronuclei with mutation inductioo as measured by trißuorothymidine (TFI') resistance in mouse L5178Y cells using four aneugens: colcemid, diethylstilbestrol, griseofulvin and vioblastine. AU four compounds induced micronuclei which appeared in the first cell cycle after treatment. More than 85\% of the micronuclei induced by each compound stained positive for the presence of kinetochores implying that the micronuclei contained wbole cbromosomes. However, these same compounds were unable to induce TFf resistance under tbree different treatment regimes. We concluded that tbese compounds, under conditions where tbey induce primarily kinetochore positive micronuclel, were not able to induce mutations. Thus, the induction of micronuclei containing wbole chromosomes barborlog a select.able gene is not an early event leadlog to mutations in these cells.}, subject = {Toxikologie}, language = {en} } @article{StopperKoerberSpenceretal.1993, author = {Stopper, Helga and K{\"o}rber, C. and Spencer, D. L. and Kirchner, S. and Caspary, W.J. and Schiffmann, D.}, title = {An investigation of micronucleus and mutation induction by oxazepam in mammalian cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63404}, year = {1993}, abstract = {Tbe benzodiazepines are a class of d.rugs that are widely used in the treatment of various psychiatric disorders. One member of um ~' oxazepam, is also a common metabolite of sevmd other benzod.iazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is incol:lsb1ent, we investigated the oxazepam-induced fonnation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and LS178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all tbree ceU llnes. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were fonned in the first mitosis after treatment. Kinetochore staining (CREST -antiserum) revealed the presence of kinetochores in -SO\% of the micronuclei in aU tbree ceU types. ThJs resu1t was further confinned by in situ bybridization in LS178Y cells and indicates tbe presence of wbole Chromosomes or centric fragments as weU as acentric fragments in the oxazepam-induced micronuclei. The LS178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used.}, subject = {Toxikologie}, language = {en} } @article{StopperKoerberSchiffmannetal.1993, author = {Stopper, Helga and K{\"o}rber, C. and Schiffmann, D. and Caspary, W. J.}, title = {Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63411}, year = {1993}, abstract = {5-Azacytidine was originally developed to treat human myelogenous leukemia. However, interest in this compound has expanded because of reports of its ability to affect cell differentiation and to alter eukaryotic gene expression. In an ongoing attempt to understand the biochemical effects of this compound, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. In L5178Y mouse cells, 5-azacytidine induced micronuclei at concentrations at which we and others have already reported its mutagenicity at the tk locus. Using CREST staining and C-banding studies, we showed that the induced micronuclei contained mostly chromosomal fragments although some may have contained whole chromosomes. By incorporating BrdU into the DNA of SHE cells, we determined that micronuclei were induced only when the compound was added while the cells were in S phase. Microscopically visible effects due to 5-azacytidine treatment were not observed until anaphase of the mitosis following treatment or thereafter. 5-Azacytidine did not induce micronuclei via interference with formation of the metaphase chromosome arrangement in mitosis, a common mechanism leading to aneuploidy. SupravitalUV microscopy revealed that chromatid bridges were observed in anaphase and, in some cases, were sustained into interphase. In the first mitosis after 5-azacytidine treatment we observed that many cells were unable to perform anaphase separation. All of these observations indicate that 5-azacytidine is predominantly a clastogen through its incorporation into DNA.}, subject = {Toxikologie}, language = {en} } @article{AdamAhrweilerSahaMoelleretal.1993, author = {Adam, W. and Ahrweiler, M. and Saha-M{\"o}ller, C. R. and Sauter, M. and Sch{\"o}nberger, A. and Epe, B. and M{\"u}ller, E. and Schiffmann, D. and Stopper, Helga and Wild, D.}, title = {Genotoxicity studies of benzofuran dioxetanes and epoxides with isolated DNA, bacteria and mammalian cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63420}, year = {1993}, abstract = {1.2-Dioxetanes, very reactive and high energy molecules. are involved as labile intermediates in dioxygenase- activated aerobic metabolism and in physiological processes. Various toxico1ogica1 tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonucleasesensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, fonnamidopyrimidines). Pyrimidinedimersand sites ofbase loss (AP sites) which were probed by UV endonuclease and exonuclease 111 are minor lesions in this system. While the alky1-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA I 00. DNA adducts formed with an intermediary alkyJating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter. since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TAIOO and they form DNA adducts. as detected by the 212Ppostlabelling technique. Our results imply that the type of D NA darnage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA darnage by energy transfer. i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA. while radical darnage and alkylation prevail in the cellular system.}, subject = {Toxikologie}, language = {en} } @phdthesis{Fischer2010, author = {Fischer, Thomas Horst}, title = {Die transkriptionelle Regulation der microRNA-21 im Herzen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-50702}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {MicroRNAs sind kleine, nicht kodierende RNA-Molek{\"u}le, die posttranskriptionell die Genexpression regulieren. Sie binden hierf{\"u}r spezifisch an 3'-UTRs von messenger-RNAs und f{\"u}hren entweder direkt zu deren Abbau oder inhibieren deren Translation. {\"U}ber die Mechanismen, die die Expression von microRNAs regulieren, ist jedoch noch wenig bekannt. Die Tatsache, dass sie als lange Vorl{\"a}ufermolek{\"u}le (pri-microRNAs) durch die RNA-Polymerase-II transkribiert werden, legt die Existenz eines Promotorbereiches nahe, der dem proteinkodierender Gene {\"a}hnelt. Mit Hilfe von microRNA-Arrays konnten wir im linksventrikul{\"a}ren Myokard mehrere bei Herzinsuffizienz deutlich ver{\"a}ndert exprimierte microRNAs identifizieren. Die microRNA-21 ist dabei bereits im Fr{\"u}hstadium der Herzinsuffizienz verst{\"a}rkt exprimiert (Northern Blot). Auch in prim{\"a}ren, kardialen Zellen (Fibroblasten, Kardiomyozyten) wird die microRNA-21 nach Induktion einer Hypertrophie verst{\"a}rkt exprimiert. Weiterf{\"u}hrendes Ziel dieser Arbeit war es nun, diejenigen Mechanismen aufzukl{\"a}ren, die der starken Induktion der microRNA-21 im erkrankten Myokard zu Grunde liegen. Durch bioinformatische Analyse des zugeh{\"o}rigen Promotorbereiches (Trans-Spezies-Konservierung) und Klonierung danach ausgerichteter Fragmente in Luciferase-basierte Reporter-Plasmide konnte ein 118 Basen langer Bereich identifiziert werden, der maßgeblich die Expression der microRNA-21 im Herzen bedingt. Durch Deaktivierung einzelner cis-Elemente konnte die kardiale Expression auf zwei essentielle Transkriptionsfaktorbindungsstellen zur{\"u}ckgef{\"u}hrt werden. Es handelt sich dabei um Erkennungssequenzen f{\"u}r die im Herz bedeutsamen Transkriptionsfaktoren CREB und SRF. Sie liegen in enger r{\"a}umlicher Nachbarschaft ungef{\"a}hr 1150 bp vor der Transkriptionsstartstelle. Die Suppression der Expression dieser beiden Transkriptionsfaktoren mittels geeigneter siRNAs f{\"u}hrte jeweils zu einer signifikanten Aktivit{\"a}tsminderung des microRNA-21-Promotors und konnte somit die vorangehenden Ergebnisse validieren. Durch Generierung einer transgenen Tierlinie, die lacZ unter der Kontrolle des microRNA-21-Promotors exprimiert, werden in naher Zukunft n{\"a}here Aufschl{\"u}sse {\"u}ber die gewebsspezifische Verteilung der microRNA-21-Expresssion in vivo m{\"o}glich sein. Zusammenfassend beschreiben wir hier erstmals den Mechanismus der transkriptionellen Regulation der microRNA-21 im Herzen. Dieser Mechanismus bedingt wahrscheinlich die starke Induktion dieser microRNA bei kardialer Hypertrophie und Herzinsuffizienz.}, subject = {Small RNA}, language = {de} } @phdthesis{Moro2011, author = {Moro, Sabrina}, title = {Identification of target proteins of furan reactive metabolites in rat liver}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-57617}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Furan was recently found to be present in a variety of food items that undergo heat treatment. It is known to act as a potent hepatotoxin and liver carcinogen in rodents. In a 2-year bioassay, chronic furan administration to rats was shown to cause hepatocellular adenomas and carcinomas and very high incidences of cholangiocarcinomas even at the lowest furan dose tested (2.0 mg/kg bw). However, the mechanisms of furan-induced tumor formation are poorly understood. Furan is metabolized by cytochrome P450 (CYP) enzymes, predominantly CYP2E1, to its major metabolite cis-2-butene-1,4-dial (BDA). BDA is thought to be the key mediator of furan toxicity and carcinogenicity and was shown to react with cellular nucleophiles such as nucleosides and amino acid residues in vitro. It is well known that covalent protein binding may lead to cytotoxicity, but the cellular mechanisms involved remain to be elucidated. Since covalent binding of reactive intermediates to a target protein may result in loss of protein function and subsequent damage to the cell, the aim of this study was to identify furan target proteins to establish their role in the pathogenesis of furan-associated liver toxicity and carcinogenicity. In order to identify target proteins of furan reactive metabolites, male F344/N rats were administered [3,4-14C]-furan. Liquid scintillation counting of protein extracts revealed a dose-dependent increase of radioactivity covalently bound to liver proteins. After separation of the liver protein extracts by two-dimensional gel electrophoresis and subsequent detection of radioactive spots by fluorography, target proteins of reactive furan intermediates were identified by mass spectrometry and database search via Mascot. A total of 61 putative target proteins were consistently found to be adducted in 3 furan-treated rats. The identified proteins represent - among others - enzymes, transport proteins, structural proteins and chaperones. Pathway mapping tools revealed that target proteins are predominantly located in the cytosol and mitochondria and participate in glucose metabolism, mitochondrial β-oxidation of fatty acids, and amino acid degradation. These findings together with the fact that ATP synthase β subunit was also identified as a putative target protein strongly suggest that binding of furan reactive metabolites to proteins may result in mitochondrial injury, impaired cellular energy production, and altered redox state, which may contribute to cell death. Moreover, several proteins involved in the regulation of redox homeostasis represent putative furan target proteins. Loss of function of these proteins by covalent binding of furan reactive metabolites may impair cellular defense mechanisms against oxidative stress, which may also result in cell death. Besides the potential malfunction of whole pathways due to loss of functions of several participating proteins, loss of function of individual proteins which are involved in various cellular processes such as transport processes across the mitochondrial membranes, cell signaling, DNA methylation, blood coagulation, and bile acid transport may also contribute to furan-induced cytotoxicity and carcinogenicity. Covalent binding of reactive metabolites to cellular proteins may result in accumulation of high amounts of unfolded or damaged proteins in the endoplasmic reticulum (ER). In response to this ER stress, the cell can activate the unfolded protein response (UPR) to repair or degrade damaged proteins. To address whether binding of furan reactive metabolites to cellular proteins triggers activation of the UPR, semiquantitative PCR and TaqMan® real-time PCR were performed. In the case of UPR activation, semiquantitative PCR should show enhanced splicing of X-box binding protein-1 (XBP1) mRNA (transcription factor and key regulator of the UPR) and TaqMan® real-time PCR should determine an increased expression of UPR target genes. However, our data showed no evidence for activation of the UPR in the livers of rats treated either with a single hepatotoxic dose or with a known carcinogenic dose for 4 weeks. This suggests either that furan administration does not induce ER stress through accumulation of damaged proteins or that activation of the UPR is disrupted. Consistent with the latter, glucose-regulated protein 78 (GRP78), identified as a target protein in our study, represents an important mediator involved in activation of the UPR whose inhibition was shown to impair induction of the UPR. Thus, adduct formation and inactivation of GRP78 by furan metabolites may disturb activation of the UPR. In addition to impaired activation of UPR, protein repair and degradation functions may be altered, because several proteins involved in these processes also represent target proteins of furan and thus may show impaired functionality. Taken together...}, subject = {Furan}, language = {en} } @phdthesis{Queisser2010, author = {Queisser, Nina}, title = {Oxidative and nitrosative stress induced by the mineralocorticoid aldosterone - Mechanism of induction and role of signal transduction pathways and transcription factors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-53566}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Several epidemiological studies found that hypertensive patients have an increased risk to develop kidney cancer. Hyperaldosteronism frequently results in arterial hypertension and contributes to the development and progression of kidney injury, with reactive oxygen species (ROS) playing an important role. ROS are thought to be associated with many pathological conditions such as cancer and other disorders, like cardiovascular complications , which often go along with hypertension. The aim of the present work was to investigate whether the effects of elevated aldosterone concentrations might be involved in the increased cancer incidence of hypertensive individuals. First, the potential capacity of aldosterone to induce oxidative stress and DNA damage was investigated in vitro and in vivo. In LLC-PK1 porcine kidney cells and MDCK canine kidney cells the significant formation of ROS, and especially of superoxide (O2˙ˉ) was assessed. With two genotoxicity tests, the comet assay and the micronucleus frequency test, the DNA damaging potential of aldosterone was quantified. In both genotoxicity tests a dose-dependent increase in aldosterone-induced structural DNA damage was observed. Oxidative stress and DNA damage were prevented by antioxidants, suggesting ROS as a major cause of DNA damage. Furthermore, the oxidatively modified DNA lesion 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG), was found to be significantly elevated. In kidneys of rats with desoxycorticosterone acetate (DOCA)/salt-induced hypertension, which is a model of severe mineralocorticoid-dependent hypertension, elevated levels of ROS and superoxide were found, compared to kidneys of sham rats. Also DNA strand breaks, measured with the comet assay and double strand breaks, visualized with antibodies against the double strand break-marker gamma-H2AX were significantly elevated in kidneys of DOCA/salt-treated rats. In addition, significantly increased amounts of 8-oxodG were detected. Proliferation of kidney cells was found to be increased, which theoretically enables the DNA damage to manifest itself as mutations, since the cells divide. Second, the effects of aldosterone on the activation of transcription factors and signaling pathways were investigated. A significant activation of the potentially protective transcription factor Nrf2 was observed in LLC-PK1 cells. This activation was triggered by an increase of ROS or reactive nitrogen species (RNS). In response to oxidative stress, glutathione synthesis and detoxifying enzymes, such as the subunits of the glutathione-cysteine-ligase or heme oxygenase 1 were rapidly induced after 4 h. Nevertheless, after 24 h a decrease of glutathione levels was observed. Since ROS levels were still high after 24 h, but Nrf2 activation decreased, this adaptive survival response seems to be transient and quickly saturated and overwhelmed by ROS/RNS. Furthermore, Nrf2 activation was not sufficient to protect cells against oxidative DNA damage, because the amounts of double strand breaks and 8-oxodG lesions steadily rose up to 48 h of aldosterone treatment. The second transcription factor that was time- and dose-dependently activated by aldosterone in LLC-PK1 and MDCK cells was NF-kappaB. Furthermore, a significant cytosolic and nuclear activation of ERK was detected. Aldosterone induced the phosphorylation of the transcription factors CREB, STAT1 and STAT3 through ERK. Third, the underlying mechanisms of oxidant production, DNA damage and activation of transcription factors and signaling pathways were studied. Aldosterone exclusively acted via the MR, which was proven by the MR antagonists eplerenone, spironolactone and BR-4628, whereas the glucocorticoid receptor (GR) antagonist mifepristone did not show any effect. Furthermore, aldosterone needed cytosolic calcium to exert its negative effects. Calcium from intracellular stores and the influx of calcium across the plasma membrane was involved in aldosterone signaling. The calcium signal activated on the one hand, the prooxidant enzyme complex NAD(P)H oxidase through PKC, which subsequently caused the generation of O2˙ˉ. On the other hand, nitric oxide synthase (NOS) was activated, which in turn produced NO. NO and O2˙ˉ can react to the highly reactive species ONOO- that can damage the DNA more severely than the less reactive O2˙ˉ. In the short term, the activation of transcription factors and signaling pathways could be a protective response against aldosterone-induced oxidative stress and DNA damage. However, a long-term NF-B and ERK/CREB/STAT activation by persistently high aldosterone levels could unfold the prosurvival activity of NF-kappaB and ERK/CREB/STAT in aldosterone-exposed cells. DNA damage caused by increased ROS might become persistent and could be inherited to daughter cells, probably initiating carcinogenesis. If these events also occur in patients with hyperaldosteronism, these results suggest that aldosterone could be involved in the increased cancer incidence of hypertensive individuals.}, subject = {Aldosteron}, language = {en} } @phdthesis{Hommers2011, author = {Hommers, Leif}, title = {{\"U}ber die Interaktion aktivierter G-Proteine mit G-Protein gekoppelten Rezeptoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-56576}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Aktivierte G-Protein gekoppelte Rezeptoren aktivieren heterotrimere GProteine, in dem sie den Austausch von GDP zu GTP am G-Protein katalysieren. Theoretische Untersuchungen mittels eines vereinfachten kinetischen Modells des Gi/o-Protein Zyklus legen nahe, dass nicht nur GDP-,sondern auch GTP-gebundene Gi/o-Proteine mit aktivierten α2A-adrenergen Rezeptoren (α2A-AR) interagieren k{\"o}nnen. Demgem{\"a}ß sollten aktivierte Gi/o-Proteine mit aktivierten α2A-AR vermehrt interagieren, wenn mehr α2A-AR aktiviert werden als f{\"u}r eine maximale G-Protein Aktivierung n{\"o}tig sind. Dies sollte zu einer paradoxen Deaktivierung von Gi/o-Proteinen und deren Effektorproteinen, z.B. dem G-Protein gekoppelten, einw{\"a}rtsgleichrichtenden Kaliumkanal (GIRK-Kanal) f{\"u}hren. Mittels FRET l{\"a}sst sich in lebenden und in permeabilisierten Zellen unter Kontrolle der intrazellul{\"a}ren Nukleotide die Aktivierung von α2A-AR, die Interaktion von Gi/o-Proteinen mit α2A-AR und die Aktivierung von Gi/o-Proteinen bestimmen. Die Arbeit zeigt auf mehreren Ebenen, dass Go-Proteine mit aktivierten α2A-AR interagieren und im nukleotidfreiem Zustand sequestriert werden k{\"o}nnen: (I) Go-Proteine,irreversibel durch GTPγS aktiviert werden abh{\"a}ngig von der Rezeptor Aktivierung in Abwesenheit von Nukleotiden deaktiviert, (II) Go-Proteine interagieren in Gegenwart niedriger Nukleotidkonzentrationen in wesentlich gr{\"o}ßer Fraktion mit aktivierten α2A-AR als in Gegenwart hoher Nukleotidkonzentrationen, (III) Go Proteine k{\"o}nnen in Gegenwart niedriger GTP und GTPγS-Konzentrationen bei Aktivierung des α2A-AR inaktiviert werden. Die Arbeit zeigt exemplarisch an der Signalkaskade des α2A-AR und Go, dass der G-Protein Zyklus in lebenden Zellen reversibel ist, woraus eine Deaktivierung aktivierter G-Proteine und aktivierter G-Protein Effektoren resultieren kann. Dies erkl{\"a}rt paradoxe Befunde zur Deaktivierung von GIRK-Kan{\"a}len in Myozyten durch A1-Rezeptoren.}, subject = {G-Protein gekoppelte Rezeptoren}, language = {de} } @article{SchmaehlFrankLutzetal.1985, author = {Schm{\"a}hl, D. and Frank, HK and Lutz, WK and Stransky, M. and Ritzel, G. and Beaufort, F. and Vutuc, C.}, title = {Ern{\"a}hrung und Krebs}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-55224}, year = {1985}, abstract = {No abstract available}, subject = {Medizin}, language = {de} } @phdthesis{Koussemou2020, author = {Kouss{\´e}mou, Y{\´e}wa Bony Marthe}, title = {A\(_{2B}\) adenosine receptor signaling in MDA-MB-231 breast cancer cells: Mechanism of A\(_{2B}\)-mediated reduction of ERK1/2 phosphorylation}, doi = {10.25972/OPUS-20965}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-209655}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Recently, it was shown that MDA-MB-231 breast cancer cells express very high levels of the A2BAR as the sole adenosine receptor subtype, and stimulation of the A2BAR in MDA-MB-231 cells triggers an unusual inhibitory signal on ERK1/2 phosphorylation. The ERK1/2 pathway is reported to be associated with the control of growth, proliferation and differentiation of cells and as such might serve as a promising target for tumor treatment. The present study investigated signaling mechanisms involved in linking A2BAR to ERK1/2 phosphorylation in MDA-MB-231 cells. The A2BAR mediated reduction of ERK1/2 phosphorylation and of proliferation of MDA-MB-231 cell is in good agreement with previous results from (Dubey et al., 2005). These observations provide support to the hypothesis that activation of A2BAR could attenuate the growth of some types of cancer cell and argue against a stimulation of proliferation resulting from the activation of A2BAR as discussed by (Fernandez-Gallardo et al., 2016). AC activation by forskolin has recently been shown to enhance the activity of the chemotherapeutic agent doxorubicin in TNBC cells via a mechanism dependent on the PKA-mediated inhibition of ERK1/2 phosphorylation. Furthermore, forskolin also increased the sensitivity of MDA-MB-231 and MDA-MB-468 triple negative breast cancer cells to 5-fluorouracil and taxol (Illiano et al., 2018), and sustains the evidence of anticancer activity mediated by cAMP/PKA-mediated ERK1/2 inhibition. Similar to these studies, a reduced amount of pERK1/2 was also observed after stimulation of AC with FSK, application of cAMP-AM or inhibition of PDE-4. The inhibition of ERK1/2 phosphorylation was mimicked by UTP and abolished with the PLC inhibitor U73122 or by chelating intracellular Ca2+ with BAPTA-AM. These results point to an important role for both cAMP and Ca2+ signaling in the pathway leading to a decrease in ERK1/2 phosphorylation. This study encourages the idea that A2BAR could be used as target in cancer therapy. But A2BAR did not only stimulate signaling cascades associated with cell survival and proliferation reduction, but also key phases relevant in angiogenesis like Ca2+ mobilization (Kohn et al., 1995). Whereas the potency toward AC and Ca2+ are similar for the diverse agonists, the potency to promote ERK1/2 reduction is much higher. Interestingly, the proliferation of MDA-MB-231 cells is inhibited by low nanomolar agonist concentration which is inactive in Ca2+ mobilization. This means that it is certainly possible to reduce the proliferation without promoting angiogenesis. LUF6210 is particularly interesting when considering that it preferentially stimulates a reduction in ERK1/2 phosphorylation over Ca2+ and therefore may not promote angiogenesis. LUF6210 is therapeutically appealing as adjuvant in treatment of cancer. Given that stimulation of AC can activate a reduction of ERK1/2 phosphorylation and proliferation in cancer cells, agonist bias toward Gs-AC-PKA-mediated ERK1/2 inhibition represent a potential therapy of various malignancies. The fact that the reduction of ERK1/2 phosphorylation followed by reduced proliferation observed in MDA-MB-231 cells were mediated by the activation of the A2BAR illustrates the importance of this receptor subtype in cancer. A2BARs must be considered as a key factor in cancer treatment and deserve attention for the development of new therapeutic strategies.}, subject = {Adenosinrezeptor}, language = {en} } @article{GonzalesCaleroCuberoKlotz1992, author = {Gonzales-Calero, G. and Cubero, A. and Klotz, Karl-Norbert}, title = {G protein coupled A\(_1\) adenosine receptors in coated vesicles of mammalian brain. Characterization by radioligand binding and photoaffinity labeling}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60435}, year = {1992}, abstract = {A\(_1\) adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaflinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1 ,3-[\(^3\)H]dipropyl-xanthine ([\(^3\)H]DPCPX) gave a Kdvalue of 0.7 nM and a Bmax value of 82± 13 fmol/mg protein. For the highly A\(_1\)-selective agonist 2-chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [\(^3\)H]DPCPX binding gave a pharmacological profile with R-N\(^6\)-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCP A to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N\(^6\)- \(^{125}\)I-p-hydroxyphenylisopropyladenosine ([\(^{125}\)I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A\(_1\) receptors labelled in brain membranes. Therefore, coated vesicles contain A\(_1\) adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A\(_1\) receptors in these vesicles are not a processed receptor fonn. These results confirm that A\(_1\) receptors in coated vesicles are coupled to a G-protein, and it appears that the A\(_1\) receptor systems in coated vesicles andin plasma membranes are identical.}, subject = {Toxikologie}, language = {en} } @article{JesaitisEricksonKlotzetal.1993, author = {Jesaitis, A. J. and Erickson, R. W. and Klotz, Karl-Norbert and Bommakanti, R. K. and Siemsen, D. W.}, title = {Functional molecular complexes of human N-formyl peptide chemoattractant receptors and actin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60445}, year = {1993}, abstract = {When human neutrophils become desensitized to formyl peptide chemoattractants, the receptors (FPR) for these peptides are converted to a high affinity, GTP-insensitive form that is associated with the Triton X-1 00- insoluble membrane skeleton from surface membrane domains. These domains are actin and fodrin-rich, but G protein-depfeted suggesting that FPR shuttling between G protein-enriched and depleted domains may control signal transduction. Todetermine the molecular basis for FPR interaction with the membrane skeleton, neutrophil subcellular fractions were screened for molecules that could bind photoaffinity-radioiodinated FPR solubilized in Triton X-1 00. These receptors showed a propensity to bind to a 41- to43-kDa proteinband on nitrocelluloseoverlays of SOS-PAGE-separated cytosol and plasma membrane fractions of neutrophils. This binding, as weil as FPR binding to purified neutrophil actin, was inhibited 50\% by 0.6 \(\mu\)M free neutrophil cytosolic actin. Addition of greater than 1 \(\mu\)M G-actin to crude or lectin-purified Triton X-1 00 extracts of FPR from neutrophil membranes increased the sedimentationrate of a significant fraction of FPR two to three fold as measured by velocity sedimentation in Triton X-1 00-containing linear sucrose density gradients. Addition of anti-actin antibodies to FPR extracts caused a concentration-dependent immunoprecipitation of at least 65\% of the FPR. More than 40\% of the immunoprecipitated FPR was specifically retained on protein A affinity matrices. Membrane actin was stabilized to alkaline washing when membranes were photoaffinity labeled. Conversely, when purified neutrophil cytosolic actinwas added to membranes or their digitonin extracts, after prior depletion of actin by an alkaline membrane wash, photoaffinity labeling of FPR was increased two- to fourfold with an EC\(_{50}\) of approximately 0.1 \(\mu\)M actin. We conclude that FPR from human neutrophils may interact with actin in membranes to form Triton X-1 00-stable physical complexes. These complexes can accept additional G-actin monomers to form higher order molecular complexes. Formation of FPR-actin complexes in the neutrophil may play a role in the regulation of chemoattractantinduced activation or actin polymerization.}, subject = {Toxikologie}, language = {en} } @article{BommakantiKlotzDratzetal.1993, author = {Bommakanti, R. K. and Klotz, Karl-Norbert and Dratz, E. A. and Jesaitis, A. J.}, title = {A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60456}, year = {1993}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{KlotzKrotecGripentrogetal.1994, author = {Klotz, Karl-Norbert and Krotec, K. L. and Gripentrog, J. and Jesaitis, A. J.}, title = {Regulatory interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton in human neutrophils}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60466}, year = {1994}, abstract = {The cytoskeleton and/or membrane skeleton has been implicated in the regulation of N-formyl peptide receptors. The coupling of these chemotactic receptors to the membrane skeleton was investigated in plasma membranes from unstimulated and desensitized human neutrophils using the photoreactive agonist N-formyl-met-leu-phelys-N\(^6\)-[\(^{125}\)I]2(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (fMLFK-[\(^{125}\)I]ASD). When membranes of unstimulated cells were solubilized in Triton-X 100, a detergent that does not disrupt actin filaments, only 50\% of the photoaffinity-labeled receptors were solubilized sedimenting in sucrose density gradients at a rate consistent with previous reports. The remainder were found in the pellet fraction along with the membrane skeletal actin. Solubilization of the membranes in the presence of p-chloromercuriphenylsulfonic acid, elevated concentrations of KCI, or deoxyribonuclease I released receptors in parallel with actin. When membranes from neutrophils, desensitized by incubation with fMLFK-e 251]ASD at 15°C, were solubilized, nearly all receptors were recovered in the pellet fraction. lncubation of cells with the Iigand at 4°C inhibited desensitization partially and prevented the conversion of a significant fraction of receptors to the form associated with the membrane skeletal pellet. ln these separations the photoaffinity-labeled receptors not sedimenting to the pellet cosedimented with actin. Approximately 25\% of these receptors could be immunosedimented with antiactin antibodies suggesting that N-formyl peptide receptors may interact directly with actin. These results are consistent with a regulatory role for the interaction of chemotactic N-formyl peptide receptors with actin of the membrane skeleton.}, subject = {Toxikologie}, language = {en} } @article{KlotzJesaitis1994, author = {Klotz, Karl-Norbert and Jesaitis, A. J.}, title = {Neutrophil chemoattractant receptors and the membrane skeleton}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60471}, year = {1994}, abstract = {Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process which involves participation of cytoskeletal elements. Evidence exists suggesting that the cytoskeleton and/or the membrane skeleton controls the distributJon of FPR in the plane of the plasma membrane, thus controlling the accessibility of FPR to different proteins in functionally distinct domains. In desensitized cells, FPR are restricted todomains which are depleted of G proteins but enriched in cytoskeletal proteins such as actin and fodrin. Thus, the G protein signal transduction partners of FPR become inaccessible to the agonist-occupied receptor, preventing cell activation. The mechanism of interaction of FPR with the membrane skeleton is poorly understood but evidence is accumulating that suggests a direct binding of FPR (and other receptors) to cytoskeletal proteins such as actin.}, subject = {Toxikologie}, language = {en} } @article{CantoreggiDietrichLutz1993, author = {Cantoreggi, S. and Dietrich, D. R. and Lutz, Werner K.}, title = {Induction of cell proliferation in the forestomach of F344 rats following subchronic administration of styrene 7,8-oxide and butylated hydroxyanisole}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60669}, year = {1993}, abstract = {The question addressed was whether Stimulation of cell proliferation could be responsible for tumor induction in the torestornach by styrene 7,8-oxide (SO). Male F344 rats were treated for 4 weeks with 0, 137,275, and 550 mglkg SO by p.o. gavage 3 times/week. Positive controls received 0, 0.5, I, and 2\% butylated hydroxyanisole (BHA) in the diet for 4 weeks. Twenty-four h before termination of the experlment, the rats were implanted s.c. with an osmotic minipump deliverlog S-bromo-2'-deoxyuri· dine (BrdU). Cell proliferation in the forestomach was assessed by immunohistochemistry for BrdU incorporated into DNA. Cell number/mm section length and fraction of replicating cells (labeling Index) were determined in 3 domains of the forestomach, the saccus caecus, the midregion, and the prefundic region. With the exception of the prefundic reglon of the low-dose SO group, a significant increase of the labeling index was found in all regions both with SO and BHA. Rats treated with BHA showed, in addition, a dose-dependent increase in number and size of hyperplastic lesions. This was most pronounced in the prefundic region where carcinomas were reported to be localized. In this region, the number of dividing cells/mm section length was increased up to 17-fold. With SO, only marginal morphological changes were occasionally observed, despite the fact that the respective long-term treatment bad been reported to result in a higher carcinoma incidence than treatment with BHA. It ls concluded that the rate of replicating cells alone, numerically expressed by the labeling Index, is an lnsufficient tool for interpretlog the role of cell division in carcinogenesis. It is postulated that SO and BHA induce forestomach tumors via different mechanisms. While hyperplasia in the prefundic region most likely dominates the carcinogenicity of BHA, a mechanism combining marginal genotoxicity with strong promotion by increased cell proliferation appears to be involved in the tumorigenic action of SO.}, subject = {Toxikologie}, language = {en} } @article{FischerBelandLutz1993, author = {Fischer, W. H. and Beland, P. E. and Lutz, Werner K.}, title = {DNA adducts, cell proliferation and papilloma latency time in mouse skin after repeated dermal application of DMBA and TPA}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60673}, year = {1993}, abstract = {'lbe mouse skin tumor model was used to investigate whether the Ievel of DNA 8dducts and/or the rate of cell division in the epidermis are indicators of the risk of cancer formation for an individual in an outbred animal popul8tion. A high risk was considered to be reftected by 8 short latency period for the 8ppearance of 8 papilloma. Fernale NMRI mice were treated twice weekly with 2.5 nmol 7 ,12-dimethylbenz[a]antbracene (DMBA) and 3 nmoi12-0-tetradecanoylphorbol-13- 8cetate (TPA) and the appearance of papillomas was registered. The first papilloma 8ppeared after 7.5 weeks. After 17 weeks, when 12 of 14 mice bad 8t least one papilloma, an osmotic minipump deliverlog 5-bromo-2'deoxyuridine (BrdU) was implanted into eacb mouse for 24 h. The mice were killed after 24 h ~d the epidermis was analyzed for D:MBA-nucleotide 8dducts by 32p.postlabeling, for the cell number per unit skin length, and for the labeling index for DNA synthesls. Unexpectedly, D:MBA-nucleotide 8dduct Ievels were highest in those anima1s wbich showed the Iongest latency periods. Adduct Ievels were negatively correlated with the 18beling index, indicating that dilution of adducts by cell division was a predominant factor in determining average adduct concentrations. Individual tumor-latency time was not corTelated with either cell ntunber or labeling index. This could be due to the fact that the measurements only provided 8veraged data and gave no infonnation on the specific situation in clones of premalignant cells. Under the conditions of tbis assay, therefore, neither DNA adduct Ievels nor information on the average kinetics of cell division bad a predidive value for the individual amcer risk withln a group of outbred animals receiving the same treatment}, subject = {Toxikologie}, language = {en} } @article{HuberLutz1984, author = {Huber, K. W. and Lutz, Werner K.}, title = {Methylation of DNA in stomach and small intestine of rats after oral administration of methylamine and nitrite}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60984}, year = {1984}, abstract = {Young adult male Sprague-Dawley rats were given 30 \(\mu\)mol/kg body weight [\(^{14}\)C]methylamine hydrochloride and 700 \(\mu\)mol/ kg body weight sodium nilrite by oral gavage. DNA isolated from the stomach and from the first 15 cm of the smaß intestine was methylated, containing 7-methylguanine (7mG) at a level of one 7mG molecule per 5x10\8^6\) and lx10\(^7\) nucleotides, respectively. No 7mG was found fn the liver at a limit of detection of one 7mG molecule per 2xl0\(^8\) nucleotides. ln a second experiment, the excised stomachs were incubated with deoxyribonuclease before the isolation of the DNA in order to degrade DNA in the Iumen and in the uppermost lining cells. This treatment resulted in a 30\% decrease in the yield of DNA and a 90\% reduction in the level of 7mG formation. The results show that nitrosation of a primary alkylamine yields a precursor of an alkylating agent which has a long enough lifetime to diffuse towards and react with intracellular DNA. A correlation of DNA methylation in the stomach with the corresponding tumor formation by the methylating carcinogen N-methyi-N'-nitro-N-nitroso-guanidine was used to estimate the roJe of DNA damage resulting from endogenous nitrosation of dietary methylamine in man. It was concluded that the risk resulting from this single amine must be negligible bot that a similar evaluation of other primary amines is required before the over-aU role of primary amine nitrosation in the etiology of human gastric cancer can be assessed.}, subject = {Toxikologie}, language = {en} } @article{CaviezelLutzMininietal.1984, author = {Caviezel, M. and Lutz, Werner K. and Minini, U. and Schlatter, C.}, title = {Interaction of estrone and estradiol with DNA and protein of liver and kidney in rat and hamster in vivo and in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60995}, year = {1984}, abstract = {(6,7-\(^3\)H] Estrone (E) and [6,7-\(^3\)H]estradiol-17ß (E\(_2\)) have been synthesized by reduction of 6-dehydroestrone and 6-dehydroestradiol with tritium gas. Tritiated E and E\(_2\) were administered by oral gavage to female rats and to male and female hamsters on a dose level of about 300 \(\mu\)g/kg (54 mCi/kg). After 8 h, the liver was excised from the rats; liver and kidneys were taken from the hamsters. DNA was purified either directly from an organ homogenate or via chromatin. The radioactivity in the DNA was expressed in the units of the Covalent Binding Index, CBI = (\(\mu\)mol chemical bound per mol Similar considerations can be made for the liver where any true covalent DNA binding must be below a Ievel of 0.01. It is concluded that an observable tumor induction by estrone or estradiol is unlikely to be due to DNA binding. DNA-P)/(mmol chemical administered per kg b.w.). Rat liver DNA isolated via chromatin exhibited the very low values of 0.08 and 0.09 for E and E\(_2\) respectively. The respective figures in hamster liver were 0.08 and 0.11 in females and 0.21 and 0.18 in the males. DNA isolated from the kidney revealed a detectable radioactivity only in the female, with values of 0.03 and 0.05 for E and E\(_2\) respectively. The values for male hamster kidney were < 0.01 for both hormones. The minute radioactivity detectable in the DNA samples does not represent covalent binding to DNA, however, as indicated by' two sets of control experiments. (A) Analysis by HPLC of the nucleosides prepared by enzyme digest of liver DNA isolated directly or via chromatin did not reveal any consistent peak which could have been attributed to a nucleoside-steroid adduct. (B) All DNA radioactivity could be due to protein contaminations, because the specific activity of chromatin protein was determined to be more than 3 ,000 tim es high er than of DNA. The high affinity of the hormone to protein was also demonstrated by in vitro incubations, where it could be shown that the specific activity of DNA and protein was essentially proportional to the concentration of radiolabelled hormone in the organ homogenate, regardless of whether the animal was treated or whether the hormone was added in vitro to the homogenate. Carcinogens acting by covalent DNA binding can be classified according to potency on the basis of the Covalent Binding Index. Values of 10\(^3\)-10\(^4\) have been found for potent, 10\(^2\) for moderate, and 1-10 for weak carcinogens. Since estrone is moderately carcinogenic for the kidney of the male hamster, a CBI of about 100 would be expected. The actually measured Iimit of detection of 0.01 places covalent DNA binding among the highly unlikely mechanisms of action.}, subject = {Toxikologie}, language = {en} } @article{DaenikenLutzJaeckhetal.1984, author = {D{\"a}niken, A. von and Lutz, Werner K. and J{\"a}ckh, R. and Schlatter, C.}, title = {Investigation of the potential for binding of Di(2-ethylhexyl) phthalate (DEHP) and Di(2-ethylhexyl) adipate (DEHA) to liver DNA in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61004}, year = {1984}, abstract = {Investigation of the Potential for Binding of Di(2-ethylhexyl) Phthalate (DEHP) and Di(2- ethylhexyl) Adipate (DEHA) to Liver DNA in Vivo. VON D{\"A}NIKEN, A., LUTZ, W. K., J{\"A}CKH, R., AND ScHLATTER, C. (1984). Toxico/. App/. Pharmaco/. 73, 373-387. It was the aim oftbis investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or witbout pretreatment for 4 weeks with 1\% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [\(^{14}\)H]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The Iimit of detection, CBI < 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [\(^{14}\)C]- and [\(^{3}\)H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where tbe carcinogenmodified nucleoside or base adducts are expected. The respective Iimits of detection were at 0.07 and 0.04 CBI units for the \(^{14}\)C and \(^{3}\)H Iabels, respectively. The experiments with [\(^{14}\)C]- and [\(^{3}\)H]DEHA, labeled in the alcobol moiety and administered to mice, revealed aminute radioactivity of <50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which bad been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for \(^{14}\)C and 0.01 to 0.12 for \(^{3}\)H resulted. Determination of the level· of \(^{14}\)C02 expiration revealed a linear correlation with the speciftc activity of DNA. Experiments with 2-ethyl[ 1-\(^{14}\)C]hexanol perfonned with both rats and mice allowed the conclusion tbat most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01. Pretreatment of the animals witb unlabeled compound bad no effect on the DNA radioactivities in either species. The present negative data, in conjunction witb other negative short-term tests for mutagenicity, strongly indicate that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP and DEHA in rodents.}, subject = {Toxikologie}, language = {en} } @article{HuberLutz1984, author = {Huber, K. W. and Lutz, Werner K.}, title = {Methylation of DNA by incubation with methylamine and nitrite}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61011}, year = {1984}, abstract = {DNA was incubated in septum-closed reaction vials with [\(^{14}\)C]methylamine and nitrite. The DNA was purified, hydrolysed with hydrochloric acid, and the purines were analysed by h.p.l.c. 7-Methylguanine was detectable as a result of DN A methylation in experiments perfonned in 100 mM acetate at pH 4. Using different concentrations of amine and nitrite a first order reaction for total amine and a second order for total nilrite could be shown. A study on the pH dependence using 100 mM malonate buffer, pH 2.0-6.0, revealed a maximum rate at pH 3.5, with steep slopes above and below this pH value, in agreement with a mathematical analysis of the reaction equations. The data show that the alkylating agent fonned spontaneously by nitrosation and deamination of a primary amine has a long enough lifetime to react with DNA in vitro. Using the reactioil orders established here, an extrapolation to lower concentrations found in the stomach can now be perfonned. Future in vivo experiments on the methylation of gastro-intestinal DNA then would show to what extent DNA in a cell is protected from alkylation.}, subject = {Toxikologie}, language = {en} } @article{LutzBuesserSagelsdorff1984, author = {Lutz, Werner K. and B{\"u}sser, M. T. and Sagelsdorff, P.}, title = {Potency of carcinogens derived from covalent DNA binding and stimulation of DNA synthesis in rat liver}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61026}, year = {1984}, abstract = {~n order to investigate the role of the stimu~ation of ceU division for the initiation (and possi:bly promotion) of live·r tumors by chemical carcinogens, the incorporation of radiolabeUed thymidine into liver DNA was dete:rmined in male rats. Single doses of various level!s of af.latoxin 81, benzidine and carbon tetrachloride (aU known to be genotoxic via DNA binding} did not affect cell division, whereas several hepatoca:rcinogens known not to bind to DNA (alphaHCH, dofibrate, and 2,3;7,8-t!etrachlorodiibenzo~p~dioxin) gave rise to a dosedependent stimulation of Ii ver DNA synthesis within 24 h. An equation combining the infl.uences of mitotic stimu:lation, expressed as dose required to double the contro~ Ievei of DNA synthesis, and DNA binding potency, exp:ressed as t.he Covalent Binding Index, correliated weil with the cardnogenk potency for both dasses of hepatocardnogens.}, subject = {Toxikologie}, language = {en} } @article{SagelsdorffLutzSchlatter1983, author = {Sagelsdorff, P. and Lutz, Werner K. and Schlatter, C.}, title = {The relevance of covalent binding to mouse liver DNA to the carcinogenic action of hexachlorocyclohexane isomers}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61039}, year = {1983}, abstract = {[\(^3\)H]Hexachlorocyclohexane (HCH) was synthesized by chlorination of [\(^3\)H]benzene prepared by catalytic tritiation of benzene with tritiated water. The isomers of HCH were separated by adsorption chromatography on silica gel. In order to determine the covalent binding to DNA, [\(^3\)H]HCH was administered to male mice by oral gavage, and liver DNA was isolated via cbromatin. The specific radioactivity of the DNA was nonnalized by the dose administered and expressed in the molar units of the Covalent binding index, CBI = DNA damage/dose = (\(\mu\)mol bound HCH/mol DNA nucleotide)/(mmol HCH administered/kg body weight). CBI values of - 0.2 were found 10 h after the administration of alpha- and gamma-HCH. Enzymatic digestion of the DNA to the nucleosides and h.p.l.c. analysis revealed that - 40\% of the radioactivity co-migrated with the natural nucleosides. At elution volumes known to contain the more lipophilic carcinogen-nucleoside adducts, - 10\% of the radioactivity could be detected. The remaining 50\% of th,e radioactivity eluted with the front, representing a mixture of oligonucleotide- HCH adducts and/or hydrophilic degradation products which were strongly bot not covalently associated with intact DNA. Therefore, a true CBI of 0.02-0.1 must be expected both for alpha- and gamma-HCH. This CBI is by a factor of 10\(^5\) -10\(^6\) below the value found with the strongest DNAbinding carcinogens like aflatoxin B1 or dimethylnitrosamine and is unlikely to be decisive for the liver tumor induction in mice because of the foUowing additional findings: (i) both isomers gave rise to similar Ievels of DNA darnage although the alpha-isomer is a much morepotent tumor inducer. This similarity was seen not only at the time of m{\"a}ximum binding but up to 10 days after oral administration; (ii) three mouse strains with apparently different susceptibility to tumor induction by gamma-HCH could not be distinguished with respect to DNA binding; (iii) the level of DNA binding of alpha-HCH (CBI = 0.02-0.1) is more than three orders of magnitude lower than would be expected if the mechanism of tumor induction was by genotoxicity mediated by DNAbinding. For a preliminary investigation on a potential stimulatory effect on liver DN A replication and ceU division, [\(^{14}\)]thymidine was admlnistered i.p. 3.5 h before sacrifice of the [\(^3\)H]HCH-treated mice. The alpha-isomer was found to be more potent than the gamma-isomer in this respect. Taken together, our data allow the conclusion that the non- mutational processes must be more important for the carcinogenicity of HCH.}, subject = {Toxikologie}, language = {en} } @misc{SchlatterLutz1990, author = {Schlatter, J. and Lutz, Werner K.}, title = {The carcinogenic potential of ethyl carbamate (urethane): risk assessment at human dietary exposure levels}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60826}, year = {1990}, abstract = {Ethyl carbamate is found in fermented foods: bread contains 3-15 ng/g, stone-fruit brandies 200-20,000 ngfg, and about one-third of table-wine samples analysed contained more than 10 ng/g. In animals, ethyl carbamate is degraded to C02, H20 and NH3, with intermediate formation ofethanol. This degradation has been shown tobe inhibited (postponed) in the mouse by ethanol concentrations in the blood of about 0.15\% and higher. A quantitatively minor pathway involves a two-step oxidation of the ethyl group to vinyl carbamate and epoxyethyl carbamate, the postulated electrophilic moiety that reacts with DNA. This reaction is probably the mode of the mutagenic action observed in many cellular and animal systems. The fact that only vinyl carbamate, but not ethyl carbamate, is mutagenic in a standard Ames test is probably because there is insufficient production of the intermediate oxidation product in the standard test. Consistent with this metabolism is the carcinogenic activity of ethyl carbamate in various animal species and in different organs; this activity can be seen even after a single high dose in early life. Quantitative analysis of the total tumour incidences after chronic exposure of rats and mice to 0.1-12.5 mg ethyl carbamate/kg body weightjday in the drinking-water showed a dose-related increase. The main target organs were the mammary gland (female rats and mice having similar susceptibilities) and the Jung (mice only). On the basis of sex- and organ-specific tumour data and with a linear extrapolation to a negligible increase of the lifetime tumour incidence by 0.0001\% ( one additional tumour in one mil{\"u}on individuals exposed for life), a "virtually safe dose .. of 20 to 80 ng/kg body weight/day was estimated. The daily burden reached under normal dietary habits without alcoholic beverages is in the range of about 20 ng/kg body weightfday. Regular table-wine consumption would increase the risk by a factor of up to five. Regular drinking of 20 to 40 ml stone-fruit brandy per day could raise the calculated lifetime tumour risk to near 0.01\%.}, subject = {Toxikologie}, language = {en} } @article{AlldrickLutz1989, author = {Alldrick, A. J. and Lutz, Werner K.}, title = {Covalent binding of [2-\(^{14}\)C]2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) to mouse DNA in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60832}, year = {1989}, abstract = {Fernale BALB/c mice were administered intragastrically with equimolar amounts of either [2-\(^{14}\)C]2-amino-3,8-dimethyi[ 4,5-J]qulnoxaline (MeiQx) or 2-acetylamino[9-\(^{14}\)C]fluorene (2AAF). DNA was isolated from tissues of mice killed either 6 or 24 h after administration. Analysis of liver DNA nucleotide digests by HPLC analysis revealed that all of the radioactivity was attributable to adduct formation. Tbe specific activities of DNA samples were converted to covalent bindlog indices (CBI, J.LIDOI adduct per mol DNA nucleotides/mmol chemical app6ed per kg animal body weight). CBI values of 25 and 9 were detennined for 2AAF and MeiQx in tbe llvers of mice killed 6 h after dosing. The values were in general agreement with the moderate carcinogenic potency of these compounds. The specific activities of DNA preparations obtained from the lddneys, spleens, stomachs, small intestines and large intestlnes of mice treated witb MeiQx and killed 6 h after doslng were S- to 35-times less tban those obtained witb the llver. DNA isolated from tbe lungs (a target organ for MeiQx tumorigenicity) of MeiQx-treated mice was not radiolabeUed at tbe limit of detection (CBI <0.3). With tbe exception of tbe gastrolntestinal tract, the specific activities of DNA samples isolated from mice killed 6 h after administration were higher than those from mice killed after 24 h.}, subject = {Toxikologie}, language = {en} } @misc{ParodiLutzColaccietal.1989, author = {Parodi, S. and Lutz, Werner K. and Colacci, A. and Mazzullo, M. and Taningher, M. and Grilli, S.}, title = {Results of animal studies suggest a nonlinear dose-response relationship for benzene effects}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60843}, year = {1989}, abstract = {Considering the very large industrial usage of benzene, studies in risk assessment aimed at the evaluation of carcinogenic risk at low Ievels of exposure are important. Animal data can offer indications about what could happen in humans and provide more diverse information than epidemiological data with respect to doseresponse consideration. We have considered experiments investigating metabolism, short·term genotoxicity tests, DNA adduct formation, and carcinogenicity long-term tests. According to the different experiments, a Saturation of benzene metabolism and benzene effects in terms of genotoxicity seems evident above 30 to 100 ppm. Below 30 to 60 ppm the initiating effect ofbenzene seems tobe linear fora large intervaJ ofdosages, at least judging from DNA adduct formation. Potentiallack of a promoting effect of benzene (below 10 ppm) could generate a sublinear response at nontox.ic levels of ex.posure. This possibility was suggested by epidemiological data in humans and is not confirmed or excluded by our observations with animals.}, subject = {Toxikologie}, language = {en} } @article{KuglerSteigmeierFriederichGrafetal.1989, author = {Kugler-Steigmeier, M. E. and Friederich, U. and Graf, U. and Lutz, Werner K. and Maier, P. and Schlatter, C.}, title = {Genotoxicity of aniline derivatives in various short-term tests}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60857}, year = {1989}, abstract = {Various substituted aniline derivatives were tested for genotoxicity in several short-term tests in order to examine the hypothesis that a Substitution at both ortho positions (2,6-disubstitution) could prevent genotoxicity due to steric hindrance of an enzymatic activation to electrophilic intermediates. In the Salmonellajmicrosome assay, 2,6-dialkylsubstituted anilines and 2,4,6-trimethylaniline (2,4,6-TMA) were weakly mutagenic in strain TA100 when 20\% S9 mixwas used, although effects were small compared to those of 2,4-dimethylaniline and 2,4,5-trimethylaniline (2,4,5-TMA). In Drosophila me/anogaster, however, 2,4,6-TMA and 2,4,6-trichloroaniline (TCA) were mutagenic in the wing spottestat 2-3 times lower doses than 2,4,5-TMA. In the 6-thioguanine resistance test in cultured fibroblasts, 2,4,6-TMA was again mutagenic at lower doses than 2,4,5-TMA. Two methylene-bis-aniline derivatives were also tested with the above methods: 4,4'-methylene-bis-(2-chloroaniline) (MOCA) was moderately genotoxic in al1 3 test systems whereas 4,4'-methylene-bis-(2-ethyl-6-methylaniline) (MMEA) showed no genotoxicity at all. DNA binding sturlies in rats, however, revealed that both MOCA and MMEA produced DNA adducts in the liver at Ievels typically found for moderately strong genotoxic carcinogens. These results indicate that the predictive value of the in vitro test systems and particularly the Salmonellajmicrosome assay is inadequate to detect genotoxicity in aromatic amines. Genotoxicity seems to be a general property of aniline derivatives and does not seem to be greatly influenced by substitution at both ortho positions.}, subject = {Toxikologie}, language = {en} } @article{KlotzJesaitis1994, author = {Klotz, Karl-Norbert and Jesaitis, A. J.}, title = {Physical coupling of N-formyl peptide chemoattractant receptors to G protein is not affected by desensitization}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60483}, year = {1994}, abstract = {Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils results in association of these receptors to the membrane skeleton. This is thought to be the critical event in the lateral segregation of receptors and guanyl nucleotide-binding proteins (G proteins) within the plane of the plasma membrane resulting in an interruption of the signaling cascade. In this study we probed the interaction of FPR with G protein in human neutrophils that were desensitized to various degrees. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phelys- N\(^\epsilon\)-[\(^{125}\)I]2(p-azidosalicylamido )ethyl-1 ,3 '-dithiopropionate (/MLFK-[\(^{125}\)I]ASD). The interaction if FPR with G protein was studied via a reconstitution assay and subsequent analysis of FPR-G protein complexes in sucrose density gradients. FPR-G protein complexes were reconstituted with solubilized FPR from partially and fully desensitized neutrophils with increasing concentrations of Gi purified from bovine brain. The respective EC\(_{50}\) values for reconstitution were similar to that determined for FPR from unstimulated neutrophils (Bommakanti RK et al., J Bio[ Chem 267: 757~7581, 1992). We conclude, therefore, that the affinity of the interaction of FPR with G protein is not affected by desensitization, consistent with the model of lateral segregation of FPR and G protein as a mechanism of desensitization.}, subject = {Toxikologie}, language = {en} } @article{KlotzJesaitis1994, author = {Klotz, Karl-Norbert and Jesaitis, A. J.}, title = {The interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton is energy-dependent}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60499}, year = {1994}, abstract = {Desensitization of N-fonnyl peptide chemoattractant receptors (FPR) in human neutrophils is thought to be achieved by lateral segregation of receptors and G proteins within the plane of the plasma membrane resulting in an interruption of the signalling cascade. Direct coupling of FPR to membrane skeletal actin appears to be the basis of this process~ however, the molecular mechanism is unknown. In this study we investigated the effect of energy depletion on formation of FPR-membrane skeleton complexes. In addition the effect of the protein kinase C inhibitor stauroporine and the phosphatase inhibitor okadaic acid on coupling of FPR to the membrane skeletonwas studied. Human neutrophils were desensitized using the photoreactive agonist N-formy1-met-leu-phe-1ys-N'[\(^{125}\)I]2(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (fMLFK-[\(^{125}\)I]ASD) after ATP depletion with NaF or after incubation with the respective inhibitors. The interaction of FPR with the membrane skeleton was studied by Sedimentation of the membrane skeleton-associated receptors in sucrose density gradients. Energy depletion of the cells markedly inhibited the formation of FPR-membrane skeleton complexes. This does not appear tobe related to inhibition of protein phosphorylation due to ATP depletion because inhibition of protein kinases and phosphatases bad no significant effect on coupling of FPR to the membrane skeleton. We conclude, therefore, that coupling of FPR to the membrane skeleton is an energy,dependent process which does not appear to require modification of the receptor protein by phosphorylation.}, subject = {Toxikologie}, language = {en} } @article{ShephardSengstagLutzetal.1993, author = {Shephard, S. E. and Sengstag, C. and Lutz, Werner K. and Schlatter, C.}, title = {Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60683}, year = {1993}, abstract = {2-Acetylaminofluorene (2-AAF) was administered at Ievels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic micc bearing the lacl genein a Iambda vector (Big Blue® mice). The Iambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10\(^5\) plaques wcre screened per animal for the appearance of a bluc colour, indicative of mutations in the lac/ gcnc which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10\(^{-5}\) (pooled results of two animals, 8 mutant plaques/289 530 plaques). At 300 ppm in the diet, the rate of 3.5 X 10\(^{-5}\)(8/236 300) was not significantly increased over background. At 600 ppm in the dict, the rate increased approximately 3 fold to 7.7 x 10\(^{-5}\) (17 /221240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose Ievels 00-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2·AAF.}, subject = {Toxikologie}, language = {en} } @phdthesis{Duraphe2010, author = {Duraphe, Prashant}, title = {Identification and characterization of AUM, a novel human tyrosine phosphatase}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-44256}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Protein Phosphatasen werden aufgrund der Aminos{\"a}uresequenzen ihrer aktiven Zentren in drei große Familien unterteilt. In einer neu entdeckten Familie von Phosphatasen ist das aktive Zentrum durch die Sequenz DXDX(T/V) charakterisiert. Diese Aspartat-abh{\"a}ngigen Phosphatasen geh{\"o}ren zu der Superfamilie der Hydrolasen vom Haloazid Dehalogenase(HAD)-Typ, einer evolution{\"a}r konservierten und ubiquit{\"a}r verbreiteten Enzymfamilie. Bislang konnten 58 menschliche HAD Enzyme durch Datenbankanalysen identifiziert werden. Ihre Funktionen sind jedoch nach wie vor nur rudiment{\"a}r verstanden. Im Rahmen dieser Arbeit wurde zun{\"a}chst das Komplement aller menschlichen HAD Phosphatasen durch Datenbank-Recherchen erfasst. Zusammen mit phylogenetischen Analysen gelang es, eine zum damaligen Zeitpunkt unbekannte, putative Phosphatase zu identifizieren, die eine vergleichsweise hohe Sequenz-Homologie zu der Zytoskelettregulierenden HAD Phosphatase Chronophin aufweist. Dieses neuartige Enzym wurde kloniert und mit biochemischen und zellbiologischen Methoden charakterisiert. Auf der Basis dieser Befunde bezeichnen wir dieses neuartige Protein als AUM (actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase).Mittels Northern blot, real-time PCR und Western blot Analysen konnte gezeigt werden, dass AUM in allen untersuchten menschlichen und murinen Geweben exprimiert wird. Die h{\"o}chste Expression konnte in Hodengewebe nachgewiesen werden. Durch immunohistochemische Untersuchungen konnte gezeigt werden, dass AUM spezifisch in reifenden Keimzellen mit einem Expressionsmaximum zum Zeitpunkt der Spermiogenese exprimiert wird. Um die Substratpr{\"a}ferenz von AUM zu charakterisieren, wurde zun{\"a}chst ein peptidbasierter in vitro Phosphatase-Substrat-Screen durchgef{\"u}hrt. Hierbei wurden 720 aus menschlichen Phosphoproteinen abgeleitete Phosphopeptide untersucht. Interessanterweise dephosphorylierte AUM ausschließlich Phosphotyrosin (pTyr)-enthaltende Peptide. Nur 17 pTyr-Peptide (~2\% aller untersuchten Peptide) fungierten als AUM-Substrate. Diese Daten legen eine hohe Substratspezifit{\"a}t von AUM nahe. Zu den putativen AUM Substraten geh{\"o}ren Proteine, die in die Dynamik der Zytoskelett-Reorganisation sowie in Tyrosin Kinasevermittelte Signalwege eingebunden sind. In {\"U}bereinstimmung mit den Ergebnissen dieses Phosphopeptid-Screens konnte mittels Phosphatase overlay assays sowie in Zellextrakten aus Pervanadat-behandelten HeLa Zellen demonstriert werden, dass AUM eine begrenzte Anzahl Tyrosin-phosphorylierter Proteinen dephosphorylieren kann.In zellul{\"a}ren Untersuchungen wurde die m{\"o}gliche Rolle von AUM im Rahmen der durch den epidermalen Wachstumsfaktor (EGF) ausgel{\"o}sten Tyrosin-Phosphorylierung in einer Spermatogonien Zelllinie (GC-1 spg-Zellen) analysiert. So konnte nachgewiesen werden, dass die {\"U}berexpression von AUM zu einer moderaten Abnahme Tyrosin phosphorylierter Proteine nach EGF-Stimulation f{\"u}hrte. Im Gegensatz dazu l{\"o}ste jedoch die durch RNAInterferenz vermittelte Depletion von endogenem AUM einen robusten Anstieg Tyrosinphosphorylierter Proteine aus, zu denen auch der EGF-Rezeptor selbst z{\"a}hlt. Zus{\"a}tzlich zu dem EGF-Rezeptor wurde die Src-Kinase im Zuge des Phosphopeptid- Screens als m{\"o}gliches AUM Substrat identifiziert. Daher wurden in vitro Kinase/Phosphatase-Assays mit gereinigtem Src und AUM durchgef{\"u}hrt. Mit diesem Ansatz konnte erstmals gezeigt werden, dass AUM in der Lage ist, die Src-Kinase zu aktivieren, w{\"a}hrend Src AUM phosphoryliert und die AUM Phosphatase-Aktivit{\"a}t blockiert. Diese Ergebnisse deuten auf eine gekoppelte, wechselseitige Regulation von AUM und Src hin. Obwohl die Details dieser Regulation derzeit noch unklar sind, zeigen unsere initialen Ergebnisse, dass AUM die Src-Aktivit{\"a}t unabh{\"a}ngig von seiner Phosphatase Aktivit{\"a}t steigert, w{\"a}hrend Src die AUM Phosphatase-Aktivit{\"a}t Kinase-abh{\"a}ngig vermindert. Auf zellul{\"a}rer Ebene sind AUM-depletierte Zellen durch Ver{\"a}nderungen der Aktin- Zytoskelett-Dynamik und der Zelladh{\"a}sion charakterisiert. So weisen AUM-defiziente Zellen stabilisierte Aktin Streßfasern und vergr{\"o}ßerte fokale Adh{\"a}sionen auf. Weiterhin sind AUMdepletierte Zellen durch ein beschleunigtes spreading auf Fibronektin gekennzeichnet. Wir haben mit AUM ein bisher nicht beschriebenes Mitglied der Familie Aspartat-abh{\"a}ngiger Phosphatasen entdeckt. In dieser Arbeit ist es gelungen, AUM phylogenetisch, biochemisch und zellbiologisch zu charakterisieren. Unsere Ergebnisse legen nahe, dass AUM einen wichtigen, neuartigen Regulator der Src-vermittelten Zytoskelett-Dynamik im Rahmen der Zelladh{\"a}sion und Migration darstellt.}, subject = {Tyrosin}, language = {en} } @article{CantoreggiLutz1993, author = {Cantoreggi, S. and Lutz, Werner K.}, title = {Covalent binding of styrene to DNA in rat and mouse}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60693}, year = {1993}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{GunzShephardLutz1993, author = {Gunz, D. and Shephard, S. E. and Lutz, Werner K.}, title = {Can nongenotoxic carcinogens be detected with the lacI transgenic mouse mutation assay?}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60707}, year = {1993}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{LutzSchlatter1992, author = {Lutz, Werner K. and Schlatter, J.}, title = {Chemical carcinogens and overnutrition in diet-related cancer [commentary]}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60712}, year = {1992}, abstract = {The intake of known dietary carclnogens was compiled and the cancer risk was estlmated on the basis of carcinogenic potencies in animals as derived from the Carcinogenic Potency Database by Gold and co-workers. The total cancer risk was compared with the number of cancer cases attributed by epidemiologists to dietary factors (one-third of all cancer cases, i.e. -80 000 per one million Jives). Except for alcohol, the known dietary carcinogens could not account for more than a few bundred cancer cases. Tbis was seen both with tbe DNA-reactive carcinogens (beterocyclic aromatic amines, polycyclic aromatic hydrocarbons, N-nitroso compounds, estragole, aflatoxin B., ethyl carbamate, to name the most important factors) as wen as with those carclnogens wbich have not been shown to react with DNA (e.g. caffelc acid and the carcinogeruc metals arsenic and cadmium). Residues and contaminants turned out to be negligible. Among the various pmsibilities to explain the discrepancy we investigated the roJe of ovemutritlon. Dietary restriction in animals is weil known for its strong reducing effect on spontaneous tumor formation. These data can be used to derive a carcinogenic potency for excess macronutrients: tbe tumor incidence seen with the restrlcted animals is taken as a control value and the increased tumor incidence in the animals fed ad libitum is attributed to the additional feed iotake. For excess standard diet in rats, a carcinogenic potency TD50 of 16 glkg/day was deduced from a recent study. Ovemutrition in Switzerland, estimated to be 5.5 kcallkg/day, was converted to excess food (1.9 g/kg/day) and tbe cancer incidence was calculated. The result, 60 000 cancer cases per one million Jives, is provocatively close to the number of cases not explained by the known dietary chemical carcinogens. Mechanistic studies will be required to test our hypothesis and investigate the role of different types of macronutrients in ovemutrition.}, subject = {Toxikologie}, language = {en} } @article{CantoreggiLutz1992, author = {Cantoreggi, S. and Lutz, Werner K.}, title = {Investigation of the covalent binding of styrene-7,8-oxide to DNA in rat and mouse}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60721}, year = {1992}, abstract = {Styrene-7,8-oxide (SO), the main intennediate metabolite of styrene, induces hyperkeratosis and tumors in the forestomach of rats and mice upon chronic administration by gavage. The aim of this study was to investigate wbether DNA binding could be responsible for the carcinogenic effect observed. [7-\(^3\)H]SO was administered by oral gavage in com oll to male CD rats at two dose levels (1.65 or 240 mg/kg). After 4 or 24 h, forestomach, glandular stomach and Uver were exclsed, DNA was isolated and its radioactivity detennined. At the 4 h time polnt, the DNA radioactivity was below the Iimit of detection in the torestornach and the liver. Expressed in the units of the covalent bindlng Index, CBI = (pmol adduct/mol DNA nucleotide)/(mmol cbemical administeredlkg body wt), the DNA-binding potency was below 2.6 and 2.0 respectively. In the glandular stomach at 4 b, and in most 24 b samples, DNA was slightly radiolabeled. Enzymatic degradation of the DNA and separation by HPLC ofthe normal nucleotides sbowed that the DNA rad.ioactivity represented biosynthetic incorporation of radlolabel into newly synthesized DNA. The Iimit of detection of DNA adducts in the glandular stomach was 1.0. In a second experlment, [7-\(^3\)H]SO was administered by i.p. injection to male 86C3Fl rnice. Liver DNA was analyzed after 2 h. No radloactivity was detectable at a Iimit of detection of CBI < 0.6. In agreement with the relatively long half-life of SO in animals, the cbemical reactivity of SO appears to be too low to result in a detectable production of DNA adducts in an in vivo situation. Upon comparison with the DNA-binding of other carcinogens, a purely genotoxic mechanism of tumorigenJc action of SO is unlikely. The observed tumorigenic potency in the forestomach could be the result of strong tumor promotion by high-dose cytotoxicity foUowed by regenerative hyperplasia.}, subject = {Toxikologie}, language = {en} } @phdthesis{Vogl2011, author = {Vogl, Silvia}, title = {Investigation of individual differences in the metabolic elimination of drugs by the polymorphic enzymes CYP2C9, 2C19 and 2D6 based on metabolite profiling by LC-MS/MS}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67216}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Mit der vorliegenden Studie sollte zu dem wichtigen Forschungsfeld der Pharmakogenetik beigetragen werden, indem zum einen eine einfache und sichere kombinierte Ph{\"a}notypisierung der drei zuvor erw{\"a}hnten CYPs (CYP2D6, CYP2C9 und CYP2C19) entwickelt, und zum anderen die Vorhersagekraft des Genotyps f{\"u}r den gemessenen Ph{\"a}notyp n{\"a}her untersucht werden sollte. Es ist uns gelungen eine sichere, einfache, schnelle und kombinierte Ph{\"a}notypisierung der beiden wichtigen Monooxygenasen CYP2D6 und CYP2C9 zu etablieren. Zun{\"a}chst wurden dazu Wechselwirkungsstudien mit den ausgew{\"a}hlten Testsubstanzen Dextromethorphan (DEX, CYP2D6), Flurbiprofen (FLB, CYP2C9) und Omeprazole (OME, CYP2C19) durchgef{\"u}hrt. Es konnte gezeigt werden, dass DEX und FLB als Kombination verabreicht werden k{\"o}nnen. Die Gabe von OME gemeinsam mit FLB ver{\"a}ndert jedoch das Ergebnis der CYP2C9 Ph{\"a}notypisierung. Dies ist eine neue Erkenntnis, denn noch 2004 wurde ein Ph{\"a}notypisierungscocktail ver{\"o}ffentlicht, der die Kombination von FLB und OME enthielt. Bei der genannten Studie wurden jedoch, unseres Wissens nach, keine Wechselwirkungsstudien zu den einzelnen Testsubstanz-Kombinationen durchgef{\"u}hrt. Die von uns entwickelte Ph{\"a}notypisierungsmethode wurde durch Wechselwirkungsstudien verifiziert. Sie ist jedoch auch in anderen Bereichen den bisher ver{\"o}ffentlichten ph{\"a}notypisierungscocktails {\"u}berlegen. Zum einen wurden nur sehr kleine Dosen sicherer Testsubstanzen verwendet. Dies wurde durch Entwicklung neuer, sensitiver LC-MS/MS Methoden erm{\"o}glicht. Zum anderen ist diese neue Prozedur schnell und nicht-invasiv durchf{\"u}hrbar. Nach Verabreichung der Testsubstanz muss der Urin nur f{\"u}r zwei Stunden gesammelt werden. Zudem weisen unsere Ergebnisse darauf hin, dass die normalerweise durchgef{\"u}hrte, aufwendige Glucuronidspaltung des CYP2D6 abh{\"a}ngigen DEX-Metaboliten, Dextrorphan, vermutlich vernachl{\"a}ssigt werden kann. Die wichtigsten Ergebnisse dieser Studie sind jedoch die Einblicke, die in die Vorhersagekraft der CYP2D6 und CYP2C9 Genotypen f{\"u}r die entsprechenden Ph{\"a}notypen gewonnen werden konnten. Fast 300 ph{\"a}notypisierte Kaukasier wurden auch in Hinsicht auf die wichtigsten varianten Allele von CYP2D6, CYP2C9 und CYP2C19 mithilfe bekannter und neu etablierter Methoden genotypisiert. Aufgrund der parallelen Ph{\"a}no- und Genotypisierung konnten Geno- und Ph{\"a}notyp direkt korreliert werden. Mit linearen Modellen war es m{\"o}glich, allen detektierten varianten CYP2D6- und CYP2C9-Allelen Aktivit{\"a}tskoeffizienten zuzuweisen. Diese k{\"o}nnen nun verwendet werden, um den Beitrag der einzelnen Allele zur resultierenden Enzymaktivit{\"a}t zu bestimmen, wodurch sich die Vorhersage dieser Aktivit{\"a}t ausgehend vom Genotyp verbessern lassen sollte. Besonders f{\"u}r CYP2D6 erm{\"o}glicht das neue Korrelationsmodel pr{\"a}zisere Vorhersagen des Ph{\"a}notyps als bisher ver{\"o}ffentlichte Modelle. Zusammengefasst leistet diese Studie durch die Entwicklung eines sicheren und einfachen Ph{\"a}notypisierungsprozesses f{\"u}r CYP2D6 und CYP2C9 und durch die Bestimmung von Aktivit{\"a}tskoeffizienten f{\"u}r alle einbezogenen CYP2D6 und CYP2C9 Allele und der damit verbundenen pr{\"a}ziseren Vorhersage des Ph{\"a}notyps ausgehend vom Genotyp einen wesentlichen Beitrag zum Forschungsfeld der Pharmakogenetik.}, subject = {Pharmakogenetik}, language = {en} } @article{HegiSagelsdorffLutz1989, author = {Hegi, M.E. and Sagelsdorff, P. and Lutz, Werner K.}, title = {Detection by \(^{32}\)P-postlabeling of thymidine glycol in gamma-irradiated DNA}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60863}, year = {1989}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{SagelsdorffLutzSchlatter1988, author = {Sagelsdorff, P. and Lutz, Werner K. and Schlatter, C.}, title = {DNA methylation in rat liver by daminozide, 1,1-dimethylhydrazine, and dimethylnitrosamine}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60875}, year = {1988}, abstract = {DNA Methylation in Rat Li ver by Daminozide, 1, 1-Dimethylhydrazine, and Dimethylnitrosamine. SAGELSDORFF, P., LUTZ, W. K., AND ScHLAITER C. (1988). Fundam. Appl. Toxico/. 11, 723-730. [methyP4C]Daminozide (succinic acid 2',2'-dimethylhydrazide; 37 mgjkg), l,l( 14C]dimethylhydrazine (UDMH; 19 mgtkg), and (14C]dimethylnitrosamine (DMNA; 0.1 mg/ kg) were administered by oral gavage to male Sprague-Dawley rats. After 24 hr, the animals were killed and DNA was purified from the livers to constant specific radioactivity. After enzymatic degradation of the DNA to the 3'-deoxynucleotides the Ievel of DNA methylation was determined by HPLC analysis. Radiolabeled 7-methylguanine (7mG) was identified by cochromatography with unlabeled 7mG added as standard after acidic depurination of DNA and HPLC analysis ofpurines and apurinic acid. All three compounds were found to methylate DNA. The relative potencies were 1:47:4900 for daminozide:UDMH:DMNA. With [methyPH]UDMH, the formation of7mG was investigated as a function of dose administered, at 20, 2, and 0.2 mgj kg. The methylation ofDNA was strictly proportional to the dose. The data were used to compare the Ievel of DNA alkylation derived from residues of daminozide and UDMH in treated apple with the genotoxicity of the intake of N-nitroso compounds in Germany and Japan. It is estimated that these residues could Iead to a DNA methylation in the Ii ver of about 6\% of an average exposure to DMNA}, subject = {Toxikologie}, language = {en} } @article{BaertschLutzSchlatter1991, author = {Baertsch, A. and Lutz, Werner K. and Schlatter, C.}, title = {Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60743}, year = {1991}, abstract = {1 ,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-tenn bioassay using gavage in com oil (24 and 48 mg/kg/day), but not by inhalation (up to 150-250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Fernale F-344 rats (183-188 g) were exposed to [1,2-14C]DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l = 80 ppm for 4 h) or to a peak concentration (up to 18 mg/1 = 4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/k:g. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymaticaBy hydrolyzed to the 3' -nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density' indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The Ievel of DNA adducts was expressed in the dose-nonnalized units ofthe Covalent Binding Index, CBI = f.Lmol adduct per mol DNA nucleotide/ mmol DCE per kg body wt. In liver DNA, the different exposure regimens resulted in markedly different CBI values of 1.8 and 69, for "constant-low" and ''peak" DCE exposure Ievels. In the Jung, the respective values were 0.9 and 31. It is concluded that the DNA darnage by DCE depends upon the concentration-time profile and that the carcinogenic potency determined in the gavage study should not be used for low-Ievel inhalation exposure.}, subject = {Toxikologie}, language = {en} } @article{OhgakiLudekeMeieretal.1991, author = {Ohgaki, H. and Ludeke, B. I. and Meier, I. and Kleihues, P. and Lutz, Werner K. and Schlatter, C.}, title = {DNA methylation in the digestive tract of F344 rats during chronic exposure to N-methyl-N-nitrosourea}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60759}, year = {1991}, abstract = {The formation of \(O^6\)-methyldeoxyguanosine (\(O^6\)-MedGuo) was determined by an immuno-slot-blot assay in DNA of various tissues of F344 rats exposed to N-methyl-N-nitrosourea (MNU) in the drinking waterat 400 ppm for 2 weeks. Although the pyloric region of the glandular stomach is a target organ under these experimental conditions, the extent of DNA methylation was highest in the forestomach (185 \(\mu\)mol \(O^6\)-MedGuojmol guanine). Fundus (91 J.!moljmol guanine) and pylorus (105 J.!moljmol guanine) of the glandular stomach, oesophagus (124 \(\mu\)mol/mol guanine) and duodenum (109 )lmoljmol guanine) showed lower Ievels of \(O^6\) - MedGuo but differed little between each other. Thus, no correlation was observed between target organ specificity and the extent of DNA methylation. This is in contrast to the gastric carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which preferentially alkylates DNA of the pylorus, the main site of induction of gastric carcinomas by this chemical. In contrast to MNU, the nonenzymic decomposition of MNNG is accelerated by thiol compounds (reduced glutathione, L-cysteine), which are present at much higher concentrations in the glandular stomach than in the forestomach and oesophagus. During chronic exposure to MNNG (80 ppm), mucosal cells immunoreactive to 0 6-MedGuo are limited to the luminal surface [Kobori et al. (1988) Carcinogenesis 9:2271-2274]. Although MNU (400 ppm) produced similar Ievels of \(O^6\)-MedGuo in the pylorus, no cells containing methylpurines were detectable by immunohistochemistry, suggesting a more uniform methylation of mucosal cells by MNU than by MNNG. After a single oral dose of MNU (90 mg/kg) cells containing methylpurines were unequivocally identified using antibodies to \(O^6\)-MedGuo and the imidazole-ring-opened product of 7-methyldeoxyguanosine. In the gastric fundus, their distribution was similar to those methylated by exposure to MNNG, whereas the pyloric region contained immunoreactive cells also in the deeper mucosallayers. After a 2-week MNU treatment, the rate of cell proliferation, as determined by bromodeoxyuridine immunoreactivity, was only slightly enhanced in the oesophagus andin the fundus, but markedly in the forestomach and the pyloric region of the glandular stomach. lt is concluded that the overall extent of DNA methylation, the distribution of alkylated cells within the mucosa and the proliferative response all contribute to the organ-specific carcinogenicity of MNU.}, subject = {Toxikologie}, language = {en} } @article{Lutz1991, author = {Lutz, Werner K.}, title = {Dose-response relationship for chemical carcinogenesis by genotoxic agents}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60766}, year = {1991}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{BussCaviezelLutz1990, author = {Buss, P. and Caviezel, M. and Lutz, Werner K.}, title = {Linear dose-response relationship for DNA adducts in rat liver from chronic exposure to aflatoxin B1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60779}, year = {1990}, abstract = {Male F-344 rats were given eH]aßatoxin B1 (AFB1) in the drinking water at three exposure Ievels (0.02, 0.6, 20 J,Lgll, resulting in average dose Ievels of 2.2, 73, 2110 nglkg per day). After 4, 6 and 8 weeks, DNA was ~ted frorn the livers and analyzed for aßatoxin-DNA adducts. Tbe Ievel of DNA adducts did not increase significantly after 4 weeks, indicating that a steady-state for adduct formation and removal had nearly been reached. At 8 weeks, the adduct Ievels were 0.91, 32 and 850 nucleotide-aßatoxin adducts per to' nucleotides, i.e. clearly proportional to the dose. At the high dose Ievel, a near SO\% tumor incidence would be expected in a 2-year bioassay with F -344 rats while the low dose used is within the range of estlmated human dietary exposures to aßatoxin in W estem countries. The proportionality seen between exposure and steady-state DNA adduct Ievel is discussed with respect to a linear extrapolation of the tumor risk to low dose.}, subject = {Toxikologie}, language = {en} } @article{Lutz1990, author = {Lutz, Werner K.}, title = {Dose-response relationship and low dose extrapolation in chemical carcinogenesis [commentary]}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60789}, year = {1990}, abstract = {Data supporting various dose-respome relationships in chemical carcinogenesis are summarized. General principles are derived to explain the relationships between exposure dose, JI>NA adduct Ievel, induction of genetic changes, and tumor incidence. Some mechanistic aspects of epigenetic carcinogens (stimulation of ceU division and maldlfl'erentlation) are analyzed in a similar way. In a bomogeneous pnpulation, non-linearities are frequent. They are due to pbenomena of induction or saturation of enzymatic activities and to the multi-step nature of carcinog~: if a carcinogen acce1erates more than one step, the SUperposition of the dose- response curves for the indJvidual steps can result in an exponential relationship. A fourth power of the dose was the maximum seen in animals (fonnaldehyde). At the lowest dose Ievels, a proportionality between dose and tumor induction is postulated independent of the mechanism of action if the carcinogen aceeierotes the endogenous proass responsible for spootaneous tumor formation. Low-dose thresholds are expected only for situations where the carcinogen acts in a way that has no endogenous counterpart. Epidemiologfcal studies in humans show linear dose- response curves in all but two investigations. The difference from the strongly nonlinear slopes ·seen in animal studies could be due to the heterogeneity of the human population: if the individual sensitivity to a carcinogen is governed by a large number of genetic and Iife-style factors, the non-linea.rities will tend to cancel each other out and the dose- response curve becomes 'quasi-linear'.}, subject = {Toxikologie}, language = {en} } @article{HegiUlrichSagelsdorffetal.1990, author = {Hegi, M. E. and Ulrich, D. and Sagelsdorff, P. and Richter, C. and Lutz, Werner K.}, title = {No measurable increase in thymidine glycol or 8-hydroxydeoxyguanosine in liver DNA of rats treated with nafenopin or choline-devoid low-methionine diet}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60790}, year = {1990}, abstract = {Male rats were treated for 2 months with 1000 ppm nafenopin in the diet or for 4 or 7 days with a choline-devoid low-methionine diet. DNA was isolated from the livers and analyzed for the presence of cis-thymidine glycol-3'-phosphate (cis-dTGp) by 32P-postlabeling and for the Ievel of 8-hydroxy-deoxyguanosine (8-0H-dG) by electrochemical detection (ECD). In no DNA sample was the Ievel of cis-dTGp above the Iimit of detection of 1 modified thymidine per 106 nucleotides. With 8-0H-dG, a background Ievel of this modification of 20 8-0H-dG per 106 nucleosides was found in liver DNA of control rats, which was not affected by either treatment. It is postulated for thymidine glycol that a potential increase was below the Iimit of detection or was rapidly repaired in vivo and that the steady-state Ievel of endogenous 8-hydroxydeoxyguanosine appears not tobe influenced by the treatments chosen.}, subject = {Toxikologie}, language = {en} } @article{MeierShephardLutz1990, author = {Meier, I. and Shephard, S. E. and Lutz, Werner K.}, title = {Nitrosation of aspartic acid, aspartame, and glycine ethylester. Alkylation of 4-(p-nitrobenzyl)pyridine (NBP) in vitro and binding to DNA in the rat}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60804}, year = {1990}, abstract = {In a colorimetric assay using 4-( p-nitrobenzyl)pyridine (NBP) as a nucleophilic scavenger of alkylating agents, the nitrosation and alkylation reactions were investigated for a number of amino acids and derivatives. The alkylating activity increased with the square of the nitrite concentration. The nitrosation rate constants for aspartic acid, aspartame, and glycine ethylester ( = precursors C) were 0.08, 1.4 and ~ 0.2, respectively, expressed in terms of the pH-dependent \(k_2\) rate constant of the equation dNOCjdt = \(k_2\) • (C]· [nitrite]\(^2\) • The rates correlated inversely with the basicity of the amino group. The stability of the alkylating activity was astonishingly high, both in acid and at neutral pH. Half-lives of 500, 200, and 30 min were determined for aspartic acid (pH 3.5), aspartame (pH 2.5), and glycine ethylester (pH 2.5). Values of 60, 15, and 2 min; respectively, were found at pH 7. It is concluded that rearrangement of the primary N-nitroso product to the ultimate alkylating agent could be rate-limiting. The potential of nitrosated a-amino acids to bind to DN A in vivo was investigated by oral gavage of radiolabelled glycine ethylester to rats, followed irnmediately by sodium nitrite. DNA was isolated from stomach and liver and analysed for radioactivity and modified nucleotides. No indication of DNA adduct formation was obtained. Based on an estimation of the dose fraction converted from glycine ethylester to the nitroso product under the given experimental conditions, the maximum possible DNA-binding potency of nitroso glycine ethylester is about one order of magnitude below the methylating potency of N-nitrosomethylurea in rat stomach. The apparent discrepancy to the in vitro data could be due to efficient detoxification processes in mammalian cells.}, subject = {Toxikologie}, language = {en} } @article{Lutz1990, author = {Lutz, Werner K.}, title = {Endogenous genotoxic agents and processes as a basis of spontaneous carcinogenesis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60816}, year = {1990}, abstract = {A list ofendogenaus DNA·damaging agents and processes is given. Endogenaus e/ectrophiles are found with the cosubstrates of physiological transfer reactions (S-adenosylrnethionine for methylation, A TP for phosphorylation, NAD\(^+\) for ADP-ribosylation, acetyl CoA for acetylation). Aldehyde groups (glyceraldehyde- 3-phosphate, formaldehyde, open forms of reducing sugars, degradation products of peroxidation) or alkylating degradation products derived from endogenaus nitrose compounds represent additional possibilities. Radical-forming reactions include leakage of the superoxide anion radical from terminal cytochromes and redox cycles, hydroxyl radical formation by the Fenton reaction from endogenaus hydrogen peroxide, and the formation of lipid peroxides. Genetic instability by spontaneaus deaminations and depurinations as well as replicative instability by tautomer errors andin the presence of mutagenic metal ions represent a third important dass of endogenaus genotoxic processes. The postulated endogenaus genotoxicity could form the mechanistic basis for what is called 'spontaneous' tumor incidence and explain the possibility of an increased tumor incidence after treatment of animals with non-genotoxic compounds exhibiting tumor-promoting activity only. Individual differences are expected to be seen also with endogenaus DNA damage. The presence of endogenaus DNA darnage implies that exogenaus DNAcarcinogen adducts give rise to an incremental darnage which is expected to be proportional to the carcinogen dose at lowest Ievels. An increased tumor risk due to exposure to exogenaus genotoxic carcinogens could therefore be assessed in terms of the background DNA damage~ for instance in multiples of the mean Ievel or of the interindividual variability in a population.}, subject = {Toxikologie}, language = {en} }