Institut für Pharmakologie und Toxikologie
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Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing
(2015)
The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program “Predict-IV”; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of “omics” technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling.
This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day.
Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors.
Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling.
Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions.
Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo.
Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound’s distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.
The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains.
INTRODUCTION: Recently, we could show that angiotensin II, the reactive peptide of the blood pressure-regulating renin-angiotensin-aldosterone-system, causes the formation of reactive oxygen species and DNA damage in kidneys and hearts of hypertensive mice. To further investigate on the one hand the mechanism of DNA damage caused by angiotensin II, and on the other hand possible intervention strategies against end-organ damage, the effects of substances interfering with the renin-angiotensin-aldosterone-system on angiotensin II-induced genomic damage were studied.
METHODS: In C57BL/6-mice, hypertension was induced by infusion of 600 ng/kg • min angiotensin II. The animals were additionally treated with the angiotensin II type 1 receptor blocker candesartan, the mineralocorticoid receptor blocker eplerenone and the antioxidant tempol. DNA damage and the activation of transcription factors were studied by immunohistochemistry and protein expression analysis.
RESULTS: Administration of angiotensin II led to a significant increase of blood pressure, decreased only by candesartan. In kidneys and hearts of angiotensin II-treated animals, significant oxidative stress could be detected (1.5-fold over control). The redox-sensitive transcription factors Nrf2 and NF-κB were activated in the kidney by angiotensin II-treatment (4- and 3-fold over control, respectively) and reduced by all interventions. In kidneys and hearts an increase of DNA damage (3- and 2-fold over control, respectively) and of DNA repair (3-fold over control) was found. These effects were ameliorated by all interventions in both organs. Consistently, candesartan and tempol were more effective than eplerenone.
CONCLUSION: Angiotensin II-induced DNA damage is caused by angiotensin II type 1 receptor-mediated formation of oxidative stress in vivo. The angiotensin II-mediated physiological increase of aldosterone adds to the DNA-damaging effects. Blocking angiotensin II and mineralocorticoid receptors therefore has beneficial effects on end-organ damage independent of blood pressure normalization.
Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.
Im Rahmen dieser Arbeit wurden immortalisierte humane Keratinozyten (HaCaT-Zelllinie) über eine Dauer von 24 Stunden mit Terahertzstrahlung der Frequenz 0,106 THz und einer Leistungsflussdichte von 2 mW/cm² behandelt und anschließend mittels „cytokinesis-block micronucleus cytome assay“ ausgewertet. Ferner wurden Proben der gleichen Zelllinie über einen Zeitraum von 24 Stunden Temperaturen zwischen 37 °C und 42 °C ausgesetzt und zum einen auf Hinweise für Gentoxizität mit Hilfe des Mikrokerntests untersucht und zum anderen mittels Western Blot die Expressionsrate von Hitzeschockproteinen der 70 kDa Familie bestimmt.
Die der Terahertzstrahlung ausgesetzten Zellen zeigten gegenüber den Kontrollen keine signifikanten Veränderungen der Marker für chromosomale Aberrationen oder der Zellteilung. Die der erhöhten Temperatur ausgesetzten Zellen zeigten einen signifikanten Anstieg der Mikrokernraten und weiterer Marker für Gentoxizität sowie eine damit korrelierende Zunahme der synthetisierten Proteinmenge der Hsp70 Proteine im Rahmen der Hitzeschockreaktion.
Adenosin steuert seine physiologische Funktionen über die vier G-Protein gekoppelten Adenosinrezeptoren A1, A2A, A2B und A3 und kann enormen Einfluss auf die Zellphysiologie und das Immunsystem haben. Hohe Adenosinkonzentrationen in Tumorgeweben haben das Interesse vieler Forschungsgruppen geweckt und den Nachweis von AR und dessen tumorfördernde sowie -hemmende Wirkung auf diverse Krebszellen nach sich gezogen. Melanome sind für 90% der letal ausgehenden Hautkrebsformen verantwortlich, da bereits kleinste Formen metastasieren können und vielmals eine rein chirurgische Therapie keine Heilung verspricht. Bei ungünstiger Prognose schränken adjuvante und systemische Therapieergänzungen die Lebensqualität erheblich ein. Auf der Suche nach gezielteren und schonenderen Therapieansätzen gilt es, die Rolle des Adenosins im Tumorgeschehen weiter aufzuklären, was den Nachweis und die Charakterisierung von AR und deren rezeptorspezifisch-gekoppelte Effekte voraussetzt. Daher sollten in der hier vorliegenden Arbeit die fünf humanen Melanomzelllinien A375, Brown, MV3, SK MEL23 und MEL2A auf die Expression von AR untersucht werden.
Zu Beginn wurden die Zellen mit Hilfe von Bindungsexperimenten unter Einsatz radioaktiv markierter Liganden ([3H]HEMADO, [3H]CCPA, [3H]NECA und [3H]ZM 241385) auf das Vorliegen der vier AR-Subtypen untersucht. A1- und A3-AR wurden nach Bindungsstudien mit [3H]CCPA bzw. [3H]HEMADO ausgeschlossen und nicht weiter untersucht. Durch Bindungsstudien mit [3H]ZM 241385 konnten in den Zellen Brown, MV3, SK-MEL23 und MEL2A geringe Mengen von A2B-Rezeptoren detektiert werden.
Es folgten funktionelle Untersuchungen zur Stimulation der AC mit den Liganden NECA und CGS 21680 unter besonderem Blick auf die Differenzierung zwischen einer A2A- oder A2B vermittelten Stimulation. Ein NECA vermittelter Anstieg des c[32P]AMP im AC-Versuch bestätigte die Anwesenheit von A2B-Rezeptoren in den Zellen A375, Brown, MV3 und SK-MEL23. Durch CGS 21680 kam es hingegen zu keiner AC-Stimulation, was eine A2A-AR-Beteiligung ausschloss. Zur Bestätigung einer A2B gekoppelten AC-Aktivität wurde zum Abschluss der Versuche die Wirkung von drei selektiven Antagonisten (DPCPX, SCH 58261 und MRE 3008 F20) auf ein NECA induziertes cAMP-Signal überprüft. Mit den drei Antagonisten konnte an den Zelllinien A375, Brown und MV3 die für A2B-AR zu erwartende Wirkung nachgewiesen und damit die Expression von A2B-AR in diesen Zellen bestätigt werden.
Die gewonnenen Daten können als Ausgangspunkt weiterführender Untersuchungen genutzt werden, um die Rolle von AR in Krebszellen besser zu verstehen. Sie könnten als Grundlage für die Suche nach potenziellen Angriffspunkten für neue Therapieansätze dienen und so einen Beitrag für Fortschritte in der Behandlung von Tumoren leisten.
Der Nachweis von oxidativen Stressmarkern hat bei der Untersuchung von Krankheiten wie Diabetes, Krebs und Hypertonie an großer Bedeutung gewonnen. Vor allem 8-Oxo-2’-desoxyguanosin (8-oxodG) wird gezielt mit verschiedenen Methoden gemessen und als Marker für oxidativen Stress herangezogen. Daneben haben 8 Oxoguanin (8-oxoGua), als Produkt aus der Basenexzisionsreparatur der DNA, sowie 8-Oxoguanosin (8-oxoGuo), als Biomarker für oxidativ geschädigte RNA, bisher weniger Aufmerksamkeit bekommen. Das Renin-Angiotensin Aldosteron System (RAAS) spielt eine wichtige Rolle in der Regulierung des Blutdrucks. Im Falle einer Hypertonie werden Angiotensin II (Ang II) und Aldosteron (Aldo) über einen langen Zeitraum in erhöhter Konzentration ausgeschüttet. Dieser Umstand bewirkt eine nicht physiologische Wirkung der Hormone des RAAS, welche zu einer Induktion von oxidativem Stress führt. Die Zielsetzung dieser Arbeit ist es, die oxidative Schädigung, ausgelöst durch Ang II und Aldo, in der DNA und der RNA in vitro und in vivo nachzuweisen und dabei speziell den Biomarker 8-oxodG zu untersuchen.
In-vitro-Experimente wurden mit LLC PK1-Zellen, einer Schweinenierenzelllinie, durchgeführt. Ang II und Aldo lösten einen dosisabhängigen Anstieg der DNA Schäden in LLC PK1 Zellen aus. Eine Zeitabhängigkeit wurde für die ersten 30 Minuten gezeigt. Für die restliche Zeit (4 h) blieb der nachgewiesene DNA Schaden konstant. Der FPG Comet-Assay und die immunzytochemische Färbung zeigten jeweils eine signifikante Zunahme von 8-oxodG in LLC-PK1-Zellen an, während die HPLC MS/MS Messung nur geringe Veränderungen nachwies. Das FPG Enzym erkennt neben 8-oxodG auch andere oxidierte Purine und sorgte so für eine Überbestimmung des DNA-Schadens. Bei der immunzytochemischen Färbung entsteht die Überbestimmung durch Kreuzreaktionen des 8 oxodG Antikörpers mit oxidierten Strukturen in der DNA. Der Vorteil beider Analysemethoden ist die direkte Messung von Schädigungen in der Zelle, während die HPLC-MS/MS eine Isolierung der Nukleinsäuren voraussetzt. Bei diesem Schritt kann es zur Oxidation der Marker für oxidativen Stress kommen, welche einen genauen Nachweis erschwert.
In vivo-Versuche hatten zum Ziel, die oxidativen Stressmarker 8-oxoGua, 8-oxodG und 8-oxoGuo im Urin nachzuweisen. Die Behandlung der C57BL/6-Mäuse und Sprague Dawley-Ratten (SD-Ratten) mit den Hormonen des RAAS zeigten einen Anstieg des Blutdrucks, erhöhte DNA Schäden durch oxidativen Stress sowie erhöhte Exkretionsraten der oxidativen Stressmarker. Durch eine Inhibierung des Angiotensin II-Typ1- oder Mineralkortikoidrezeptors sowie die Mutation des Gens AT1a konnte gezeigt werden, dass die Schädigungen unabhängig vom Blutdruck sind. Zudem konnte gezeigt werden, dass neben NOX4 auch andere NADPH Oxidasen für den oxidativen Stress verantwortlich sein müssen. Eine Aktivierung des Nrf2 Signalweges in den SD-Ratten hat Einfluss auf die Wirkung von Aldo.
Die Exkretionsrate der oxidativen Biomarker im 20-h-Urin der behandelten Tiere zeigen, wie sich das Gleichgewicht zwischen DNA-Reparatur und oxidativem Stress verändert. Da 80 % der DNA in RNA umgeschrieben werden, ist der Nachweis von 8 oxoGuo in den Fokus gerückt. In der praktischen Anwendung kann mit der Messung von 8 oxodG und 8-oxoGuo ein Krankheits- oder Heilungsprozess auf nicht invasive Weise verfolgt werden. Der Nachweis von 8-oxodG und 8-oxoGuo in den Nukleinsäuren stellt einen Einstieg für die Grundlagenforschung dar, da sie nur eine Momentaufnahme der Nukleinsäureschädigung in der Zelle zeigen. Meist findet eine Überbestimmung, ausgelöst durch die Messmethode, statt. In Gewebeproben kann eine Unterbestimmung vorliegen, falls nicht alle Zelltypen vom oxidativen Stress betroffen sind. Daher sollte es ein vorrangiges Ziel sein, ein stabileres Oxidationsprodukt des Guanins nachzuweisen, um das Gleichgewicht der DNA-Oxidation und Reparatur besser zu verstehen.
Reaktive Sauerstoffradikale spielen eine große Rolle bei der Entstehung von Karzinomen, im Alterungsprozess und bei kardiovaskulären Erkrankungen (Valko et al., 2007). Solche Sauerstoffradikale können unter anderem durch die NADPH-Oxidase gebildet werden (Finkel and Holbrook, 2000).
Ziel der Arbeit war es, die Rolle von ROS im Alterungsprozess und bei Bluthochdruck aufzuzeigen. Dafür wurden zwei Tiermodelle, eines mit einer geringen ROS-Konzentration und eines mit einer hohen ROS-Konzentration, verwendet. Zum einen handelt es sich um ein p47-Knockout-Mausmodell für die geringere ROS-Konzentration, im Vergleich zu alten und jungen Wildtyp-Tieren. Für die Rolle von ROS bei der Alterung wurden junge mit alten Wildtyp-Tieren verglichen. Um die Rolle der NOX2 in den ROS-Spiegeln der Organe zu ermitteln wurden gleichaltrige Wildtyp- mit p47-Knockout-Tieren verglichen. Zum anderen nutzten wir ein ren2-Tiermodell für die hohe ROS-Konzentration. Hierbei handelt es sich um ein Bluthochdruckmodell, in dem der Blutdruck mit Medikamenten normalisiert werden kann. Die Tiere wurden daher mit Ramipril (ACE-Inhibitor), Apocynin (NADPH-Oxidase-Inhibitor) und Tempol (Radikalfänger) behandelt. Als Maß für die ROS-Konzentration wurden die Superoxidionenspiegel mithilfe einer Dihydroethidium-Färbung ermittelt. Die DNA-Doppelstrangbrüche wurden mit einer γH2AX-Antikörper-Färbung nachgewiesen und die Expression von Sirtuin1 und Hsp70, welche als Anti-Aging Proteine bekannt sind, mittels Western Blot bestimmt.
Kardiomyopathien sind Erkrankungen des Herzmuskels, die mit einer kardialen Funktionsstörung einhergehen. Formen, die ohne erkennbare Ursache zu einer progredienten Dilatation und Reduktion der Kontraktilität des linken Ventrikels führen, werden als idiopathische dilatative Kardiomyopathie (DCM) bezeichnet. Sie ist der Hauptgrund für schwere Herzinsuffizienz und die damit assoziierten Einschränkungen der Lebensqualität bei jungen Erwachsenen. Neben der beeinträchtigten kardialen Funktion weisen diese Patienten oftmals auch Veränderungen im Bereich der humoralen und zellulären Immunität auf. Ein Teil der Patienten entwickelt Autoantikörper, die sich gegen den kardialen β1-adrenergen Rezeptor richten und ihn ähnlich wie der natürliche Ligand Adrenalin aktivieren. Hieraus resultiert eine chronische Überstimulation des Rezeptors, die über eine initiale Hypertrophie dann zu einer eingeschränkten Pumpfunktion führt.
Nachdem sich die Therapie der Antikörper-vermittelten Immunkardiomyopathie bisher auf die Behandlung der Herzinsuffizienz und die Kontrolle der Herz-insuffizienzsymptome beschränkt, könnten β1-ECII-homologe Peptide als Antikörper-Fänger bei Antikörper-positiven Patienten nun einen kausalen Therapieansatz darstellen. In diesem Zusammenhang wurden ein aus 25 Aminosäuren bestehendes zyklisches Peptid, eine aus 18 Aminosäuren bestehende Zyklopeptid-Mutante und ihre jeweiligen linearen Äquivalente im Rattenmodell auf Antikörper-neutralisierende Effekte und potentielle therapeutische Wirksamkeit getestet. Das Rattenmodell ist hierfür besonders geeignet, da die Aminosäuresequenz der funktionell wichtigen zweiten extrazellulären Domäne des β1-adrenergen Rezeptors (β1-ECII) bei Mensch und Ratte absolut identisch ist.
Auf immunologischer Ebene konnte der Titer der krankheitsinduzierenden β1-ECII-Antikörper bereits nach der ersten Applikation zyklischer Peptide relevant gesenkt werden und nahm im weiteren Verlauf der Behandlung kontinuierlich ab. Nach Zyklopeptidgabe kam es am Herzen zu einer Reduktion des linksventrikulären Durchmessers und zu einer fast vollständigen Normalisierung der anatomischen Proportionen. Auf die Morphologie der Myozyten selbst und auch den Kollagengehalt des Gewebes hatte die Zyklopeptidtherapie keinen wesentlichen Einfluss. Die funktionellen Eigenschaften des Herzens ließen sich durch die Neutralisation stimulatorischer β1-ECII-Antikörper mittels intravenöser Zyklopeptidapplikation deutlich verbessern: Die Verkürzungsfraktion des linken Ventrikels und der Herzindex als Parameter für die kardiale Leistungsfähigkeit konnten durch die Behandlung wieder weitgehend normalisiert werden.
Diese im Tiermodell erzielten Ergebnisse lassen einen therapeutischen Effekt der Zyklopeptide vermuten. Der Ansatz einer spezifisch gegen Antikörper gerichteten Therapie zur Behandlung von Patienten mit β1-Antikörper-positiver Herzinsuffizienz erscheint daher vielversprechend.
Adenosinrezeptoren werden auf nahezu allen Körperzellen exprimiert und übernehmen dort vielfältige und wichtige Funktionen. Auch auf diversen Tumorzelllinien konnten bereits Adenosinrezeptoren nachgewiesen und – je nach Subtyp – mit Pro- oder Anti-tumor-Effekten in Zusammenhang gebracht werden.
In dieser Arbeit wurden Gebärmutterhalskrebszellen sowie endometriale und triple-negative Brustkrebszellen auf Expression und mögliche Funktionen von Adenosinrezep-toren untersucht. Da spezifische Antikörper bis heute nicht verfügbar sind, wurde ein pharmakologischer Ansatz mit subtypspezifischen Agonisten und Antagonisten gewählt.
In Radioliganden-Bindungsassays, konnte nachgewiesen werden, dass sich auf der Zer-vixkarzinom-Zelllinie SiHa und der Brustkrebs-Zelllinie HCC1806 Adenosinrezeptoren des Subtyps A1 befinden. Die endometrialen Krebszelllinien Ishikawa und HEC-1-A exprimieren Rezeptoren vom Subtyp A1 und A2A. A3-Adenosinrezeptoren wurden auf keiner der untersuchten Zelllinien gefunden.
Der Nachweis von A2B-Rezeptoren kann mit dem Radioliganden-Bindungsassay nicht erbracht werden, da bislang kein Radioligand bekannt ist, der eine ausreichende Affini-tät besitzt, um diesen Subtyp zweifelsfrei nachweisen zu können.
Obwohl die Mehrheit der untersuchten Zelllinien Adenosinrezeptoren exprimiert, konnte ein signifikanter Effekt auf die Adenylatcyclase bei Stimulation der auf den Zellen vorhandenen Adenosinrezeptoren nur bei den HEC-1-A-Zellen festgestellt werden. Auch auf funktionelle A2B-Rezeptoren fand sich im Adenylatcyclaseassy kein Hinweis.
Im durchgeführten Kristallviolettassay zeigte sich ein proapoptotischer Effekt auf Ishi-kawa- und HEC-1-A-Zellen bei hohen Adenosin-Konzentrationen (100 µM). Die im BrdU-Assay gemessene Proliferationsrate hingegen änderte sich nach Vorbehandlung mit Adenosin nicht. Das metabolisch stabilere NECA (in Kombination mit ADA) hatte im Kristallviolettassay einen stärkeren Einfluss auf die Apoptoserate der jeweiligen Zelllinie als Adenosin und auch im BrdU-Assay sank die Menge an inkorporiertem BrdU. Ein Synergismus zwischen Stimulation von Adenosinrezeptoren und diversen Todesliganden bzw. Chemotherapeutika konnte nicht nachgewiesen werden.
Freies extrazelluläres Adenosin kann auch aus dem Abbau von ATP generiert werden, wenn Zellen die Ektonukleotidasen CD39 und CD73 exprimieren. Aufgrund der im-munsuppressiven Wirkung von Adenosin können diese Enzyme T-Zell- und NK-Zellantworten im Mikromilieu von Tumoren hemmen. Die durchflusszytometrische Analyse von HEC-1-A- und Ishikawa-Zellen zeigte zwar, dass die Expression von CD39 und CD73 nach Stimulation der Adenosinrezeptoren unverändert blieb. Die Ex-pression von Enzymen, lässt aber vermuten, dass die Zellen in vivo von Adenosin profi-tieren könnten. Angesichts der in vitro Daten, die allenfalls einen wachstumshemmen-den Effekt von Adenosin zeigten, könnte die vorrangige Wirkung von Adenosin im Tumormikromilieu tatsächlich auf der Inhibition von Immunantworten beruhen. Mög-licherweise würden die Rezeptoren dann in erster Linie als Sensoren dienen.
Weitere Forschungsarbeit wird helfen, die Rolle der Adenosinrezeptoren im Tumorge-schehen vollständig zu verstehen und möglicherweise für die Krebstherapie nutzbar zu machen.
Wben irradiated at 360 nm, furocoumarins with a hydroperoxide group in a side chain effciently give rise to a type of DNA damage that can best be explained by a photoinduced generation of hydroxyl radicals from the excited pbotosensitizers. The observed DNA damage profiles, i.e. the ratios of single-strand breaks, sites of base loss (AP sites) and base modifications sensitive to fonnamidopyrimidine-DNA glycosylase (FPG protein) and endonuclease m, are similar to the DNA damage profile produced by hydroxyl radicals generated by lonizing radiation or by xanthine and xanthine oxidase in the presence of Fe(III)-EDTA. No such damage is observed with the corresponding furocoumarin alcohols or in the absence of near-UV radiation. The damage caused by the photo-excited hydroperoxides is not influenced by superoxide dismutase (SOD) or catalase or by D2O as solvent. The presence of t-butanol, however, reduces both the formation of single-strand breaks and of base odifications sensitive to FPG protein. The cytotoxicity caused by one of the hydroperoxides in L5178Y mome lymphoma cells is found to be dependent on the near-UV irradiation and to be much higher than that of the corresponding alcohol. Therefore the new type of photoinduced damage occurs inside cells. Intercalating photosensitizers with an attached hydroperoxide group might represent a novel and versatile class of DNA damaging agents, e.g. for phototherapy.
No abstract available.
Diethylstilbestrol alters the morphology and calcium levels of growth cones of PC12 cells in vitro
(1993)
Diethylstilbestrol (DES) is a synthetic estrogen with carcinogenic properties. DES is known to alter cytoskeletal components, including the organization of actin stress fibres in C6 rat glioma cells. ln a test of the hypothesis that DES disrupts actin Filaments of growth cones in neuron-like cells, DES-induced changes in filopodial lengths were quantified in rat pheochromocytoma (PC12) cells in vitro. DES significantly altered growth cone morphology, with collapse of growth cone filopodia and neurite retraction invariably occurring at a concentration of 10 MikroM. At 5 MikroM DES, transient reductions in total filopodiallengths occurred. At DES concentrations of 0.1 nM and 1 nM, reductions in total filopodiallengths occurred in a fraction of growth cones. Evidence exists which shows that growth cone activity and morphology are intimately linked to Ieveis of intracellular, free calcium and that DES increases such levels. Measurements of free intracellular calcium levels by fluorescence microscopy, at times concurrent with the DES-induced reduction in total filopodial lengths, showed that calcium levels were indeed significantly increased by 10 MirkoM DES. Labelling of filamentaus actin (f-actin) with FITC-phalloidin showed that the f-actin distribution in growth cones exposed to DES could not be differentiated from the distribution found in spontaneously retracting growth cones. Tagether with evidence which showed that growth cone motility was not affected, the results are taken to indicate that DES, rather than acting directly on the cytoskeleton, exerts its effects indirectly, by a calcium-induced destabilization of actin filaments in the growth cone.
Tbe alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, 1)rr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, l)T-'I)T, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present durlog the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artiticial sweetener aspartame); only Met under these conditions bad a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Metproduces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the tirst-order reaction rate for nitrite. A decrease in nitrite concentration from the millimolar concentrations ofthe in-vitro assay to the micromolar concentrations in the stomach reduces the reaction rate by a factor of 1000 for the side-chain nitrosation, whereas a million-fold reduction will be observed for nitrosation of the amino group.
Known mutagens and carcinogens in the dict were compiled and the risk of cancer was estimated on the basis of average exposure Ievels in Switzerland and carcinogenic potencies from rodent bioassays. The analysis showed that, except for a1cohol, the sum of all known dietary carcinogens could only explain a few percent of the cancer deaths attributed by epidemiologists to dietary factors. The discrepancy was explained by a "carcinogenicity" of excess macronutrients. This hypothesis was based on an evaluation of dietary restriction experiments in rats and mice, where a dramatic reducing effect on spontaneaus tumour formation was seen. From these experiments, a "carcinogenic potency" was deduced for food in excess (TD50 approximately 16 g/kg per day). Ovemutrition in Switzerland was converted into excess food intake and the cancer risk estimated on the basis ofthe TD50 value. The resulting risk of60,000 cases per one million lives wou1d aJlow to explain by overnutrition almost all "diet-related" cancer deaths in humans.
An improved 32P-postlabelling assay for detection and quantitation of styrene 7,8-oxide-DNA adducts
(1993)
Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P 1-enriched adducts were 32P-labelled and purified by high-salt ( 4.0 M ammonium formate, pH 6.1} C1s reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H20 (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected. The efficiency of the kinase reaction depended on the ATP concentration. Use of standard labelling conditions (['Y· 32P]ATP, <3000 Ci/mmol; <2 Mikromol) resulted in poor ( 4-7%) adduct recovery. An ATP concentration of 40 Mikromol, however, increased the labeJling efficiency by a factor of 5-8 (35-55% based on 3H-SO labelied DNA). The results indicate that the new separation technique is suitable for the relatively polar SO-DNA adducts and that high labelling efficiency can be achieved.
Polymorphonuclear leukocyte (PMNL) infiltration is an important characteristic in psoriatic lesions. Elevated concentrations of the chemoattractant eicosanoid leukotriene B4 (L TB4) are present in psoriatic skin. Its chemotactic activity is mediated via high affinity receptors on PMNL. The goal of our work was to ascertain whether PMNL infiltration in psoriasis can be accounted for by functional abnormalities of the circulating PMNL due to alterations in the LTB4 receptor density or affinity (or both). No significant difference was found between patients with psoriasis, healthy controls and patients with another inflammatory dermatosis (atopic eczema) with regard to the binding parameters of LTB4 receptors on PMNL. Our findings suggest that PMNL accumulation in psoriatic skin may be the result of an excess of cutaneous hemoattractant rather than the increased readiness of psoriatic PMNL to migrate towards L TB4 due to altered LTB4 receptor density or affinity.
Adenosine modulates a variety of physiological functions via membrane-bound receptors. These receptors couple via G proteins to adenylate cyclase and K+channels. The A1 subtype mediates an inhibition of adenylate cyclase and an opening of K+-channels, and the A2 subtype a Stimulation of adenylate cyclase. Both subtypes have been characterized by radioligand binding. This has facilitated the development of agonists and antagonists with more than 1000-fold A1 selectivity. A1-selective photoaffinity labels have been used for the biochemical characterization of A1 receptors and the study of their coupling to adenylate cyclase. Such selective ligands allow the analysis of the involvement of adenosine receptors in physiological functions. Selective interference with adenosine receptors provides new pharmacological tools and eventually new therapeutic approaches to a number of pathophysiological states.
Nitrosation of dietary components has been combined with the 4-(para-nitrobenzyl)pyridine (NBP) colorimetric test for screening alkylating agents and with the Ames test for the detection of mutagenic activity. This allowed the investigation of short-hved nitrosation products of dietary components which generate electrophilic degradation products requiring no metabolic activation (natural amino acids and some derivatives, ureas, guanidines, primary alkyl and aryl amines). In a first system, precursor, nitrous acid and NBP were present simultaneously. All amino acids tested, except glutamic acid and glutamine, gave positive results. The reactivities spanned more than three orders of magnitude, with the aromatic amino acids and methionine the most active; two primary amines, tryptamine and histamine, were also strongly reactive. All guanidines tested, except the amino acid arginine, gave negative results. A second system consisted of two phases: NBP was added only after destruction of residual nitrite and adjustment of the pH to neutrality. This system was useful for the study of ureas, which are stable in acid but not in neutral media. The range of responses covered more than two orders of magnitude. Most amino acids and primary amines also gave positive results, but could be assessed only after analysing the kinetics of the competing reactions and choosing appropriate reaction times. In a third system, Salmonella typhimurium strain TA1OO replaced NBP. Representatives of the class of amino acids, ureas, the primary amine tryptamine, and aniline became higbly mutagenic upon nitrosation. Methylguanidine was only weakly mutagenic under the present assay conditions. The results indicate that further studies with unstable nitrosation products of dietary components are required to understand more thoroughly the role of endogenous nitrosation in gastric cancer.
The potential health risk posed by the endogenous formation of N-nitroso compounds (NOC) from nitrosation of dietary ureas, guanidines, amides, amino acids and amanes (primary, secondary and aromatic) was estimated according to the model:
Risk = ( daily intake of precursor] X (gastric concentration of nitrite ]n X [nitrosatability rate constant] X [cilrcinogenicity of derivative].
The daily intakes ofthese compound classes span five orders ofmagnitude (100 g/day amides, top; 1-10 mg/day secondary amines, ureas, bottom); the nitrosation rate constants span seven orders of magnitude (aryl amines, ureas, top; amides, secondary amines, bottom); and the carcinogenicity estimates span a 10 000-fold range from 'very strong' to 'virtually noncarcinogenic'. The resulting risk estimates likewise span an enormous range (nine orders of magnitude ): dietary ureas and aromatic amines combined with high nitrite concentration could pose as great a risk as the intake of preformed N-nitrosodimethylamine in the diet. In contrast, the risk posed by the in-vivo nitrosation of primary and secondary amines is probably negligible. The risk contributed by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes.
The ligand-binding subunit ofthe A1 adenosine receptor has been identified in membranes with the photoaffinity Iabel R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA). Covalent labelling ofthe A1 receptor can also be achieved in intact cells. The dissociation of the radioiodinated label (1251-AHPIA) from isolated rat fat cells was incomplete after UV irradiation, leaving about 20°/o of irreversible specific binding. Such covalent labelling of the receptor led to a concentration-dependent reduction of cellular cyclic AMP levels. This persistent effect of covalent labeHing occurred with an IC50 value of 9 nM, as compared to an IC50 value of 0.9 nM for the direct reduction of cyclic AMP Ievels by the ligand. The difference in the IC5o values can be explained by assuming spare receptors. This hypothesis was verified in binding studies using [ 3HJPIA as a radioligand. R-AHPIA inhibited binding of [3H)PIA to intact fat cells with a K1 value of about 20 nM, which is about 20 tim es high er than the corresponding IC50 value of cyclic AMP reduction. These data show that the A1 receptor is activated according to the occupancy theory. The high sensitivity of the activation in intact ceJis is due to a large number of spare receptors.
We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both uf these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A 1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist f:1H]DPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of [: 1H]DPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent Kd value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of [: 1H]DPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A 1 receptor population in 28A cells has similar agonist binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine ;)' -triphosphate ( 100 ,uM) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic. indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins. In the absence of evidence for subpopulations of the A1 receptor, it appears that in 28A cells. A single A1 receptor population. As defined by ligand binding characteristics, couples via one or more pertussis toxin-sensitive guanine nucleotide binding proteins to two different biological signaling mechanisms.
It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxyphenytisopropyladenosine [(R)-AHPIA] into the A, adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of celular cAMP Ieveis [Mol. Pharmacol. 30:403-409 (1986)]. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenytate cyclase Stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies in-dicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase Stimulation of up to SOOk of the control value. Similarly, the activation via these 10-20% of receptors occurs with a halflife that is only 2 times Ionger than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenytate cyclase Stimulation. These observations require a modification of the models of receptor-adenytate cyclase coupling, which is described in the accompanying paper [Mol. Pharmacol. 39:524-530 (1991)].
The binding of agonists and antagonists to a2-adrenergic receptors of human platelets was studied. The receptors showed homogeneaus affinities for antagonists but two affinity states for the agonist (-)-epinephrine, which were modulated by guanine nucleotides. Van't Hoffplots of antagonist binding had a break point at about 18° and considerable diversity between 18° and 0°. Agonist binding to both affinity states showed a similar break point; agonist binding to the high affinity state was characterized by a large entropy component compared to the low affinity state. This entropy component was reduced at higher concentrations of sodium, indicating that it may be due to Iiberation of sodium ions. Measurements of the fluorescence of 1-anilin-8-naphthalenesulfonate showed thermotropic phase transitions of theplatelet membranes at about 17°. The transition temperature was decreased to about 12° by addition of 1 0 mM octanoic acid. Octanoic acidalso shifted the break points of the van't Hoffplot of antagonist and low affinity agonist binding from 18° to 12°. High affinity agonist binding, however, remained unchanged. It is concluded that agonist-specific thennodynamic characteristics of ligand binding to a2-receptors of human platelets can only be investigated by regarding differences between high and low affinity agonist binding. These differences include an entropy increase upon Iigand binding, which is in part due to enhanced liberation of sodium ions, and a loss of sensitivity to fluidity changes in the outer layer of the plasma membrane.
Mechanistic possibilitles responsible for nonlinear shapes of the dose-response relationship in chemical carcinogenesis are discussed. (i) Induction and saturation of enzymatic activation and detoxification processes and of DNA repair affect the relationship between dose and steady-state DNA adduct Ievel; (ii) The fixation of DNA adducts in the form of mutations is accelerated by stimulation of the cell division, for Jnstance due to regenerative hyperplasia at cytotoxic dose Ievels; (iii) The rate of tumor formation results from a superposition of the rates of the individual steps. It can become exponential with dose if more than one step is accelerated by the DNA damage exerted by the genotoxic carcinogen. The strongly sigmoidal shapes often observed for dose-tumor incidence relationships in animal bioassays supports this analysis. A power of four for the dose in the su~linear part of the curve is the maximum observed (formaldehyde). In contrast to animal experiments, epidemiological data ln humans rarely show a slgnificant deviation from linearity. The discrepancy might be explained by the fact that a I arge nu mber of genes contribute to the overall sensitivity of an individual and to the respective heterogeneity within the human population. Mechanistic nonlinearities are flattened out in the presence of genetic and life-style factors which affect the sensitivity for the development of cancer. For a risk assessment, linear extrapolation from the high-dose lncidence to the spontaneaus rate can therefore be approprlate in a heterogeneous population even if the mechanism of action would result in a nonlinear shape of the dose-response curve in a homogeneaus population.
The diet contains a large number of constituents which can be nitrosated in the gastrointestinal tract (especially in the stomach) to potentially carcinogenic nitroso compounds (NOC). The nitrosation of food mixtures has been investigated with a number of assays, such as chemical analysis or detection of alkylating potential, mutagenicity and carcinogenicity. Relatively good information is available on the formation of stable nitrosamines using high nitrite concentrations. Little is known, however, about the formation of chemically unstable NOC at low nitrite concentration and their genotoxicity in target cells. A comparison of the precursor classes, alkylamines, aromatic amines, amino acids, amides and peptides, ureas and guanidines, reveals a vast range, both with respect to daily intake (105-fold) and nitrosation rate (104-fold both for 1st and 2nd order nitrite dependence). A total span of 108 results for the relative yield of NOC in the stomach. The endogenous NOC burden from dietary ureas and aromatic amines may represent as large a hazard as the intake of preformed NOC. Recent evidence also indicates that heterocyclic amines and phenols must be considered and that the half-life of nitrosated a-amino acids can be much longer than that of nitrosated primary alkylamines. In these classes, more information should be collected on dietary concentrations, on the nitrosation under realistic conditions and on the genotoxicity in stomach lining cells. Within a chemical precursor class, a wide range is seen with respect to alkylating potency. It cannot, therefore, be excluded that individual precursors within the top ranking classes might become more important than single preformed NOC. Not considered in the above analysis but probably just as important for a risk evaluation in a population is the knowledge of the nitrosation conditions and target cell susceptibility in individuals.
[7-3H)Styrene 7,8-oxide was administered by oral gavage to male CD rats at a dose of 1.3 mg/kg. After 4 h, the forestomach was excised, DNA was isolated, purified to constant specific radioactivity and degraded nzymatically to the 3 '-nucleotides. Highperformance liquid chromatography fractions with the normal nucleotides contained most of the radiolabel, but a minute level of adduct label was also detccted. Using the units of the covalent binding index (micromoles adduct per mole DNA nucleotide)/(millimole chemical administered per kilogram body weight), a DNA binding potency of 1.0 was derived. A comparison of the covalent binding indices and carcinogenic potencies of other genotoxic forestarnach carcinogens showed that the tumorigenic activity of styrene oxide is unlikely to be purely genotoxic. Therefore, styrene oxide was compared with 3-tbutylhydroxyanisole (BHA) with respect to stimulation of cell proliferation in the forestomach. Male Fischer 344 rats were treated for four weeks at three dose levels of styrene oxide (0, 137, 275 and 550 mg/kg, three times per week by oral gavage) and BHA (0, 0.5, 1 and 2% in the diet); the highest doses had been reported to result in 84% and 22% carcinomas in the forestomach, respectively. Cell proliferation was assessed by incorporation of bromodeoxyuridine into DNA and immunohistochemical analysis. An increase in the lablling indexwas found in a11 treated animals. In the prefundic region of the forestomach, the labeHing index increased significantly, from 42% (controls) to 54% with styrene oxide and from 41 to 55% with BHA. Rats treated with BHA also had severe hyperplastic lesions in the prefundic region, i.e., at the location of BHA-induced forestomach carcinomas. The number of cells per millimetre of section length was increased up to 19 fold. Hyperplastic lesions were not seen with styrene oxide, despite the higher tumour incidence reported with this compound. We conclude that the carcinogenicity of styrene oxide to the forestomach most probably involves a mechanism in which marginal genotoxicity is combined with promotion by increased cell proliferation.
Barbiturates inhibit binding of radioligands to A 1(Ri) adenosine receptors of rat brain membranes. This inhibition is dose-dependent and stereospecific and occurs in the range of pharmacologically active concentrations. The displacement of radiolabelled A1antagonists by barbiturates is not modified by GTP, indicating that barbiturates might act as antagonists at this receptor. This action of barbiturates does not seem to be related to the binding of barbiturates to plasma membranes, as the latter process has different characteristics. Barbiturates also inhibit the binding of radioligands to solubilized A1receptors, and saturation and kinetic experiments suggest that this is due to a competitive antagonism. These results indicate that barbiturates interact with the recognition site of the A1adenosine receptor.
Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in chronic renal failure (CRF) in rats. The aim of this work was to study the role of inflammation and oxidative stress in adenine-induced CRF and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in feed (0.75%, w/w), GA in drinking water (15%, w/v) and adenine+GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration). In addition, the concentrations of the pro-inflammatory cytokine TNF-a and the oxidative stress markers glutathione and superoxide dismutase, renal apoptosis, superoxide formation and DNA double strand break frequency, detected by immunohistochemistry for
c-H2AX, were measured. Adenine significantly increased the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance and induced significant increases in the concentration of the measured inflammatory mediators.
Further, it caused oxidative stress and DNA damage. Treatment with GA significantly ameliorated these actions. The mechanism of the reported salutary effect of GA in adenine-induced CRF is associated with mitigation of the adenine-induced inflammation and generation of free radicals.
Spatiotemporale Organisation der Interaktion von Gq Protein-Untereinheiten und der Phospholipase Cβ3
(2012)
Die G-Protein vermittelte Aktivierung der Phospholipase Cβ (PLCβ) stellt einen primären Mechanismus dar, um eine Vielzahl von physiologischen Ereignissen zu regulieren, z.B. die Kontraktion glatter Muskelzellen, Sekretion oder die Modulation der synaptischen Transmission. Sowohl Gαq- als auch Gβγ-Untereinheiten sind dafür bekannt mit PLCβ Enzymen zu interagieren und diese zu aktivieren. Über die Dynamik dieser Interaktion und den relative Beitrag der G-Protein Untereinheiten ist jedoch nur wenig bekannt. Unter Verwendung Fluoreszenz Resonanz Energie Transfer (FRET)- basierter Methoden in lebenden Zellen, wurde die Kinetik der Rezeptor-induzierten Interaktion zwischen Gβγ und Gαq Untereinheiten, die Interaktion von sowohl der Gαq als auch der Gβγ-Untereinheit mit der PLCβ3 und die Interaktion des regulator of G-Protein signaling 2 (RGS2) mit Gαq-Untereinheiten untersucht. Um die Untersuchung der Protein-Protein-Interaktion auf die Zellmembran zu beschränken, wurde die Total-Internal Reflection Fluorescence (TIRF) Mikroskopie angewandt. Zeitlich hoch auflösendes, ratiometrisches FRET-Imaging offenbarte eine deutlich schnellere Dissoziation von Gαq und PLCβ3 nach Entzug purinerger Agonisten verglichen mit der Deaktivierung von Gq Proteinen in der Abwesenheit der PLCβ3. Dieser offensichtliche Unterschied in der Kinetik kann durch die GTPase-aktivierende Eigenschaft der PLCβ3 in lebenden Zellen erklärt werden. Weiterhin zeigte es sich, dass PLCβ3 die Gq Protein Kinetik in einem ähnlich Ausmaß beeinflusst wie RGS2, welches in vitro deutlich effizienter darin ist, die intrinsische GTPase Aktivität der Gαq-Untereinheit zu beschleunigen. Als Antwort auf die Rezeptorstimulation wurde sowohl eine Interaktion von Gαq-Untereinheiten als auch von Gq-abstammende Gβγ-Untereinheiten mit der PLCβ3 beobachtet. Darüber hinaus zeigte sich auch eine Agonist-abhängige Interaktion von Gαq und RGS2. In Abwesenheit einer Rezeptorstimulation konnte kein spezifisches FRET-Signal zwischen Gq Proteinen und der PLCβ3 oder RGS2 detektiert werden. Zusammengefasst ermöglichte das ratiometrische FRET-Imaging in der TIRF Mikroskopie neue Einsichten in die Dynamik und Interaktionsmuster des Gq-Signalwegs.
Hyperinsulinemia, a condition with excessively high insulin blood levels, is related to an increased cancer incidence. Diabetes mellitus, metabolic syndrome, obesity and polycystic ovarian syndrome are the most common of several diseases accompanied by hyperinsulinemia. Since an elevated cancer risk especially for colon and kidney cancers, was reported for those patients, we investigated for the first time the induction of genomic damage by insulin mainly in HT29 (human colon cells), LLC-PK1 (pig kidney cells), HK2 (human kidney cells) and peripheral lymphocytes, and to confirm the genotoxicity of insulin in other cells from different tissues. To ascertain that the insulin effects were not only limited to permanent cell lines, rat primary colon, kidney, liver and fatty tissue cells were also studied. To connect the study and the findings to in vivo conditions, two in vivo models for hyperinsulinemia were used; Zucker diabetic fatty rats in a lean and diabetic state infused with different insulin concentrations and peripheral lymphocytes from type 2 diabetes mellitus patients. First, the human colon adenocarcinoma cells (HT29) showed significant elevation of DNA damage using comet assay and micronucleus frequency analysis upon treatment with 5 nM insulin in standard protocols. Extension of the treatment to 6 days lowered the concentration needed to reach significance to 0.5-1 nM. Insulin enhanced the cellular ROS production as examined by the oxidation of the dyes 2´,7´-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium (DHE). The FPG modified comet assay and the reduction of damage by the radical scavenger tempol connected the insulin-mediatedDNA damage to ROS production. To investigate the sources of ROS upon insulin stimulation, apocynin and VAS2870 as NADPH oxidase inhibitors and rotenone as mitochondrial inhibitor were applied in combination with insulin and all of them led to a reduction of the genomic damage. Investigation of the signaling pathway started by evaluation of the binding of insulin to its receptor and to the IGF-1 receptor. The results showed the involvement of both receptors in the signaling mechanism. Following the activation of both receptors, PI3K activation occurs leading to phosphorylation of AKT which in turn activates two pathways for ROS production, the first related to mitochondria and the second through activation of Rac1 , resulting in the activation of Nox1. Both pathways could be activated through AKT or through the mitochondrial ROS which in turn could activates Nox1. Studying another human colon cancer cell line, Caco-2 and rat primary colon cells in vitro confirmed the effect of insulin on cellular chromatin. We conclude that pathophysiological levels of insulin can cause DNA damage in colon cells, which may contribute to the induction or progression of colon cancer. Second, in kidney cells, insulin at a concentration of 5 nM caused a significant increase in DNA damage in vitro. This was associated with the formation of reactive oxygen species (ROS). In the presence of antioxidants, blockers of the insulin and IGF-1 receptors, and a phosphatidylinositol 3-kinases (PI3K) inhibitor, the insulin mediated DNA damage was reduced. Phosphorylation of AKT was increased and p53 accumulated. Inhibition of the mitochondrial and NADPH oxidase related ROS production reduced the insulin mediated damage. In primary rat cells insulin also induced genomic damage. HK2 cells were used to investigate the mechanistic pathway in the kidney The signaling is identical to the one in the colon cells untill the activation of the mitochondrial ROS production, because after the activation of PI3K activation of Nox4 occurs at the same time across talk between mitochondria and Nox4 activation has been suggested and might play a role in the observed effects. In the in vivo model, kidneys from healthy, lean ZDF rats, which were infused with insulin to yield normal or high blood insulin levels, while keeping blood glucose levels constant, the amounts of ROS and p53 were elevated in the high insulin group compared to the control level group. ROS and p53 were also elevated in diabetic obese ZDF rats. The treatment of the diabetic rats with metformin reduced the DNA oxidation measured as 8-oxodG as well as the ROS production in that group. HL60 the human premyelocytic cells and cultured lymphocytes as models for the hemopoietic system cells showed a significant induction for DNA damage upon treatment with insulin. The diabetic patients also exhibited an increase in the micronucleus formation over the healthy individuals. In the present study, we showed for the first time that insulin induced oxidative stress resulting in genomic damage in different tissues, and that the source of the produced ROS differs between the tissues. If the same mechanisms are active in patients, hyperinsulinemia might cause genomic damage through the induction of ROS contributing to the increased cancer risk, against which the use of antioxidants as well as mitochondrial and NADPH oxidase inhibitors might exert protective effects with cancer preventive potential under certain conditions. Normal healthy human plasma insulin concentrations are in the order of 0.04 nM after overnight fasting and increase to less than about 0.2 nM after a meal. Pathophysiological levels can reach 1 nM and can stay above 0.2 nM for the majority of the daytime yielding condictions close to the insulin concentrations determined in the present study. Whether the observed effects also occur in vivo and whether they actually initiate or promote tumor formation remains to be determined. However, if proof of that can be obtained, our experiments with inhibitors indicate chances for pharmacological intervention applying antioxidants or enzyme inhibitors. It will not be the aim to reduce ROS in any case or as much as possible because ROS have now been recognized as important signaling molecules and participatants in immune defense, but a reduction to physiological levels instead of pathophysiological levels in the context of a disease associated with ROS overproduction might be beneficial.
Unsere Arbeitsgruppe verfolgt im Rattenmodell der Antikörper-induzierten DiCM einen Antikörper-spezifischen Therapieansatz durch die Gabe eines synthetischen β1-ECII homologen zyklischen „Fänger“-Peptids. Die Applikation dieses Peptids führte zu einer deutlichen Verbesserung wichtiger Herzparameter wie z.B. des Durchmessers des linken Ventrikels (LV) sowie der LV-Verkürzungsfraktion und des kardialen Indexes. Des Weiteren kam es unter der Peptid-Therapie zu einer signifikanten Reduktion des anti-β1-ECII Antikörpertiters trotz kontinuierlich durchgeführter Immunisierungen mit dem die Erkrankung induzierenden Antigen.
Es lag daher nahe, dass durch die Peptid-Therapie auch eine Art immunologischer Toleranz induziert wird, der eine Depletion bzw. funktionelle Beeinträchtigung von Zellen des Immunsystems, die an der Produktion von anti-β1-ECII Antikörpern beteiligt sind, zugrunde liegen könnte. Im Rahmen der vorliegenden Doktorarbeit war es daher Aufgabe, die verschiedenen Zellkomponenten der humoralen und zellulären Immunantwort zu untersuchen, um den immunologischen Effekt der Peptid-Applikation besser zu verstehen.
So scheinen in unserem Modell die β1-ECII-spezifischen CD4+T-Zellen durch die Peptid-Therapie
nicht beeinflusst zu werden; ebenso konnten unsere Experimente keinen Zyklopeptideffekt auf die langlebigen Plasmazellen nachweisen. Die erzielten Ergebnisse sprechen jedoch dafür, dass die Rückbildung des DiCM Phänotyps und der Abfall des anti-β1-ECII Antikörpertiters trotz kontinuierlicher Immunisierung zum einen auf der direkten Neutralisierung der anti-β1-ECII Antikörper durch die Ring-Peptide (Scavenger-Effekt)und zum anderen auf einer Depletion bzw. funktionellen Beeinträchtigung der β1-ECII-spezifischen Memory B-Zellen beruhen könnte. Der für die Reduktion der Memory B-Zellen letztlich verantwortliche molekulare Mechanismus muss jedoch noch in weiteren Experimenten untersucht werden.
Die im Rahmen der vorliegenden Arbeit erzielten Ergebnisse tragen aber wesentlich zum besseren Verständnis der Wirkungsweise dieses neuartigen Therapiekonzepts zur Behandlung der immuninduzierten Kardiomyopathie bei.
In addition to its tumor-promoting activity in honnone-receptive tissue, the carcinogenic estrogen diethylstilbestrol (DES) has been found to induce cell transformation, aneuploidy and micronucleus formation in mammalian cells. The majority of these micronuclei contained whole chromosomes and were fonned during mitosis. Here a possible relationship between a disturbance in cell cycle progression and micronucleus fonnation is investigated by exposing Syrian hamster embryo (SHE) cells to DES. Continuous bromodeoxyuridine labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry was employed for analysis of cell cycle transit and related to the time course of micronucleus formation. Treatment of SHE cells with DES resulted in delayed and impaired cell activation (exit from the GO/G 1 phase), impaired S-phase transit and, mainly, G2-phase traverse. Cells forming micronuclei, on the other hand, were predominantly in G2 phase during DES treatment. These results suggest that impairment of Sand G2 transit may involve a process ultimately leading to micronucleus formation.
Dilated cardiomyopathy (DCM) represents an important subgroup of patients suffering from heart failure. The disease is supposed to be associated with autoimmune mechanisms in about one third of the cases. In the latter patients functionally active conformational autoantibodies directed against the second extracellular loop of the β1-adrenergic receptor (AR, β1ECII-aabs) have been detected. Such antibodies chronically stimulate the β1-AR thereby inducing the adrenergic signaling cascade in cardiomyocytes, which, in the long run, contributes to heart failure progression. We analyzed the production of cAMP after aab-mediated β1-AR activation in vitro using a fluorescence resonance energy transfer (FRET) assay. This assay is based on HEK293 cells stably expressing human β1-AR as well as the cAMP-sensor Epac1-camps. The assay showed a concentration-dependent increase in intracellular cAMP upon stimulation with the full agonist (-) isoproterenol. This response was comparable to results obtained in isolated adult murine cardiomyocytes and was partially blockable by a selective β1-AR antagonist. In the same assay poly- and monoclonal anti-β1ECII-abs (induced in different animals) could activate the adrenergic signaling cascade, whereas isotypic control abs had no effect on intracellular cAMP levels. Using the same method, we were able to detect functionally activating aabs in the serum of heart failure patients with ischemic and hypertensive heart disease as well as patients with DCM, but not in sera of healthy control subjects. In patients with DCM we observed an inverse correlation between the stimulatory potential of anti-β1-aabs and left ventricular pump function. To adopt this assay for the detection of functionally activating anti-β1ECII-aabs in clinical routine we attempted to establish an automated large-scale approach. Neither flow cytometry nor FRET detection with a fluorescence plate reader provided an acceptable signal-to-noise ratio. It was possible to detect (-) isoproterenol in a concentration-dependent manner using two different FRET multiwell microscopes. However, due to focus problems large-scale detection of activating anti-β1ECII-abs could not be implemented. Neutralization of anti-β1-aabs with the corresponding epitope-mimicking peptides is a possible therapeutic approach to treat aab-associated autoimmune DCM. Using our FRET assay we could demonstrate a reduction in the stimulatory potential of anti-β1ECII-abs after in vitro incubation with β1ECII-mimicking peptides. Cyclic (and to a lesser extent linear) peptides in 40-fold molar excess acted as efficient ab-scavengers in vitro. Intravenously injected cyclic peptides in a rat model of DCM also neutralized functionally active anti-β1ECII-abs efficiently in vivo. For a detailed analysis of the receptor-epitope targeted by anti-β1ECII-abs we used sequentially alanine-mutated β1ECII-mimicking cyclic peptides. Our data revealed that the disulfide bridge between the cysteine residues C209 and C215 of the human β1-AR appears essential for the formation of the ab-epitope. Substitution of further amino acids relevant for ab-binding in the cyclic scavenger peptide by alanine reduced its affinity to the ab and the receptor-activating potential was blocked less efficiently. In contrast, the non-mutant cyclic peptide almost completely blocked ab-induced receptor activation. Using this ala-scan approach we were able to identify a “NDPK”-epitope as essential for ab binding to the β1ECII. In summary, neutralization of conformational activating anti-β1ECII-(a)abs by cyclic peptides is a plausible therapeutic concept in heart failure that should be further exploited based on the here presented data.
Cell adhesion and migration are essential for development and homeostasis. Adhesion to the extracellular matrix occurs at specialized plasma membrane domains where transmembrane adhesion receptors, signaling proteins such as kinases and phosphatases, and a large number of adaptor proteins interact with the cytoskeleton in a tightly regulated and synchronized fashion. Whereas altered cell adhesion and migration are known to be important in cardiovascular disease and malignant tumors, the target proteins and molecular interactions that regulate these complex processes still remain incompletely understood. Whereas numerous kinases are known to regulate cell adhesion dynamics, information about the involved protein phosphatases is still very limited. A newly emerging phosphatase family contains the unconventional active site sequence DXDX(T/V) and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases. Our laboratory has recently discovered AUM, a novel phosphatase that belongs to this poorly characterized enzyme family. Initial findings pointed toward a potential involvement of AUM in the regulation of cell adhesion to the extracellular matrix. The objective of the present study was to study the potential role of AUM in cell adhesion. We could show that cells stably depleted of AUM are characterized by accelerated adhesion on immobilized fibronectin. To confirm these findings, we used an siRNA-based approach for the acute depletion of AUM and observed a similar phenomenon. Rescue experiments were performed with stably AUM-depleted cells to ensure that the above mentioned effects are indeed AUM specific. We observed that the re-addition of AUM normalizes cellular adhesion kinetics on fibronectin. These results clearly show that AUM exerts important functions in cell-matrix adhesion. To investigate the molecular basis of these effects, we have characterized integrin expression patterns using flow cytometry. Interestingly, fibronectin-stimulated AUM-depleted cells are characterized by an increase in the cell surface expression of conformationally active 1-integrins. Consistent with the important role of 1-integrins in the regulation of RhoA activity, we also observed a specific increase in RhoA-GTP, but not Rac1-GTP-levels during cell adhesion to fibronectin. Consistent with these findings and with the important role of RhoA for focal adhesion maturation, AUM depleted cells showed more elongated and more centripetally oriented focal adhesions as compared to control cells when spread on fibronectin. Taken together, this study has revealed an important role of AUM for cell-matrix adhesion. Our findings strongly suggest that AUM functions as a negative regulator of 1-integrins and RhoA-dependent cytoskeletal dynamics during cell adhesion.
The binding of tritiated benzo(a)pyrene (BP) to liver DNA of 25 adult male rats (SIV 50) has been determined 50 h after a single intraperitoneal injection of doses between 40 ug/kg and 4; mg/kg. The dose-response relations~ ip is linear up to i mg/kg, shows a sigmoid step towards 2 mg/kg and a shallow linear. slope above that value. TlJe 0 bserved bin ding ranges from 1.7 to 180 nmoles BP per mole DNA phosphate. The non-linearity between 1 and 2 mg/kg could be explained 0):1 the basis of an induction of metabolizing enzymes. A pure1y mathematical extrapolation of therumour incidence from a carcinogenic dose (1 x 40mg/kg for a 20% hepatoma incidence in newborn mice) to human exposure levels (aboilt 0.1 ug/kg per day) would never have followed a step like the on~ found in our experiments. Our dose-effect study therefore shows how carcinogenitity data could be extrapolated in a biologically founded way to low doses.
In the inhalation system described an animal can be kept in the same atmosphere of a 2-liter desiccator for up to 24 h. The expired carbon dioxide is adsorbed with soda lime and the resulting reduced pressure is balanced by a supply of oxygen also used for the inflow of the chemical to be investigated. Urine and faeces can be collected ~eparately and the system allows a periodical control of the concentration of the chemical by sampling the air with needle and syringe.
b-adrenergic receptors (b-ARs) participate strongly in the development of cardiac hypertrophy and human heart failure. Stimulation of b-adrenergic receptors with catecholamines as well as cardiac overexpression of b1-ARs or of Gas-proteins in transgenic mice induces cardiac hypertrophy. However, direct activation of their downstream targets, such as adenylyl cyclase (AC) or protein kinase A do not promote a significant degree of cardiac hypertrophy. These findings suggest that additional events may occur and that these events require Gas-protein activation. A hypertrophic pathway involving Gaq-protein coupled receptors has recently been described. Upon activation of Gaq-coupled receptors Gbg-subunits are released from Gaq and bind directly to the activated Raf/Mek/Erk cascade. Direct interaction between bg-subunits and activated Erk1/2 leads to an additional autophosphorylation of Erk2 at threonine 188, which mediates cardiac hypertrophy. Murine hearts, as well as isolated cardiomyocytes present an increase in Erk2Thr188-phosphorylation upon b-AR activation. Similarly overexpression of phosphorylation deficient Erk2 mutants (Erk2T188S and Erk2T188A) reduces b-AR mediated cardiomyocyte hypertrophy. Increase in left ventricular wall thickness, fibrosis and up-regulation of natriuretic peptide synthesis, which are physiological features for cardiac hypertrophy, are strongly inhibited in transgenic mice with a cardiac expression of Erk2T188S after two weeks of sustained isoproterenol treatment. It could further be shown in this work that b-AR mediated cardiac hypertrophy requires two distinct pathways initiated by Gs-protein activation: the canonical phosphorylation of Erk1/2 via adenylyl cyclase and the direct interaction of released bg-subunits with activated Erk1/2. Coincidence of both events leads to Erk2Thr188-phosphorylation, which activates then different transcription factors responsible for cardiac hypertrophy. Sequestration of bg-subunits by overexpression of the C-terminus of GRK2 bark-ct and inhibition of adenylyl cyclase efficiently reduced the hypertrophic response to isoproterenol, whereas direct activation of AC by forskolin failed to induce Erk2Thr188-phosphorylation and cardiomyocyte hypertrophy. These findings may help to develop new therapeutic strategies for the prevention of cardiac hypertrophy and maladaptive remodeling of the heart.
The determination of a covalent binding of radioactive chemieals to DNA in intact mammalian organisms is proposedas a short-term test for carcinogenicity. The effectiveness of covalent binding to rat liver DNA correlates well with the hepatocarcinogenicity known from long-term bioassays. The binding indices range over more than five orders of rriagnitude between the strongest hepatocarcinogen aflatoxin B 1 and the limit of detection of a binding with 100 f-LCi 14C-labelled chemical. The order of magnitude of binding is therefore a surprisingly good quantitative measure for carcinogenicity. The pattern of DNA binding sites is important especially for small alkylating agents where the determination of total binding might indicate a higher carcinogenic potency than is actually observed.
The covalent binding of [3H]aflatoxin B1 (AF) to liver DNA was determined, 6 h after oral administration to male rabbits. A Covalent Binding Index, CBI (flmol AF/mol DNA-P)/(mmol AF/kg b. w.) = 8,500 was found. Pretreatment of rabbits with AF coupled to bovine serum albumin in Freund's adjuvant led to the production of AF-directed antibodies. Administration of [3H]AF to such immunized rabbits resulted in a CJH of only 2,500, i.e., the iiDJ{.lUnization provided a protection by a factor of more than 3. Although this is encouraging evidence for the potential of active immunization against genotoxic carcinogens, a nurober of pointswill have to be clarified, such as the time course for the DNA binding and the question of a possible shift to other target cells.
The superfamily of G protein-coupled receptors (GPCR) regulates numerous physiological and pathophysiological processes. Hence GPCRs are of significant interest for pharmacological therapy. Embedded into cytoplasmic membranes, GPCRs represent the core of large signaling complexes, which are critical for transduction of exogenous stimuli towards activation of downstream signaling pathways. As a member of the GPCR family B, the parathyroid hormone receptor (PTHR) activates adenylyl cyclases, phospholipases C β as well as mitogen-activated protein kinase-dependent signaling pathways, thereby mediating endocrine and paracrine effects of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. This regulates, calcium homeostasis, bone metabolism and bone development. Paradoxically, PTH is able to induce both catabolic and anabolic bone metabolism. The anabolic effect of PTH is successfully applied in the therapy of severe osteoporosis. Domination of anabolic or catabolic bone-metabolism is entailed by temporal and cell-type specific determinants. The molecular bases are presumably differential arrangements of adaptor proteins within large signaling complexes that may lead to differential activation of signaling pathways, thereby regulating physiological effects. The molecular mechanisms are largely unclear; thus, there is significant interest in revealing a better understanding of PTHR-related adaptor proteins. To identify novel adaptor proteins which direct PTHR signaling pathways, a proteomic screening approach was developed. In this screening, vav2, a guanine-nucleotide exchange factor (GEF) for small GTPases which regulates cytoskeleton reorganization, was found to interact with intracellular domains of PTHR. Evidence is provided that vav2 impairs PTH-mediated phospholipase C β (PLCβ) signaling pathways by competitive interactions with G protein αq subunits. Vice versa, PTH was shown to regulate phosphorylation and subsequent GEF activity of vav2. These findings may thus shed new light on the molecular mechanisms underlying the effects of PTH on bone metabolism by PLC-signaling, cell migration and cytoskeleton organization. In addition to the understanding of intracellular molecular signaling processes, screening for ligands is a fundamental and demanding prerequisite for modern drug development. To this end, ligand binding assays represent a fundamental technique. As a substitution for expensive and potentially harmful radioligand binding, fluorescence-based ligand-binding assays for PTHR were developed in this work. Based on time-resolved fluorescence, several assay variants were established to facilitate drug development for the PTHR.
Many mutagens and carcinogens act via covalent interaction of metabolic intermediates with DNA in the target cell. This report groups those structural elements which are often found to form the basis for a metabolism to such chemically reactive metabolites. ~mpounds which are chemically reactive per se and which do not require metabolic activation form group 1. Group 2 compri~es of olefins and aromatic hydrocarbons where the oxidation via an epoxide can be responsible for the generation of reactive species. Aromatic amines, hydrazines, and nitrosamirres form group 3 requiring an oxidation of a nitrogen atom or of a carbon atom in alpha position to a nitrosated amine. Group 4 compounds are halogenated hydrocarbons which can either give rise to radicals or can form an ·olefin (group 2) upon dehydrohalogenation. Group 5 compounds depend upon some preceding enzymatic activity either not available in the target cell or acting on positions in the molecule which are not directly involved in the subsequent formation of electrophilic atoms. Examples for each group are taken from the "List of Chemieals and Irrdustrial Processes Associated with Cancer in Humans" as compiled by the International Agency for the Research on Cancer, and it is shown that 91% of the organic carcinogens would have been detected on the basis of structural elements characteristic for group 1-5. As opposed to this very high sensitivity, the specificity ( the true negative fraction) of using this approach as a short-term test for carcinogenicity is shown to be bad because detoxification pathways have so far not been taken into account. These competing processes are so complex, however, that either only very extensive knowledge about pharmacokinetics, stability, and reactivity will be required or that in vivo systems have to be used to predict, on a quantitative basis, the darnage expected on the DNA. DNA-binding experiments in vivo are presented with benzene and toluene to demonstrate one possible way for an experimental assessment and it is shown that the detoxification reaction at the methyl group available only in toluene gives rise to a reduction by at least a factor of forty for the binding to rat liver DNA. This quantitative approach available with DNA-binding tests in vivo, also allows evaluation as to whether reactive metabolites and their DNA binding are always the most important single activities contributing to the overall carcinogenicity of a chemical. With the example of the livertumor inducing hexachlorocyclohexane isomers it is shown that situations will be found where reactive metabolites are formed and DNA binding in vivo is measurable but where this activity cannot be the decisive mode of carcinogenic action. It is concluded that the lack of structural elements known to become potentially reactive does not guarantee the lack of a carcinogenic potential.
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Ich habe versucht darzulegen, daß mechanistische Überlegungen zur Extrapolation der Dosis-WirkungsBeziehung herangezogen werden können. Ein nichtlinearer Verlauf ist nicht nur bei den epigenetischen Kanzerogenen wahrscheinlich, sondern auch bei den DNA-bindenden. Echte Schwellen sind aber nur in solchen Fällen zu erwarten, wo kein endogenes Korrelat besteht. Immerhin können auch steile Nichtlinearitäten zu einer drastischen Risikoreduktion führen, so daß die Anstrengungen dahin gehen sollten, die Steigung und den Bereich des überproportionalen Abfalls experimentell zu zeigen. In einer heterogenen Population kann die 0 0- sis-Wirkungs-Kurve zusätzliche "Wellen" bekommen und wird dadurch grundsätzlich flacher. Im Extremfall ergibt sich eine lineare Dosis-Wirkungs-Beziehung unabhängig vom Wirkmechanismus des Kanzerogens. Diese Proportionalität zwischen tiefster Dosis und Effekt wird bei genotoxischen Kanzerogenen aus mechanistischen Gründen schon für eine homogene Population postuliert, doch kann dies in einer heterogenen Population auch bei epigenetischen Kanzerogenen in Frage kommen.
Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process. It involves direct interaction of receptors with heterotrimeric G-proteins and may be under thc control of cytoskeletal clemcnts. Evidencc exists suggesting that thc cytoskeleton and/or the membrane ske1eton determines the distribution of FPR in the plane of the plasma membrane, thus controlling FPR accessibility to different protcins in functionally distinct membrane domains. In desensitized cells, FPR are restricted to domains 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 inacccssible to the agonist-occupied receptor, preventing cell activation. We are investigating the molecular basis for the interaction of FPR with the membrane skeleton, and our results suggest that FPR, and possibly other receptors, may directly bind to cytoskeletal proteins such as actin.
Seit der Entdeckung, dass Adenosin auch als Botenstoff dient, beschäftigen sich Forschungsgruppen mit Adenosinrezeptoren und ihrer möglichen therapeutischen Modulation, insbesondere in Zusammenhang mit Krebserkrankungen. Bislang sind die Rezeptoren auf diversen Krebszellen nachgewiesen worden. So konnten beispielsweise in einer Brustkrebszelllinie A2B Adenosinrezeptoren nachgewiesen werden, deren Stimulation zu einer Hemmung der wachstumsfördernden MAP Kinase führt. Pharmaka zur weitgehend selektiven Aktivierung oder Hemmung einzelner Adenosinrezeptor-Subtypen stehen ebenfalls zur Verfügung. Beim Ovarialkarzinom mit seiner leider meist erst spät auftretenden Symptomatik besteht derzeit noch keine Möglichkeit zur frühen Diagnosestellung, sodass die Prognose ausgesprochen ungünstig ausfällt und die Erkrankung bei Frauen eine der häufigsten krebsbedingten Todesursachen darstellt. Daher war es ein Ziel dieser Arbeit herauszufinden, ob Adenosinrezeptoren auf diesen Zellen einen möglichen therapeutischen Angriffspunkt bieten. Dazu untersuchten wir die vier Ovarialkarzinomzelllinien OVCAR-3, SK-OV-3, PA-1 und OAW-42 auf eine mögliche Expression von allen vier Adenosinrezeptorsubtypen. Zunächst wurden mit radioaktiv markierten Liganden ([3H]CCPA, [3H]NECA und [3H]HEMADO) Bindungsstudien für den A1-, A2A- und A3-Subtyp durchgeführt. Die Expression des A2B-Rezeptors wurde mithilfe eines funktionellen Nachweises, der Stimulation der Adenylylcyclase mithilfe von NECA (einem unspezifischen Adenosinrezeptoragonisten) analysiert. Im Anschluss daran untersuchten wir an OAW-42 und SK-OV-3 Zellen, ob sich ihr Proliferationsverhalten durch eine Stimulation mit NECA verändern ließe und ob sich das Ansprechen auf gängige Chemotherapeutika bzw. einen Todesliganden ändern würde. Trotz des erfolgreichen Nachweises von Adenosinrezeptoren auf allen Zelllinien waren die Ergebnisse der Proliferationsstudien aber nicht eindeutig. OAW-42 und SK-OV-3 Zellen reagierten zwar auf eine NECA-Stimulation mit sinkendem BrdU-Einbau, OAW-42 Zellen zeigten aber nach Behandlung mit NECA eine leicht erhöhte Resistenz gegenüber Cisplatin. NECA-behandelte SK-OV-3 Zellen reagierten hingegen etwas sensitiver auf Doxorubicin und Fas-Ligand. Die Unterschiede waren aber insgesamt sehr gering und wurden daher von uns als nicht entscheidender Effekt gewertet. Auch Untersuchungen zur Expression der Adenosin-generierenden Enzyme CD39 und CD73 vor und nach NECA-Stimulation blieben ohne erkennbare Veränderung. Insofern ergaben unsere Untersuchungen keine Hinweise darauf, dass Adenosinrezeptoren eine mögliche therapeutische Zielstruktur darstellen könnten. Zukünftige Studien können aber die gewonnenen Daten als Grundlage und Ausgangspunkt nützen, um auch andere Tumorzellarten zu untersuchen und im Kampf gegen den Krebs nach neuen potenziellen pharmakologischen Angriffspunkten zu suchen.
The novel refrigerant 2,3,3,3‐tetrafluoropropene (HFO‐1234yf) as well as the novel foam blowing and precision cleaning agent trans‐1‐chloro‐3,3,3‐trifluoropropene (trans‐HCFO‐1233zd) are both chlorofluorocarbon replacements with low GWPs and a short atmospheric life time. Whereas the hydrofluoroolefin HFO‐1234yf has no negative effect on stratospheric ozone due to the lack of chlorine in its structure, the hydrochlorofluoroolefine trans‐HCFO‐1233zd exhibits a very low potential for ozone depletion (ODP). This is approximately 100 times lower than the ozone depletion potential of precursor compounds such as 1,1,2‐trichloro‐1,2,2‐trifluoroethane (CFC‐113). Principle aims of this thesis were to investigate the unknown metabolism of the new solvent trans‐HCFO‐1233zd and to further investigate a possible biotransformation based toxicity of HFO‐1234yf observed in rabbits. Therefore study specimens of different in vitro and in vivo studies with trans‐HCFO‐1233zd and HFO‐1234yf were analyzed for metabolites using 19FNMR spectroscopy, LC‐MS/MS spectrometry and GC/MS spectrometry. Metabolites were identified by comparison with purchased or synthesized standard substances. Excretion kinetics of the predominant metabolites were determined by LC‐MS/MS quantification,inorganic fluoride was determined by potentiometry. Moreover cytochrome P‐450 2E1 and 3A4 liver enzyme activities were measured in a multi‐exposure study with HFO‐1234yf. ...
Attention-deficit/hyperactivity disorder (ADHD) is the most frequent psychiatric disorder in children and adolescents and is often treated with methylphenidate (MPH), resulting in MPH exposure in more than 1% of all children in many countries. A 2005 report on cytogenetic effects in peripheral lymphocytes from 12 ADHD children treated for 3 months with MPH raised questions about its genetic toxicity and potential carcinogenicity. A healthy control group (23 individuals), a chronically MPH-treated (>12 months) group (21 patients), and a drug naïve group of ADHD-affected children (26 patients), which was analyzed again after 3 months (17 patients) and 6 months (11 patients), provided samples for analysis of micronucleated lymphocytes. No significant alteration in genomic damage as seen as micronucleus frequency in peripheral lypmphocytes was detected after MPH treatment. No indication for genomic damage induced by MPH was obtained in this study. Ongoing studies in the USA, as well as continuation of recently published epidemiological cancer incidence analysis should provide additional reassurance for MPH-treated ADHD patients.
Terahertz electromagnetic fields are non-ionizing electromagnetic fields in the frequency range from 0.1 to 10 THz. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along human exposure to these fields. Up to now, only a limited number of investigations on biological effects of terahertz electromagnetic fields have been performed. Therefore, research is strongly needed to enable reliable risk assessment. Cells were exposed for 2 h, 8 h, and 24 h with different power intensities ranging from 0.04 mW/cm2 to 2 mW/cm2, representing levels below, at, and above current safety limits. Genomic damage on the chromosomal level was measured as micronucleus formation. DNA strand breaks and alkali-labile sites were quantified with the comet assay. No DNA strand breaks or alkali-labile sites were observed as a consequence of exposure to terahertz electromagnetic fields in the comet assay. The fields did not cause chromosomal damage in the form of micronucleus induction.
Introduction: Colon cancer is one of the major human malignancies worldwide, and much effort has been applied to understand the process of colon carcinogenesis, as well as the role of potential treatments and co-therapeutical agents against it. A growing body of evidence suggests that the use of fluoxetine (FLX), an antidepressant belonging to the selective serotonin reuptake inhibitors (SSRIs), may be associated with a reduced colon cancer risk. However, controversial opinions have been published and an identification of the mechanisms of the activity of FLX on colon cells would help in the clarification of this controversy. Objectives: Using several in vitro and in vivo-based methods and analyses, we aimed to verify whether FLX has antioxidant, pro-oxidant or DNA-damaging potential in standard toxicological assays; to check whether and how FLX could prevent and reduce colon preneoplastic lesions; to ascertain whether FLX has any oncostatic potential against colon tumors; and, to investigate whether FLX activity could be comparable with a known and current applied chemotherapeutic agent against colon cancer. Results: FLX did not have any antioxidant potential in our experiments. Although it did not induce reactive oxygen species (ROS) generation or DNA-damage in fibroblast and colon tumor cell lines, FLX reduced dysplasia and proliferation in two different carcinogen models. Further, a significant decrease in colon stromal reactivity and angiogenesis was found in both carcinogen-induced preneoplasia models. In a xenograft model of colon cancer, FLX shrank tumors, reduced tumor proliferation, arrested cancer cells at the G0/G1 cell-cycle phase, and took ROS generation under control. Such effects were detected together with an intracellular acidification and loss of mitochondrial membrane potential in FLX-treated cells. Modulating mitochondrial respiratory chain, HIF-1 expression and Akt/mTOR signaling pathway, FLX was found to reduce colon tumors similar to the widely used chemotherapeutic agent 5-Fluoracil activity. Conclusion: Our collective data suggest that FLX is a remarkable chemopreventive and oncostatic agent against colon preneoplastic lesions and tumors, acting without DNA-damage or ROS generation.
Das Renin-Angiotensin-Aldosteron-System (RAAS) reguliert den Blutdruck und den Wasser- und Elektrolythaushalt des Körpers. Angiotensin II (Ang II), das aktive Peptid des RAAS, bewirkt eine Vasokonstriktion und in höheren Konzentrationen Bluthochdruck. Epidemiologische Studien haben gezeigt, dass eine Verbindung zwischen Hypertonie und dem gehäuften Auftreten von Krebs besteht. Eine Metaanalyse von 13 Fall-Kontroll-Studien konnte einen Zusammenhang zwischen Hypertonie und einem erhöhten Risiko, an einem Nierenzellkarzinom zu erkranken nachweisen. In vitro-Studien und Studien an der isolierten Niere konnten bereits genotoxische Effekte des blutdruckregulierenden Hormons Ang II zeigen. Zielsetzung dieser Arbeit war es, zunächst in vivo zu prüfen, ob steigende Ang II-Konzentrationen einen Einfluss auf die genomische Stabilität von Nieren- und Herzzellen besitzen. Hierzu wurden im Dosisversuch männliche C57BL/6-Mäuse mit osmotischen Minipumpen ausgestattet, die Ang II in vier verschiedenen Konzentrationen zwischen 60 ng/kg min und 1 µg/kg min über einen Zeitraum von 28 Tagen abgeben sollten. Während des Versuchszeitraums fanden regelmäßige, nicht-invasive Blutdruckmessungen an der Maus statt. Die Behandlung mit Ang II führte zu einem signifikanten Anstieg des Blutdrucks und zu histopathologischen Veränderungen der Glomeruli und des Tubulussystems, was sich in einer verschlechterten Albumin-Ausscheidung wiederspiegelte. Außerdem induzierte die Behandlung mit Ang II die dosisabhängige Bildung von reaktiven Sauerstoffspezies, DNA-Doppelstrangbrüchen und oxidativer DNA-Schäden. Diese Parameter waren bereits in Tieren erhöht, die keinen Bluthochdruck entwickelten und stiegen mit der höchsten Ang II-Konzentration noch an, obwohl hier im Vergleich zur Vorgängergruppe, die eine geringere Ang II-Konzentration erhielt, kein höherer Blutdruck vorlag. Diese Beobachtung deutet auf eine mögliche Unabhängigkeit des entstandenen Schadens vom Bluthochdruck hin und lenkt die Aufmerksamkeit auf Ang II als genomschädigenden Faktor. Der folgende Interventionsversuch sollte Aufschluss über die mögliche blutdruckunabhängige genomschädigende Wirkung von Ang II geben. Dazu wurden C57BL/6-Mäuse neben der Ang II-Behandlung in einer Konzentration von 600 ng/kg min zusätzlich über einen Zeitraum von 28 Tagen mit 5 verschiedenen Substanzen behandelt: Candesartan, Ramipril, Hydralazin, Eplerenon und Tempol. Candesartan ist ein Ang II-Rezeptor-Antagonist, der selektiv den AT1-Rezeptor blockiert. Ramipril wirkt als Hemmer des Angiotensin-Konversions-Enzyms und verhindert die Bildung von endogenem Ang II aus Ang I. Hydralazin, als Vasodilatator, greift nicht in das Renin-Angiotensin-Aldosteron-System ein. Eplerenon blockiert als selektiver Aldosteronantagonist den Mineralkortikoidrezeptor. Tempol wirkt als Antioxidans. Die Behandlung mit Ang II in einer Konzentration von 600 ng/kg min im Interventionsversuch führte zur Hochregulierung der NADPH-Oxidase 4 und zur Produktion reaktiver Sauerstoffspezies in der Niere und im kardiovaskulären Gewebe. Der entstandene oxidative Stress führte wiederum zu DNA-Schäden und einer Aktivierung der Transkriptionsfaktoren Nrf2 und NF-B. Nrf2-vermittelt wurde die Induktion antioxidativer Gene ausgelöst, was allerdings nicht ausreichend war, um vor Ang II-induzierten ROS und DNA-Schäden zu schützen. Eine längerfristige NF-B-Aktivierung durch hohe Ang II-Spiegel kann das Überleben und die Proliferation von Zellen, die DNA-Schäden in Form von Doppelstrangbrüchen tragen, fördern, was eine Tumor-initiierende Wirkung haben könnte. Die beschriebenen Effekte erhöhter Ang II-Spiegel konnten durch die Intervention mit dem AT1-Rezeptorblocker Candesartan verhindert werden, was die Beteiligung des Rezeptors nachweist. Eine blutdruckunabhängige, genomschädigende Wirkung von Ang II konnte leider durch die Intervention mit Hydralazin nicht verdeutlicht werden, da die erwünschte langfristige Blutdrucksenkung ausblieb. Allerdings zeigte die Intervention mit Tempol eine Abnahme an oxidativem Stress und DNA-Schäden trotz ausbleibender Blutdrucksenkung. Die Bedeutung von ROS in der Bildung von DNA-Schäden und die Unabhängigkeit dieser Schäden vom Blutdruck konnten somit hervorgehoben werden. Die Tatsache, dass die Intervention mit Ramipril den Blutdruck nicht senken konnte, der oxidative Stress und die DNA-Schäden durch mögliche antioxidative Eigenschaften aber vermindert wurden, unterstützt diese Beobachtung. Die Intervention mit Eplerenon führte zum Teil zu einer Verminderung an ROS und DNA-Schäden, brachte diese Parameter aber nicht auf Kontrollniveau zurück. Somit ist eine Beteiligung von Aldosteron nicht auszuschließen.
The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G0/G1 phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.
Idiosynkratische Leberschädigung durch Arzneimittel (z.B. Diclofenac) stellt trotz ihres seltenen Auftretens eine erhebliche Komplikation in der Arzneimittelentwicklung und -therapie dar. Die zu idiosynkratischen Reaktionen führenden, komplexen chemischen und biologischen Abläufe sind noch weitgehend unklar. Inzwischen wird jedoch vermutet, dass die Toxizität eines Arzneimittels durch Arzneistoff-unabhängige Risikofaktoren, wie Krankheiten, Entzündungsreaktionen, Co-Medikation oder Alkohol, erhöht werden kann. Mögliche Mechanismen könnten hierbei eine vermehrte Bildung reaktiver Metaboliten bzw. eine veränderte zelluläre Stress- und Immunantwort sein. Um tiefere Einblicke in die Bedeutung möglicher Arzneistoff-unabhängiger Risikofaktoren zu erhalten, wurde in der vorliegenden Arbeit der Einfluss drei verschiedener Stressfaktoren auf die Toxizität von Diclofenac (Dcl) untersucht. Bei diesen Stressfaktoren handelte es sich um Lipopolysaccharid (LPS) und Poly I:C (PIC) zur Simulation einer bakteriellen bzw. viralen Entzündung sowie um Buthionin-Sulfoximin (BSO) zur Depletion zellulären Glutathions. Zusätzlich wurde getestet, ob eine durch Stressfaktoren ausgelöste Erhöhung der Toxizität von Dcl in Ratten mit Veränderungen in der Biotransformation bzw. mit einer Hochregulation co-stimulatorischer Faktoren (z.B. Zytokine oder Alarmsignale) einhergeht. Die Kombination einer einwöchigen therapeutisch dosierten Dcl-Behandlung mit einer einmaligen LPS-Dosis erzeugte in den Tieren eine ausgeprägte Hepatotoxizität, die mit erhöhten Aktivitäten 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 Lebertoxizität beiträgt. Mittels LC-MS/MS-Messungen konnten wir jedoch nachweisen, dass die Gabe von LPS + Dcl keine erhöhte Bildung reaktiver Metaboliten oder Dcl-AG-abhängiger Proteinaddukte auslöst. Im Einklang damit wurden Enzyme, die für 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. Zusätzliche qRT-PCR-Analysen Nrf2-abhängiger Gene, als Sensor für elektrophilen oder oxidativen Stress, zeigten keine Hochregulation zytoprotektiver Faktoren und unterstützen die Schlussfolgerung, dass Arzneistoff-unabhängige Stress-faktoren keine erhöhte Bildung toxischer Dcl-Metaboliten auslösen. 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 darüber hinaus eine massive Freisetzung pro-inflammatorischer Zytokine, Chemokine sowie toxizitätsfördernder Alarmsignale (z.B. IL-1β, TNF-α, CINC-1, HMGB1, LTB4) ins Plasma. Zusätzlich waren schützende 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 Leberschädigung nicht ausschlaggebend beteiligt ist. Im Gegensatz zu PIC oder BSO führte 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 leberschädigende Wirkung von Dcl potenziert wurde.
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.
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 ermöglicht und reguliert wird. Raf1 und GRK2 sind die einzigen bekannten direkten Interaktionspartner von RKIP und wurden deshalb gewählt, 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 für die RKIP/Raf1-Dissoziation und die RKIP/GRK2-Interaktion essentiell ist. Co-Immunpräzipitationsexperimente mit einer phosphorylierungsdefizienten Mutante zeigten, dass für diese Dimerisierung die Phosphorylierung von beiden RKIP-Molekülen notwendig ist. Als Dimerinteraktionsfläche wurden die Aminosäuren 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 für 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 für 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 höhere Affinität 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 Kinaseaktivität von GRK2 und Raf implizierten, dass dimerisiertes RKIP nur die Aktivität 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 Aufklärung dieses Mechanismus erweitert unser Verständnis der spezifischen Interaktion von Kinasen mit ihren Regulatorproteinen.
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.
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.
Zahlreiche experimentelle Befunde lassen vermuten, dass G-Protein gekoppelte Rezeptoren (GPCR) nach ihrer Aktivierung einer ligandenselektiven Änderung der Rezeptorkonformation unterliegen. Ziel der vorliegenden Arbeit war es dieses Phänomen am Subtyp 2 der muskarinischen Acetylcholinrezeptoren (M2 AChR) zu untersuchen. Muskarinische Acetylcholinrezeptoren (mAChR) können in fünf 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 fünf Subtypen hoch konserviert ist, wird ihr pharmakologischer Nutzen derzeit allerdings durch die unselektive Rezeptormodulation und dem damit verbundenen Auftreten unerwünschter 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, können so gezielt einzelne Subtypen der mAChR reguliert werden. Der M2 AChR stellt hinsichtlich allosterischer Modulation ein gut charakterisiertes Modellsystem dar. Für ihn wurde bereits eine Vielzahl allosterischer Liganden entwickelt. Auch bitopische Liganden, die sowohl einen allosterischen als auch einen orthosterischen Anteil enthalten, wurden für 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) gewählt. CFP wurde in den Sensoren am Ende des C-Terminus angefügt. Die zur Markierung mit FlAsH nötige Tetracysteinsequenz wurde in verschiedenen Bereichen der dritten intrazellulären 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 bezüglich Ligandenbindung, Gi-Protein Aktivierung und β-Arrestin2 Translokation ergab keine signifikanten Unterschiede zwischen M2i3-N, M2i3 C und M2CFP oder Wildtyp M2 AChR. Zunächst wurden sowohl unterschiedliche orthosterische, als auch allosterische Liganden hinsichtlich ihrer mittleren effektiven Konzentration und ihrer maximalen Wirkstärke 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 unabhängig 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 sensorabhängig unterschiedliche mittlere effektive Konzentrationen aufwie-sen. Bei der Untersuchung der Konformationsänderung des M2 AChR konnte kein liganden-selektiver Unterschied zwischen den FRET-Signalen für 19 getestete orthosterische Liganden beobachtet werden. Dies deutet darauf hin, dass alle orthosterischen Testliganden eine dem Acetylcholin (ACh) vergleichbare Änderung der M2 AChR Konformation induzier-ten. Um zu untersuchen, ob für die orthosterischen Testliganden eine Korrelation zwischen ihrer maximalen Wirkstärke 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 äußerst geringe β-Arrestin2 Translokation hervor. Im zweiten Teil der Arbeit wurde der Einfluss verschiedener Allostere auf eine ligandenselektive Konformationsänderung des M2 AChR untersucht. Die allosterischen Liganden JK 337 und Seminaph beeinflussten den M2i3-C Sensor signifikant stärker, als das M2i3-N Konstrukt. Dagegen zeigte EHW 477 eine stärkere Beeinflussung der Rezeptorkon-formation des M2i3-N-, als des M2i3-C Sensors. Dies erlaubt die Vermutung, dass JK 337 und Seminaph eine stärkere Bewegung unterhalb von Transmembrandomäne (TM) 6, als unterhalb von TM 5 hervorriefen. Die Ergebnisse für EHW 477 legen nahe, dass TM 5 eine größere Bewegung eingeht, als TM 6. FRET-basierte Untersuchungen der Einflüsse 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 möglich. Im Folgenden wurden die bitopischen Liganden Hybrid 1 und 2 (H 1, H 2) hinsichtlich ihres Effekts auf die Konformationsänderung des M2 AChR untersucht. Während 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-Änderung detektiert werden. Um den mangeln-den Effekt der Hybridsubstanzen in FRET-mikroskopischen Untersuchungen aufzuklären, wurden verschiedene Ansätze gewählt: Mit kettenverlängerten Derivaten der Hybridsubstanzen konnte in FRET-Messungen eine Änderung des FRET-Signals detektiert werden. Die Entfernung des allosterischen Bausteins führte 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 Signaländerung des FRET-Ratio. Diese Beobachtungen führten 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 ermöglichen. Ein anderer Erklärungsansatz besteht darin, dass nach Bindung des Orthosters der Kanal zwischen orthosterischer und allosterischer Bindestelle durch die Konformationsänderung des Rezeptors verschlossen wird, weshalb keine dauerhafte, dualsterische Bindung der Hybridsubstanzen an den M2 AChR möglich ist. Im Rahmen der vorliegenden Arbeit ist es gelungen mittels FRET-Experimenten die Existenz einer ligandenselektiven Rezeptorkonformation des M2 AChR mit allosterischen Liganden nachzuweisen. Darüber hinaus konnte auch ein Bezug zum Auftreten einer funktionellen Selektivität 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 Konformationsänderung. 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 für diesen Liganden hin. Die Untersuchung der dualsterischen Liganden H 1 und 2 bezüglich ihrer Fähigkeit zur Rezeptoraktivierung ergab, dass erst durch eine Verlängerung der Linkerkette, durch die orthosterischer und alloste-rischer Baustein miteinander verbunden sind eine Konformationsänderung des M2 AChR hin zu einer aktiven Konformation erreicht werden kann. Es kann somit angenommen werden, dass in den ursprünglichen Hybridsubstanzen H 1 und H 2 eine zu kurze Linkerkette, durch die keine dualsterische Bindung der Hybride an die allosterische und orthosterische Bindestelle möglich ist, ursächlich für die mangelnde Rezeptoraktivierung des M2 AChR war.
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.
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.
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.
Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization
(1988)
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
Der Fluoreszenz-Resonanz-Energie-Transfer ist ein Phänomen, welches erstmals 1948 von Theodor Förster beschrieben wurde. Mit der Entwicklung von Fluoreszenzproteinen konnten in Kombination mit Mikroskopietechniken Einblicke in zellbiologische Vorgänge gewonnen werden, die durch biochemische oder physiologische Experimente nicht möglich sind. Dabei spielt die hohe zeitliche und räumliche Auflösung eine wichtige Rolle. Auf dem Forschungsgebiet der GPCR, welche die größte Gruppe von Membranproteinen bei den Säugetieren darstellen, wurden insbesondere Erkenntnisse über Konformationsänderungen der Rezeptoren, die Kinetik der Rezeptoraktivierung und die Interaktion mit intrazellulären Signalproteinen gewonnen. Der µ-Opioidrezeptor gehört 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. Zunächst sollte ein intramolekularer FRET-Sensor des µ-Opioidrezeptors entwickelt werden, dazu wurden basierend auf den Kenntnissen über die Tertiärstruktur 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 eingefügten Sequenzen wurden in den intrazellulären Domänen variiert, da der Rezeptor auf die Modifikationen mit beeinträchtigter Membranlokalisation reagierte. Durch die Optimierung wurden Rezeptoren konstruiert, die an der Zellmembran lokalisiert waren. Jedoch zeigte keines der Rezeptorkonstrukte Funktionalität 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 Fähigkeit der Metabolite, Gi-Proteine zu aktivieren und β-Arrestin2 zu rekrutieren, mittels FRET-basierter Messungen an lebenden Zellen untersucht. Außerdem wurde die Affinität der Metabolite zum humanen µ Opioidrezeptor anhand der Verdrängung 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, während 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 überraschenderweise höhere Potenz und Effizienz für 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.
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.
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.
It is shown by means of IR. spectroscopic methodsthat nigericin and monensin bave a cyclic conformation similar to that of their silver salts. Camplex fonnation constants with sodium and potassium ions follow the selectivity order determined by EMF. measurements on liquid membranes: nigericin: K\(^+\) >Rb\(^+\)> Na\(^+\)> Cs\(^+\) >Li\(^+\); monensin: Na\(^+\)> K\(^+\) >Li\(^+\)> Rb\(^+\)> Cs\(^+\). Transport experiments show that nigericin and monensin facilitate the diffusion of potassium ions across model membranes, although in electrolytic transport experiments the permeability is not affected.
The structure of monensin, C36H620 11 , has been deterrnined by X-ray analysis of its crystalline monohydrate (orthorhombic, a = 15.15, b = 23.61, c = 10.65 A, Z = 4, space group P212121). Phases were assigned by direct methods, malring use of the 'tangent formula'. Although the conformation of the free acid resembles that of the silver salt in being cyclic, there are differences in the hydrogen bonding pattern. These featurcs are discussed in relation to the cornplexation of metal ions by m.onensin.
The thermodynainic parameters ΔH0, ΔG0 and ΔS0 - and thereby the equilibrium constants - for the complexation of the carrier antibiotics nigericin and monensin with sodium and potassium ions in methanol at 25°C have been determined by microcalorimetry. Tbc results are discussed in terms of the nature of the interaction between ligands and cations.
Aryl hydrocarbon hydroxylase (AHH) has been measured in male rat Jiver nucJei and microsomes after treatment of adult animals with various inducers for up to 14 days. After daily i.p. injections of 3-methylcholanthrene (MC, 20 mg/kg) the nuclear activity increased to a maximum of 600 per cent of the control activity after 4 days whereas the microsomal activity was 400 per cent of control at the same date. After 12 days, both activities equilibrated at 400 per cent. A similar time course was found after a single i.p. injection of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD, 0.01 mg/kg) with an induction to .500 and 300 per cent for nuclei and microsomes, respectiveJy. after 2 days, and to 400 per cent for both after 12 days. PhenobarbitaJ (PB) was given continuously in the drinking water (I g/1) and induced the microsomal activity to 200 per cent after 8 days and 170 per cent after 14 days. The nuclear activity was only slightly induced to a constant Ievei of 130 per cent between day 8 and 14. Dieldrin did not significantly increase the microsomal activity after daiJy i.p. injections (20 mg/kg), but the nuclear activity raised to 200 per cent after 3 days and levelled down tocontrol valuesafter 12 days. Other inducers tested were benz[a)anthracene (BA), hexachlorobenzene (HCB} and 1,1.1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The induction pattern with BA was similar tothat of MC, a modeJ compound for the group of cytochrome P448 inducers. The induction by HCB and DDT resembled that by PB. a typical cytochrome P450 inducer.
Wlth radioactive compound of high specific activity, the binding of carcinogene to DNA can be measured wlth doses that are ineffective ln long-term studies. The binding of tritiated benzo(a )pyrene to liver DNA of adult male rats has been determined 50 hr after a singie l.p. injection of doses between 40 1'9/kg and 4 mg/kg. The doseresponse relationship is linear up to 1 mg/kg, shows a step towards 2 mg/kg, and gives a shallow linear slope above that value. The observed binding ranges from 1.7 to 180 nmoles benzo(a)pyrene per mole DNA phosphate. The nonlinearity could be due to an induction of metabolizing enzymes. The microsomal aryl hydrocarbon hydroxylase activity increases significantly 24 hr after a single dose of 4 mg/kg and 48 hr after doses of 2 and 4 mg/kg, but no induction Ia found with 1 mg/kg. The binding from an equimolar dose is 35 times lower than the one found on mouse skin DNA and 300 times lower than that of N,Ndlmethylnitrosamine in rat liver. A good correlatlon exiats to the respective tumor formation in long-term studles.
Thecovalent bindingof [6,7-\(^3\)H]ethinylestradiol (EE)and [6,7-\(^3\)H]estrone (E) to liver DNA of 200 g female ratswas measured 8 h after the administration of 80 \(\mu\)g (9.2 mCi) estrogen by gavage. The binding is 1.5 for EE and 1.1 for E, expressedas binding to DNA/dose, in units of \(\mu\)mol hormonefmol DNA phosphate/mmole honnone/kg body wt. It is in the same order of magnitude as for benzene and about 10 000 tim es below the binding of typical liver carcinogens, such as aflatoxin B\(_1\) or N,N-dimethylnitrosamine.
The lnfluence of mlcrosomal and nuclear aryl hydrocarbon hydroxylase (AHH) actlvlty on the covalent blndlng of [G·3H]benzo(a )pyrene to rat llver DNA was evaluated in viWJ. lnductlon of mlcrosomal AHH was obtalned alter phenobarbltal treatment (160% of control), whlch also lncreased DNA blndlng to 190%, but left the nuclear actlvlty unchanged. Nuclear AHH was lnduced wlth dleldrln (150%), and the blndlng was decreased to 75%, whereaa the mlcrosomal AHH was at control Ievei. The lncreaslng effect of mlcrosomal AHH lnductlon as weil as the decreaslng effect of nuclear AHH lnductlon on the blndlng was shown clearly when the data of the Individual rata were uaed to solve the equatlon Binding = e•(mlcroeomal AHH) + b•(nuclear AHH) + c Multiple linear regresslon analysls wlth the data from 10 anlmala reaulted ln positive valuea for a and c, a negative value for b, and a good multiple correlatlon coefflclent of r = 0.974. Pretreatment wlth 3-methylcholanthrene ln· duced mlcrosomal AHH to 380% of control and nuclear AHH to 590% and lncreased the blndlng' to 175,.-o. The blndlng was hlgher than predlcted by the formula found, probably because the lncreaslng lnfluence of lnduced mlcrosomal AHH overahadowed the decreaslng effect of the nuclear AHH. The study ahows clearly that the blndlng of a forelgn compound to DNA in viWJ Ia dependent not only on mlcrosomal enzyme actlvltles but also on nuclear actlvltles even lf the latter are conslderably lower than thoae of mlcrosomes.