TY  - JOUR
A1  - Hintzsche, Henning
A1  - Jastrow, Christian
A1  - Kleine-Ostmann, Thomas
A1  - Kärst, Uwe
A1  - Schrader, Thorsten
A1  - Stopper, Helga
T1  - Terahertz electromagnetic fields (0.106 THz) do not induce manifest genomic damage in vitro
N2  - 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.
KW  - Toxikologie
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76268
ER  - 
TY  - JOUR
A1  - Förtsch, Christina
A1  - Hupp, Sabrina
A1  - Ma, Jiangtao
A1  - Mitchell, Timothy J.
A1  - Maier, Elke
A1  - Benz, Roland
A1  - Iliev, Asparouh I.
T1  - Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity
N2  - 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.
KW  - Toxikologie
KW  - pneumolysin
KW  - pore formation
KW  - cytoskeleton
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69084
ER  - 
TY  - THES
A1  - Sieber, Maximilian
T1  - Evaluation of 1H-NMR and GC/MS-based metabonomics for the assessment of liver and kidney toxicity
T1  - Bewertung von 1H-NMR und GC/MS-Metabonomics zur Erkennung von Leber- und Nierentoxizität
N2  - For the assessment of metabonomics techniques for the early, non-invasive detection of toxicity, the nephrotoxins gentamicin (s.c. administration of 0, 60 and 120 mg/kg bw 2x daily for 8 days), ochratoxin A (p.o. administration of 0, 21, 70 and 210 µg/kg bw 5 days/week for 90 days) and aristolochic acid (p.o. administration of 0, 0.1, 1.0 and 10 mg/kg bw for 12 days) were administered to rats and urine samples were analyzed with 1H-NMR and GC/MS. Urine samples from the InnoMed PredTox project were analyzed as well, thereby focusing on 1H-NMR analysis and bile duct necrosis as histopathological endpoint. 1H-NMR analysis used water supression with the following protocol: 1 M phosphate buffer, D2O as shift lock reagent, D4-trimethylsilyl­propionic acid as chemical shift reference, noesygppr1d pulse sequence (Bruker). For multivariate data analysis, spectral intensity was binned into 0.04 ppm wide bins. GC/MS analysis of urine was carried out after protein precipitation with methanol, drying, derivatization with methoxyamine hydrochloride in pyridine and with methyl(trimethylsilyl)­trifluoroacetamide on a DB5-MS column using EI ionization. The chromatograms were prepared for multivariate data analysis using the R-program based peak picking and alignment software XCMS version 2.4.0. Principal component analysis (PCA) to detect and visualize time-point and dose-dependent differences between treated animals and controls and orthogonal projection to latent structures discriminant analysis (OPLS-DA) for identification of potential molecular markers of toxicity was carried out using SIMCA P+ 11.5 1H-NMR-based markers were identified and quantified with the Chenomx NMR Suite, GC/MS based markers were identified using the NIST Mass Spectral Database and by co-elution with authentic reference standards. PCA of urinary metabolite profiles was able to differentiate treated animals from controls at the same time as histopathology. An advantage over classical clinical chemistry parameters regarding sensitivity could be observed in some cases. Metabonomic analysis with GC/MS and 1H-NMR revealed alterations in the urinary profile of treated animals 1 day after start of treatment with gentamicin, correlating with changes in clinical chemistry parameters and histopathology. Decreased urinary excretion of citrate, 2-oxoglutarate, hippurate, trigonelline and 3-indoxylsulfate increased excretion of 5-oxoproline, lactate, alanine and glucose were observed. Ochratoxin A treatment caused decreased excretion of citrate, 2-oxoglutarate and hippurate and and increased excretion of glucose, myo-inositol, N,N-dimethylglycine, glycine, alanine and lactate as early as 2 weeks after start of treatment with 210µg OTA/kg bw, correlating with changes in clinical chemistry parameters and histopathology. Integration of histopathology scores increased confidence in the molecular markers discovered. Aristolochic acid treatment resulted in decreased urinary excretion of citrate, 2-oxoglutarate, hippurate and creatinine as well as increased excretion of 5-oxoproline, N,N-dimethylglycine, pseudouridine and uric acid. No alterations in clinical chemistry parameters or histopathology were noted.Decreased excretion of hippurate indicates alterations in the gut microflora, an effect that is expected as pharmacological action of the aminoglycoside antibiotic gentamicin and that can also be explained by the p.o. administration of xenobiotica. Decreased Krebs cycle intermediates (citrate and 2-oxoglutarate) and increased lactate is associated with altered energy metabolism. Increased pseudouridine excretion is associated with cell proliferation and was observed with aristolochic acid and ochratoxin A, for which proliferative processes were observed with histopathology. 5-oxoproline and N,N-dimethylglycine can be associated with oxidative stress. Glucose, a marker of renal damage in clinical chemistry, was observed for all three nephrotoxins studied. Single study analysis with PCA of GC/MS chromatograms and 1H-NMR spectra of urine from 3 studies conducted within the InnoMed PredTox project showing bile duct necrosis revealed alterations in urinary profiles with the onset of changes in clinical chemistry and histopathology. Alterations were mainly decreased Krebs cycle intermediates and changes in the aromatic gut flora metabolites, an effect that may result as a secondary effect from altered bile flow. In conclusion, metabonomics techniques are able to detect toxic lesions at the same time as histopathology and clinical chemistry. The metabolites found to be altered are common to most toxicities and are not organ-specific. A mechanistic link to the observed toxicity has to be established in order to avoid confounders such as body weight loss, pharmacological effects etc. For pattern recognition purposes, large databases are necessary.
N2  - Zur Bewertung von Metabonomics-Techniken zur frühen, nicht-invasiven Erkennung von Toxizität wurde Rattenurin nach wiederholter Gabe von Nephrotoxinen mit 1H-NMR und GC/MS analysiert. Untersucht wurden Gentamicin (s.c.-Gabe von 0, 60 und 120 mg/kg Körpergewicht (KG) 2x tägl. über 8 Tage), Ochratoxin A (p.o.-Gabe von 0, 21, 70 und 210 µg/kg KG 5xl wöchentlich für 90 Tage) und Aristolochiasäure (p.o.-Gabe von 0, 0.1, 1.0 und 10 mg/kg KG über 12 Tage). Proben von 16 Studien des InnoMed PredTox Projekts wurden mit 1H-NMR auf den histopathologischen Endpunkt Gallengangnekrose (BDN) untersucht. Folgende Parameter wurden zur 1H-NMR-Analyse mit Wasserunterdrückung verwendet: 1 M Phosphatpuffer, shift lock Reagenz D2O und Referenzierung der chemischen Verschiebung auf D4-Trimethylsilyl­propionsäure, noesygppr1d-Pulssequenz (Bruker). Zur multivariaten Datenanalyse wurden die Spektren in 0.04 ppm große „bins“ unterteilt. Zur GC/MS-Analyse wurden nach Proteinfällung mit Methanol die Urinproben getrocknet und mit Methoxyaminhydrochlorid in Pyridin und Methyl(trimethylsilyl)trifluoracetamid derivatisiert und auf einer DB5-MS -Säule getrennt. Die GC/MS-Chromatogramme wurden mit dem R-Programm-basierten XCMS-Softwarepaket Version 2.4.0 zur multivariaten Datenanalyse vorbereitet. Hauptkomponentenanalyse (PCA) zur Visualisierung von zeit- und dosisabhängigen Unterschieden zwischen Kontrollen und behandelten Tieren und „orthogonal projection to latent structures“-Diskriminantenanalyse (OPLS-DA) zur Identifizierung von Toxizitätsmarkern erfolgte mit SIMCA P+11.5 Die Chenomx-NMR-Suite wurde zur Identifizierung und Quantifizierung von 1H NMR-basierten Markern verwendet; GC/MS-basierte Marker wurden mit der „NIST Mass Spectral Database“ und durch Koelution mit Referenzstandards identifiziert. PCA unterschied Kontroll- von behandelten Tieren zum gleichen Zeitpunkt wie Histopathologie. Gegenüber klinisch-chemischen Parametern war Metabonomics in einigen Fällen empfindlicher. Gentamicin induzierte nach Tag 1 erniedrigte Ausscheidung von Citrat, 2-Oxoglutarat, Hippurat, Trigonellin und 3-Indoxylsulfat Urin, sowie erhöhte Ausscheidung von Lactat, Alanin, 5-Oxoprolin und Glucose, begleitet von geringfügigen Änderungen in klinisch-chemischen Parametern. Ochratoxin A verursachte nach zwei Wochen in einzelnen Tieren eine erniedrigte Ausscheidung von Citrat, 2-Oxoglutarat und Hippurat sowie eine erhöhte Ausscheidung von Glucose, myo-Inositol, N,N-Dimethylglycin, 5-Oxoprolin, Glycin, Alanin und Lactat, korrelierend mit Veränderungen in klinisch-chemischen Parametern und in der Histopathologie. Verwendung von Histopathologiedaten in multivariaten Modellen zur Markeridentifizierung erhöhte die Konfidenz der Marker. Aristolochiasäure induzierte eine erniedrigte Ausscheidung von Citrat, 2-Oxoglutarat, Hippurat und Creatinin und eine erhöhte Ausscheidung von 5-Oxoprolin, N,N-Dimethylglycin und Pseudouridin, ohne Veränderung der klinisch-chemischen Parameter oder der Histopathologie. Erniedrigte Ausscheidung von Hippurat weist auf eine veränderte Darmmikroflora hin; für das Aminoglykosid-Antibiotikum Gentamicin ist dies ein pharmakologischer Effekt, der für die perorale Gabe von Xenobiotica zu erwarten ist. Erniedrigte Ausscheidung von Citrat und 2-Oxoglutarat und erhöhte Ausscheidung von Lactat zeigt einen veränderten Energiestoffwechsel. Erhöhte Ausscheidung von Pseudouridin ist mit Zell­proliferation assoziiert und wurde nach Gabe der Kanzerogene Ochratoxin A und Aristolochiasäure beobachtet, bei denen proliferative Prozesse in der Histopathologie gefunden wurden. 5-Oxoprolin und N,N-Dimethyl­glycin deuten auf erhöhten oxidativen Stress hin. Erhöhte Glucose im Urin, ein Parameter zur Diagnose von Nierenschäden in der klinischen Chemie, wurde in allen drei Studien mit Nephrotoxinen beobachtet. GC/MS- und 1H-NMR-Daten von InnoMed-Studien mit Gallengang­nekrosen als histopathologischen Endpunkt zeigten Veränderung im Urin zeitgleich mit klinisch-chemischen Parametern und Histopathologie; hauptsächlich erniedrigte Ausscheidung von Citratzyklusintermediaten und Veränderungen bei Darmflora-assoziierten Metaboliten, – ein Effekt, der wahrscheinlich veränderten Gallenfluss zurückzuführen ist. Metabonomics ist prinzipiell zum gleichen Zeitpunkt wie klinisch-chemische Parameter und Histopathologie zur Erkennung von toxischen Veränderungen geeignet. Die veränderten Metaboliten sind jedoch zumeist nicht organspezifisch und können mit allgemeinen Toxizitätsmechanismen, wie oxidativem Stress oder Zellproliferation, in Verbindung gebracht werden. Für die Bewertung der Ergebnisse von Metabonomics-Studien ist ein mechanistisches Verständnis der Veränderungen im Urinprofil notwendig, um eine Trennung von toxischen Effekten und solchen, die auf pharmakologische Wirkung, Körpergewichtsverlust etc. zurückzuführen sind, zu erreichen. Für eine Vorhersage von toxischen Mechanismen aufgrund der Urinprofile ist eine größere Datengrundlage notwendig.
KW  - Toxikologie
KW  - Protonen-NMR-Spektroskopie
KW  - GC-MS
KW  - Multivariate Analyse
KW  - Niere
KW  - Leber
KW  - Metabonomics
KW  - Toxicology
KW  - Metabonomics
KW  - GC/MS
KW  - 1H-NMR-Spectroscopy
KW  - multivariate analysis
KW  - kidney
KW  - liver
Y1  - 2009
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-43052
ER  - 
TY  - THES
A1  - Trösken, Eva-Regina
T1  - Toxicological evaluation of azole fungicides in agriculture and food chemistry
T1  - Toxikologische Beurteilung der Anwendung von Azolfungiziden in der Landwirtschaft und Lebensmittelchemie
N2  - Azole sind wichtige Chemikalien, die als Fungizide in der Landwirtschaft und der Medizin eingesetzt werden. Auch als Zytostatika in der Humanmedizin finden sie Anwendung. Die fungizide Wirkung beruht auf der Hemmung der Lanosterol-14α-Demethylase (CYP51), die die Demethylierung von Lanosterol zum „Follicular Fluid Meiosis Activating Steroid (FF-MAS)“ katalysiert. Für Pilze ist das später resultierende Ergosterol ein essentieller Bestandteil der Zellmembran. Exponierten Pilzen fehlt Ergosterol was zu einem Zusammenbruch der Zellmembran führt. Säugetiere können Cholesterol, das spätere Produkt der Lanosterol-14α-Demethylierung, das zur Synthese von z.B. Gallensäuren und Sexualhormonen nötig ist, mit der Nahrung aufnehmen. FF-MAS und das resultierende T-MAS (Testis Meiosis Activating Steroids), die direkten Produkte der CYP51 katalysierten Reaktion, wirken als Meiose-aktivierende Steroide auf Ovarien und Hoden und werden nicht mit der Nahrung aufgenommen. Eine Hemmung der CYP51 Aktivität könnte das endokrine System beeinflussen und wird daher als unerwünschte Nebenwirkung der Azole betrachtet. Aromatase (CYP19) katalysiert die Demethylierung von Testosteron zu Östradiol und wird durch Azole gehemmt. Die Verringerung der Östrogenspiegel durch CYP19-Inhibition ist das Wirkprinzip der als Zytostatika genutzten Azole, bei den Fungiziden wird es als unerwünschte Nebenwirkung angesehen. Ein ideales Azol sollte Pilz-CYP51 stark inhibieren, aber sowohl humanes CYP19 wie auch humanes CYP51 sollten durch ein solches Azol nicht inhibiert werden. Ein ideales Azol-Zytostatikum sollte eine starke inhibitorische Potenz gegenüber humanem CYP19 aufweisen, hingegen sollten humanes und Pilz-CYP51 nicht inhibiert werden. Ziel dieser Arbeit war es nun festzustellen: sind Fungizide und Antimykotika starke Inhibitoren von Pilz-CYP51? Zeigen Fungizide und Antimykotika keine Aktivität gegenüber humanem CYP19 und humanem CYP51? Sind Zytostatika starke Inhibitoren von humanem CYP19? Zeigen Zytostatika keine Aktivität gegenüber humanem CYP51 und Pilz-CYP51? Die inhibitorische Potenz von 22 Azolen, aus den drei Anwendungsgebieten, wurden an vier Systemen getestet: i) an humanem CYP19 und einem fluoreszierenden Pseudosubstrat, ii) an CYP19 und Testosteron als Substrat, iii) an humanem CYP51 und iv) Candida albicans CYP51 und Lanosterol als Substrat. Die Produktbildung wurde mittels Hochdruckflüssigkeitschromatographie gekoppelter Tandem-Massenspektrometrie nach Photosprayionisation gemessen. Das humane CYP51 wurde von „BD Gentest Cooperation“ zur Verfügung gestellt. Ein katalytisch aktiver Enzymkomplex bestehend aus der Lanosterol-14α-Demethylase von Candida albicans und der Oxidoreduktase von Candida tropicalis, wurde im Baculovirussystem exprimiert. Ein Vergleich der inhibitorischen Wirkstärke der Substanzen auf menschliches CYP19 und CYP51 und Pilz-CYP51 zeigt, dass einige Azole das erwünschte Bild zeigen. Dazu gehören die beiden Zytostatika Fadrozol und Letrozol, sowie Fluconazol und Itraconazol, zwei Antimykotika aus der Humanmedizin, und einige Fungizide z.B. Cyproconazol und Hexaconazol. Ein unerwünschtes Bild zeigen z.B. Prochloraz, Bifonazol, Ketoconazol und Miconazol. Sieben Azole weisen ein gemischtes Bild an inhibitorischen Wirkstärken auf. Um einen modellartigen Eindruck der Rückstände von Azolen in Lebensmitteln zu erhalten, wurde eine auf LC-ESI-MS/MS basierende Rückstandsanalytik für Azole im Wein entwickelt. Alle gefunden Rückstände lagen unterhalb der behördlich festgelegten Rückstandshöchstmengen. Um die inhibitorische Wirkung der Azole auf die verschiedenen Enzymsysteme in einem größeren Zusammenhang zu bringen, wurden die IC50 Werte mit Expositionsdaten von Bauern, maximalen Plasmaspiegeln in Patienten nach der Einnahme von Antimykotika und mit Expositionsgrenzwerten für die Langzeitaufnahme von Pflanzenschutzmittelrückständen („Acceptable Daily Intake Levels“, ADI) verglichen. Basierend auf den dargestellten Ergebnissen können folgende Schlussfolgerungen gezogen werden. Das Risiko für landwirtschaftliche Arbeiter durch Exposition gegenüber Azolfungiziden kann im Bezug auf menschliches CYP19 und CYP51 als vernachlässigbar eingestuft werden, wenn die entsprechenden Sicherheitsvorkehrungen getroffen werden. Im medizinischen Bereich muss grundsätzlich der Einsatz von Bifonazol, Miconazol und Ketoconazol mit Blick auf die hohe inhibitorische Potenz gegenüber menschlichem CYP19 und 51 kritisch betrachtet werden. Unter der Annahme, dass die ADI Werte eingehalten werden, stellen Rückstände auf Lebensmitteln in Bezug auf die genannten Enzymsysteme keine Bedrohung für den Verbraucher da. Die Inhibition von CYP19 muss als Störung des Hormonsystems angesehen werden. Die Bedeutung von FF-MAS und T-MAS im endokrinen System muss noch abschließend geklärt werden und damit auch die Frage, wie viel Bedeutung der Inhibition von menschlichem CYP51 beigemessen werden muss.
N2  - Azoles are important chemicals used as antifungal agents in agriculture and human medicine, but also as cytostatic drugs in tumour chemotherapy. Antifungal activities are based on inhibition of lanosterol-14α-demethylase (CYP51). CYP51 catalyses the oxidative removal of the methyl group # 32 of lanosterol to produce follicular fluid meiosis activating steroid (FF-MAS). For fungi the later resulting ergosterol is an essential compound of the cell membrane. Exposed fungi lack ergosterol, which leads to a collapse of the cell membrane. In mammals cholesterol, the downstream product of lanosterol-14α-demethylation necessary for the synthesis of bile acids, mineral corticoids, glucocorticoids and sex steroids, can be supplemented with food intake. However FF-MAS and the resulting T-MAS (testis meiosis activating steroids), the direct products of the CYP51 reaction, act as meiosis-activating steroids on ovaries and testes and are not supplemented with food intake. Inhibition of CYP51 in humans may therefore affect the endocrine system and is an unwanted side effect of azoles. Aromatase (CYP19) catalyses the demethylation of testosterone to estradiol and is inhibited by azoles. Reduction of estrogen levels by CYP19 inhibition is the working principle of cytostatic drugs used in breast cancer therapy but is considered an unwanted side effect for azoles used to treat fungal infections. A favourable fungicide or antifungal drug should be a strong inhibitor of fungal CYP51. In contrast human CYP51 and human CYP19 should not be inhibited by an azole fungicide or antifungal agent. The favourable cytostatic drug should show a high potency towards human CYP19. Neither human CYP51 nor fungal CYP51 should be inhibited by a cytostatic drug. The aim of this work was to assess: are fungicides and antifungal drugs strong inhibitors of fungal CYP51? In return do they not inhibit human CYP51 and human CYP19? Do cytostatic drugs strongly inhibit human CYP19? And in return do they not inhibit human CYP51 or fungal CYP51? Inhibitory potencies of 22 azole compounds used for the three purposes were tested in four inhibition assays: i) on commercially available human CYP19 utilising a fluorescent pseudo substrate dibenzylfluorescein (DBF) ii) on CYP19 utilising testosterone as substrate iii) on human CYP51 and iv) Candida albicans CYP51 utilising lanosterol as substrate. Product formation was measured by liquid chromatography – tandem mass spectrometry utilising photospray ionisation (APPI). A functional human CYP51 was available from BD Gentest Cooperation. A functional enzyme complex comprising of the Candida albicans lanosterol-14α-demethylase and the Candida tropicalis oxidoreductase was expressed in the baculovirus system. When comparing inhibitory potencies on CYP19, human CYP51 and Candida albicans CYP51 a number of agents show desirable patterns of inhibition e.g. the two cytostatic drugs, or two antifungal agents used in human medicine, fluconazole and itraconazole, and a wide variety of the fungicides, e.g. cyproconazole and hexaconazole. Undesirable patterns of inhibition were exhibited by a number of compounds, e.g. prochloraz, bifonazole, ketoconazole and miconazole. Seven compounds show a more complex picture of inhibitory potencies though. To get a picture of residue levels of azoles in food in a model case an LC-ESI-MS/MS method was developed for the determination of azole compounds in wine. All residues were below the maximum residue levels set by authorities. To classify the inhibitory potencies on the different enzyme systems IC50 values obtained were compared to exposure levels measured in farmers, maximum plasma concentrations in humans reported after exposure to antifungal drugs and to acceptable daily intake levels set by authorities. Based on the findings presented, the following conclusions can be drawn. The risk for agricultural workers set by exposure to azole fungicides with respect to human CYP51 and CYP19 can be regarded as negligible when safety measures are adhered to. As a matter of principle however, the usage of bifonazole, miconazole and ketoconazole has to be viewed with caution in respect to the high level of inhibition of human CYP51 and/or CYP19. Under the assumption that the acceptable daily intake amounts set by authorities for azole compounds are not exceeded the residues do not pose a threat to consumer safety judged by our findings. Inhibition of CYP19 with the consequence of a reduction of estradiol levels has to be regarded as a possible disrupting effect of the hormone balance. The relevance of FF-MAS and T-MAS in the endocrine system however still has to be evaluated completely bringing with it the question of how much importance has to be attached to the inhibition of human CYP51.
KW  - Azole
KW  - Fungizid
KW  - Toxikologie
KW  - CYP19
KW  - CYP51
KW  - Azole
KW  - Toxikologie
KW  - CYP19
KW  - CYP51
KW  - Azoles
KW  - Toxicology
Y1  - 2005
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-17016
ER  - 
TY  - JOUR
A1  - Fischer, W. H.
A1  - Lutz, Werner K.
T1  - Short communication : Mouse skin papilloma formation by chronic dermal application of 7,12-dimethylbenz[a]anthracene is not reduced by diet restriction
N2  - No abstract available
KW  - Toxikologie
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60644
ER  - 
TY  - JOUR
A1  - Grunicke, H.
A1  - Pyerin, W.
A1  - Eisenbrand, G.
A1  - Havemann, K.
A1  - Rabes, H. M.
A1  - Molling, K.
A1  - Schwab, M.
A1  - Lutz, Werner K.
A1  - Wahrendorf, J.
A1  - Schirrmacher, V.
T1  - 7th International Symposium of the Division of Experimental Cancer Research (AEK) of the German Cancer Society : [Meeting report]
N2  - No abstract available
KW  - Toxikologie
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60651
ER  - 
TY  - JOUR
A1  - Stopper, Helga
A1  - Kühnel, A.
A1  - Podschun, B.
T1  - Combination of the chemotherapeutic agent 5-fluorouracil with an inhibitor of its catabolism results in increased micronucleus induction
N2  - 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.
KW  - Toxikologie
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63383
ER  - 
TY  - JOUR
A1  - Stopper, Helga
A1  - Eckert, I.
A1  - Schiffmann, D.
A1  - Spencer, D. L.
A1  - Caspary, W. J.
T1  - Is micronucleus induction by aneugens an early event leading to mutagenesis?
N2  - 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.
KW  - Toxikologie
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63390
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Krotec, K. L.
A1  - Gripentrog, J.
A1  - Jesaitis, A. J.
T1  - Regulatory interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton in human neutrophils
N2  - The cytoskeleton and/or membrane skeleton has been implicated in the regulation of N-formyl peptide receptors. The coupling of these chemotactic receptors to the membrane skeleton was investigated in plasma membranes from unstimulated and desensitized human neutrophils using the photoreactive agonist N-formyl-met-leu-phelys-N\(^6\)-[\(^{125}\)I]2(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (fMLFK-[\(^{125}\)I]ASD). When membranes of unstimulated cells were solubilized in Triton-X 100, a detergent that does not disrupt actin filaments, only 50% of the photoaffinity-labeled receptors were solubilized sedimenting in sucrose density gradients at a rate consistent with previous reports. The remainder were found in the pellet fraction along with the membrane skeletal actin. Solubilization of the membranes in the presence of p-chloromercuriphenylsulfonic acid, elevated concentrations of KCI, or deoxyribonuclease I released receptors in parallel with actin. When membranes from neutrophils, desensitized by incubation with fMLFK-e 251]ASD at 15°C, were solubilized, nearly all receptors were recovered in the pellet fraction. lncubation of cells with the Iigand at 4°C inhibited desensitization partially and prevented the conversion of a significant fraction of receptors to the form associated with the membrane skeletal pellet. ln these separations the photoaffinity-labeled receptors not sedimenting to the pellet cosedimented with actin. Approximately 25% of these receptors could be immunosedimented with antiactin antibodies suggesting that N-formyl peptide receptors may interact directly with actin. These results are consistent with a regulatory role for the interaction of chemotactic N-formyl peptide receptors with actin of the membrane skeleton.
KW  - Toxikologie
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60466
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Jesaitis, A. J.
T1  - Neutrophil chemoattractant receptors and the membrane skeleton
N2  - Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process which involves participation of cytoskeletal elements. Evidence exists suggesting that the cytoskeleton and/or the membrane skeleton controls the distributJon of FPR in the plane of the plasma membrane, thus controlling the accessibility of FPR to different proteins in functionally distinct domains. In desensitized cells, FPR are restricted todomains which are depleted of G proteins but enriched in cytoskeletal proteins such as actin and fodrin. Thus, the G protein signal transduction partners of FPR become inaccessible to the agonist-occupied receptor, preventing cell activation. The mechanism of interaction of FPR with the membrane skeleton is poorly understood but evidence is accumulating that suggests a direct binding of FPR (and other receptors) to cytoskeletal proteins such as actin.
KW  - Toxikologie
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60471
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Jesaitis, A. J.
T1  - Physical coupling of N-formyl peptide chemoattractant receptors to G protein is not affected by desensitization
N2  - Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils results in association of these receptors to the membrane skeleton. This is thought to be the critical event in the lateral segregation of receptors and guanyl nucleotide-binding proteins (G proteins) within the plane of the plasma membrane resulting in an interruption of the signaling cascade. In this study we probed the interaction of FPR with G protein in human neutrophils that were desensitized to various degrees. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phelys- N\(^\epsilon\)-[\(^{125}\)I]2(p-azidosalicylamido )ethyl-1 ,3 '-dithiopropionate (/MLFK-[\(^{125}\)I]ASD). The interaction if FPR with G protein was studied via a reconstitution assay and subsequent analysis of FPR-G protein complexes in sucrose density gradients. FPR-G protein complexes were reconstituted with solubilized FPR from partially and fully desensitized neutrophils with increasing concentrations of Gi purified from bovine brain. The respective EC\(_{50}\) values for reconstitution were similar to that determined for FPR from unstimulated neutrophils (Bommakanti RK et al., J Bio[ Chem 267: 757~7581, 1992). We conclude, therefore, that the affinity of the interaction of FPR with G protein is not affected by desensitization, consistent with the model of lateral segregation of FPR and G protein as a mechanism of desensitization.
KW  - Toxikologie
KW  - chemotactic receptors
KW  - G proteins
KW  - N-formyl peptides
KW  - signal transduction
KW  - receptor-G protein coupling
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60483
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Jesaitis, A. J.
T1  - The interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton is energy-dependent
N2  - Desensitization of N-fonnyl peptide chemoattractant receptors (FPR) in human neutrophils is thought to be achieved by lateral segregation of receptors and G proteins within the plane of the plasma membrane resulting in an interruption of the signalling cascade. Direct coupling of FPR to membrane skeletal actin appears to be the basis of this process~ however, the molecular mechanism is unknown. In this study we investigated the effect of energy depletion on formation of FPR-membrane skeleton complexes. In addition the effect of the protein kinase C inhibitor stauroporine and the phosphatase inhibitor okadaic acid on coupling of FPR to the membrane skeletonwas studied. Human neutrophils were desensitized using the photoreactive agonist N-formy1-met-leu-phe-1ys-N'[\(^{125}\)I]2(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (fMLFK-[\(^{125}\)I]ASD) after ATP depletion with NaF or after incubation with the respective inhibitors. The interaction of FPR with the membrane skeleton was studied by Sedimentation of the membrane skeleton-associated receptors in sucrose density gradients. Energy depletion of the cells markedly inhibited the formation of FPR-membrane skeleton complexes. This does not appear tobe related to inhibition of protein phosphorylation due to ATP depletion because inhibition of protein kinases and phosphatases bad no significant effect on coupling of FPR to the membrane skeleton. We conclude, therefore, that coupling of FPR to the membrane skeleton is an energy,dependent process which does not appear to require modification of the receptor protein by phosphorylation.
KW  - Toxikologie
KW  - Chemotactic receptors
KW  - G proteins
KW  - N-formyl peptides
KW  - signal transduction
KW  - desensitization
KW  - membrane skeleton
KW  - receptor-G protein coupling.
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60499
ER  - 
TY  - JOUR
A1  - Shephard, S. E.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - The lacI transgenic mouse mutagenicity assay: quantitative evaluation in comparison to tests for carcinogenicity and cytogenetic damage in vivo
N2  - The detection Iimit of the lacl transgenic mouse mutagenicity assay lies, in practice, at approximately a 50-100% increase in mutant frequency in treated animals over controls. The sensitivity of this assay in detecting genotoxins can be markedly improved by subchronic rather than acute application of the test compound. The lac/ transgenic mouse mutagenicity assay was compared quantitatively to rodent carcinogenicity tests and to presently used in vivo mutagenicity assays. With the genotoxic carcinogens tested thus far, a rough correlation between mutagenic potency and carcinogenic potency was observed: on average, to obtain a doubling in lacl mutant frequency the mice bad to be treated with a total dose equal to 50 times the TD50 daily dose Ievel. This total dose could be administered eilher at a high dose rate within a few days or, preferably, at a low dose rate over several weeks. This analysis also indicated that a lacl experiment using a 250-day exposure period would give a detection Iimit approximately equal to that of a long-term carcinogenicity study. In comparison to the micronucleus test or the chromosome aberration assay, acute sturlies with the presently available lacl system offered no increase in sensitivity. However, subchronic lacl sturlies (3-4-month exposure) resulted in an increase in sensitivity over the established tests by 1-2 orders of magnitude (shown with 2-acetylaminofluorene, N-nitrosomethylamine, N-nitrosomethylurea and urethane). 1t is concluded that a positive result in the lacl test can be highly predictive of carcinogenicity butthat a negative result does not provide a large margin of safety.
KW  - Toxikologie
KW  - Transgenie mice
KW  - Mutagenicity assay
KW  - Sensitivity
KW  - Chromosome aberration
KW  - Micronucleus test
KW  - Carcinogenic potency
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60638
ER  - 
TY  - JOUR
A1  - Stopper, Helga
A1  - Kirchner, S.
A1  - Schiffmann, D.
A1  - Poot, M.
T1  - Cell cycle disturbance in relation to micronucleus formation induced by the carcinogenic estrogen diethylstilbestrol
N2  - 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.
KW  - Toxikologie
KW  - Flow cytometry
KW  - Micronucleus formation
KW  - Diethylstilbestrol
KW  - Hoechst 33258 dye
KW  - Bromodeoxyuridine labeling
KW  - continuous
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82250
ER  - 
TY  - JOUR
A1  - Kirchner, S.
A1  - Stopper, Helga
A1  - Papp, T.
A1  - Eckert, I.
A1  - Yoo, H. J.
A1  - Vig, B. K.
A1  - Schiffmann, D.
T1  - Cytogenetic changes in primary, immortalized and malignant mammalian cells
N2  - 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.
KW  - Toxikologie
KW  - Micronuclei
KW  - Kinetochore
KW  - Chromosome distribution
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63439
ER  - 
TY  - JOUR
A1  - Stopper, Helga
A1  - Körber, C.
A1  - Spencer, D. L.
A1  - Kirchner, S.
A1  - Caspary, W.J.
A1  - Schiffmann, D.
T1  - An investigation of micronucleus and mutation induction by oxazepam in mammalian cells
N2  - 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.
KW  - Toxikologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63404
ER  - 
TY  - JOUR
A1  - Stopper, Helga
A1  - Körber, C.
A1  - Schiffmann, D.
A1  - Caspary, W. J.
T1  - Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells
N2  - 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.
KW  - Toxikologie
KW  - Micronuclei
KW  - L5178Y cells
KW  - 5-Azacytidine
KW  - Berenil
KW  - DES
KW  - Ethionine
KW  - Mitosis
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63411
ER  - 
TY  - JOUR
A1  - Adam, W.
A1  - Ahrweiler, M.
A1  - Saha-Möller, C. R.
A1  - Sauter, M.
A1  - Schönberger, A.
A1  - Epe, B.
A1  - Müller, E.
A1  - Schiffmann, D.
A1  - Stopper, Helga
A1  - Wild, D.
T1  - Genotoxicity studies of benzofuran dioxetanes and epoxides with isolated DNA, bacteria and mammalian cells
N2  - 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.
KW  - Toxikologie
KW  - 1
KW  - 2-Dioxetane
KW  - Benzefuran dioxetane
KW  - Benzefuran epoxide
KW  - DNA damage
KW  - Mutagenicity
KW  - DNA adduct . Repair endonuclease
KW  - FPG protein
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63420
ER  - 
TY  - JOUR
A1  - Jesaitis, A. J.
A1  - Erickson, R. W.
A1  - Klotz, Karl-Norbert
A1  - Bommakanti, R. K.
A1  - Siemsen, D. W.
T1  - Functional molecular complexes of human N-formyl peptide chemoattractant receptors and actin
N2  - When human neutrophils become desensitized to formyl peptide chemoattractants, the receptors (FPR) for these peptides are converted to a high affinity, GTP-insensitive form that is associated with the Triton X-1 00- insoluble membrane skeleton from surface membrane domains. These domains are actin and fodrin-rich, but G protein-depfeted suggesting that FPR shuttling between G protein-enriched and depleted domains may control signal transduction. Todetermine the molecular basis for FPR interaction with the membrane skeleton, neutrophil subcellular fractions were screened for molecules that could bind photoaffinity-radioiodinated FPR solubilized in Triton X-1 00. These receptors showed a propensity to bind to a 41- to43-kDa proteinband on nitrocelluloseoverlays of SOS-PAGE-separated cytosol and plasma membrane fractions of neutrophils. This binding, as weil as FPR binding to purified neutrophil actin, was inhibited 50% by 0.6 \(\mu\)M free neutrophil cytosolic actin. Addition of greater than 1 \(\mu\)M G-actin to crude or lectin-purified Triton X-1 00 extracts of FPR from neutrophil membranes increased the sedimentationrate of a significant fraction of FPR two to three fold as measured by velocity sedimentation in Triton X-1 00-containing linear sucrose density gradients. Addition of anti-actin antibodies to FPR extracts caused a concentration-dependent immunoprecipitation of at least 65% of the FPR. More than 40% of the immunoprecipitated FPR was specifically retained on protein A affinity matrices. Membrane actin was stabilized to alkaline washing when membranes were photoaffinity labeled. Conversely, when purified neutrophil cytosolic actinwas added to membranes or their digitonin extracts, after prior depletion of actin by an alkaline membrane wash, photoaffinity labeling of FPR was increased two- to fourfold with an EC\(_{50}\) of approximately 0.1 \(\mu\)M actin. We conclude that FPR from human neutrophils may interact with actin in membranes to form Triton X-1 00-stable physical complexes. These complexes can accept additional G-actin monomers to form higher order molecular complexes. Formation of FPR-actin complexes in the neutrophil may play a role in the regulation of chemoattractantinduced activation or actin polymerization.
KW  - Toxikologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60445
ER  - 
TY  - JOUR
A1  - Bommakanti, R. K.
A1  - Klotz, Karl-Norbert
A1  - Dratz, E. A.
A1  - Jesaitis, A. J.
T1  - A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein
N2  - No abstract available
KW  - Toxikologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60456
ER  - 
TY  - JOUR
A1  - Cantoreggi, S.
A1  - Dietrich, D. R.
A1  - Lutz, Werner K.
T1  - Induction of cell proliferation in the forestomach of F344 rats following subchronic administration of styrene 7,8-oxide and butylated hydroxyanisole
N2  - The question addressed was whether Stimulation of cell proliferation could be responsible for tumor induction in the torestornach by styrene 7,8-oxide (SO). Male F344 rats were treated for 4 weeks with 0, 137,275, and 550 mglkg SO by p.o. gavage 3 times/week. Positive controls received 0, 0.5, I, and 2% butylated hydroxyanisole (BHA) in the diet for 4 weeks. Twenty-four h before termination of the experlment, the rats were implanted s.c. with an osmotic minipump deliverlog S-bromo-2'-deoxyuri· dine (BrdU). Cell proliferation in the forestomach was assessed by immunohistochemistry for BrdU incorporated into DNA. Cell number/mm section length and fraction of replicating cells (labeling Index) were determined in 3 domains of the forestomach, the saccus caecus, the midregion, and the prefundic region. With the exception of the prefundic reglon of the low-dose SO group, a significant increase of the labeling index was found in all regions both with SO and BHA. Rats treated with BHA showed, in addition, a dose-dependent increase in number and size of hyperplastic lesions. This was most pronounced in the prefundic region where carcinomas were reported to be localized. In this region, the number of dividing cells/mm section length was increased up to 17-fold. With SO, only marginal morphological changes were occasionally observed, despite the fact that the respective long-term treatment bad been reported to result in a higher carcinoma incidence than treatment with BHA. It ls concluded that the rate of replicating cells alone, numerically expressed by the labeling Index, is an lnsufficient tool for interpretlog the role of cell division in carcinogenesis. It is postulated that SO and BHA induce forestomach tumors via different mechanisms. While hyperplasia in the prefundic region most likely dominates the carcinogenicity of BHA, a mechanism combining marginal genotoxicity with strong promotion by increased cell proliferation appears to be involved in the tumorigenic action of SO.
KW  - Toxikologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60669
ER  - 
TY  - JOUR
A1  - Fischer, W. H.
A1  - Beland, P. E.
A1  - Lutz, Werner K.
T1  - DNA adducts, cell proliferation and papilloma latency time in mouse skin after repeated dermal application of DMBA and TPA
N2  - 'lbe mouse skin tumor model was used to investigate whether the Ievel of DNA 8dducts and/or the rate of cell division in the epidermis are indicators of the risk of cancer formation for an individual in an outbred animal popul8tion. A high risk was considered to be reftected by 8 short latency period for the 8ppearance of 8 papilloma. Fernale NMRI mice were treated twice weekly with 2.5 nmol 7 ,12-dimethylbenz[a]antbracene (DMBA) and 3 nmoi12-0-tetradecanoylphorbol-13- 8cetate (TPA) and the appearance of papillomas was registered. The first papilloma 8ppeared after 7.5 weeks. After 17 weeks, when 12 of 14 mice bad 8t least one papilloma, an osmotic minipump deliverlog 5-bromo-2'deoxyuridine (BrdU) was implanted into eacb mouse for 24 h. The mice were killed after 24 h ~d the epidermis was analyzed for D:MBA-nucleotide 8dducts by 32p.postlabeling, for the cell number per unit skin length, and for the labeling index for DNA synthesls. Unexpectedly, D:MBA-nucleotide 8dduct Ievels were highest in those anima1s wbich showed the Iongest latency periods. Adduct Ievels were negatively correlated with the 18beling index, indicating that dilution of adducts by cell division was a predominant factor in determining average adduct concentrations. Individual tumor-latency time was not corTelated with either cell ntunber or labeling index. This could be due to the fact that the measurements only provided 8veraged data and gave no infonnation on the specific situation in clones of premalignant cells. Under the conditions of tbis assay, therefore, neither DNA adduct Ievels nor information on the average kinetics of cell division bad a predidive value for the individual amcer risk withln a group of outbred animals receiving the same treatment
KW  - Toxikologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60673
ER  - 
TY  - JOUR
A1  - Shephard, S. E.
A1  - Sengstag, C.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene
N2  - 2-Acetylaminofluorene (2-AAF) was administered at Ievels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic micc bearing the lacl genein a Iambda vector (Big Blue® mice). The Iambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10\(^5\) plaques wcre screened per animal for the appearance of a bluc colour, indicative of mutations in the lac/ gcnc which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10\(^{-5}\) (pooled results of two animals, 8 mutant plaques/289 530 plaques). At 300 ppm in the diet, the rate of 3.5 X 10\(^{-5}\)(8/236 300) was not significantly increased over background. At 600 ppm in the dict, the rate increased approximately 3 fold to 7.7 x 10\(^{-5}\) (17 /221240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose Ievels 00-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2·AAF.
KW  - Toxikologie
KW  - 2-Acetylaminofluorene
KW  - Transgenic mouse
KW  - Mutation assay
KW  - in vivo
KW  - Dose response
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60683
ER  - 
TY  - JOUR
A1  - Cantoreggi, S.
A1  - Lutz, Werner K.
T1  - Covalent binding of styrene to DNA in rat and mouse
N2  - No abstract available
KW  - Toxikologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60693
ER  - 
TY  - JOUR
A1  - Gunz, D.
A1  - Shephard, S. E.
A1  - Lutz, Werner K.
T1  - Can nongenotoxic carcinogens be detected with the lacI transgenic mouse mutation assay?
N2  - No abstract available
KW  - Toxikologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60707
ER  - 
TY  - JOUR
A1  - Stopper, Helga
A1  - Pechan, R.
A1  - Schiffmann, D.
T1  - 5-azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis
N2  - 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.
KW  - Toxikologie
KW  - 5-Azacytidine
KW  - Micronuclei
KW  - Kinetochores
KW  - Unscheduled DNA synthesis
KW  - Cell transformation
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63443
ER  - 
TY  - JOUR
A1  - Gonzales-Calero, G.
A1  - Cubero, A.
A1  - Klotz, Karl-Norbert
T1  - G protein coupled A\(_1\) adenosine receptors in coated vesicles of mammalian brain. Characterization by radioligand binding and photoaffinity labeling
N2  - A\(_1\) adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaflinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1 ,3-[\(^3\)H]dipropyl-xanthine ([\(^3\)H]DPCPX) gave a Kdvalue of 0.7 nM and a Bmax value of 82± 13 fmol/mg protein. For the highly A\(_1\)-selective agonist 2-chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [\(^3\)H]DPCPX binding gave a pharmacological profile with R-N\(^6\)-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCP A to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N\(^6\)- \(^{125}\)I-p-hydroxyphenylisopropyladenosine ([\(^{125}\)I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A\(_1\) receptors labelled in brain membranes. Therefore, coated vesicles contain A\(_1\) adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A\(_1\) receptors in these vesicles are not a processed receptor fonn. These results confirm that A\(_1\) receptors in coated vesicles are coupled to a G-protein, and it appears that the A\(_1\) receptor systems in coated vesicles andin plasma membranes are identical.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - coated vesicles
KW  - G-protein
KW  - radioligand
KW  - photoaffinity labelling
KW  - brain membranes
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60435
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Schlatter, J.
T1  - Chemical carcinogens and overnutrition in diet-related  cancer [commentary]
N2  - The intake of known dietary carclnogens was compiled and the cancer risk was estlmated on the basis of carcinogenic potencies in animals as derived from the Carcinogenic Potency Database by Gold and co-workers. The total cancer risk was compared with the number of cancer cases attributed by epidemiologists to dietary factors (one-third of all cancer cases, i.e. -80 000 per one million Jives). Except for alcohol, the known dietary carcinogens could not account for more than a few bundred cancer cases. Tbis was seen both with tbe DNA-reactive carcinogens (beterocyclic aromatic amines, polycyclic aromatic hydrocarbons, N-nitroso compounds, estragole, aflatoxin B., ethyl carbamate, to name the most important factors) as wen as with those carclnogens wbich have not been shown to react with DNA (e.g. caffelc acid and the carcinogeruc metals arsenic and cadmium). Residues and contaminants turned out to be negligible. Among the various pmsibilities to explain the discrepancy we investigated the roJe of ovemutritlon. Dietary restriction in animals is weil known for its strong reducing effect on spontaneous tumor formation. These data can be used to derive a carcinogenic potency for excess macronutrients: tbe tumor incidence seen with the restrlcted animals is taken as a control value and the increased tumor incidence in the animals fed ad libitum is attributed to the additional feed iotake. For excess standard diet in rats, a carcinogenic potency TD50 of 16 glkg/day was deduced from a recent study. Ovemutrition in Switzerland, estimated to be 5.5 kcallkg/day, was converted to excess food (1.9 g/kg/day) and tbe cancer incidence was calculated. The result, 60 000 cancer cases per one million Jives, is provocatively close to the number of cases not explained by the known dietary chemical carcinogens. Mechanistic studies will be required to test our hypothesis and investigate the role of different types of macronutrients in ovemutrition.
KW  - Toxikologie
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60712
ER  - 
TY  - JOUR
A1  - Cantoreggi, S.
A1  - Lutz, Werner K.
T1  - Investigation of the covalent binding of styrene-7,8-oxide to DNA in rat and mouse
N2  - Styrene-7,8-oxide (SO), the main intennediate metabolite of styrene, induces hyperkeratosis and tumors in the forestomach of rats and mice upon chronic administration by gavage. The aim of this study was to investigate wbether DNA binding could be responsible for the carcinogenic effect observed. [7-\(^3\)H]SO was administered by oral gavage in com oll to male CD rats at two dose levels (1.65 or 240 mg/kg). After 4 or 24 h, forestomach, glandular stomach and Uver were exclsed, DNA was isolated and its radioactivity detennined. At the 4 h time polnt, the DNA radioactivity was below the Iimit of detection in the torestornach and the liver. Expressed in the units of the covalent bindlng Index, CBI = (pmol adduct/mol DNA nucleotide)/(mmol cbemical administeredlkg body wt), the DNA-binding potency was below 2.6 and 2.0 respectively. In the glandular stomach at 4 b, and in most 24 b samples, DNA was slightly radiolabeled. Enzymatic degradation of the DNA and separation by HPLC ofthe normal nucleotides sbowed that the DNA rad.ioactivity represented biosynthetic incorporation of radlolabel into newly synthesized DNA. The Iimit of detection of DNA adducts in the glandular stomach was 1.0. In a second experlment, [7-\(^3\)H]SO was administered by i.p. injection to male 86C3Fl rnice. Liver DNA was analyzed after 2 h. No radloactivity was detectable at a Iimit of detection of CBI < 0.6. In agreement with the relatively long half-life of SO in animals, the cbemical reactivity of SO appears to be too low to result in a detectable production of DNA adducts in an in vivo situation. Upon comparison with the DNA-binding of other carcinogens, a purely genotoxic mechanism of tumorigenJc action of SO is unlikely. The observed tumorigenic potency in the forestomach could be the result of strong tumor promotion by high-dose cytotoxicity foUowed by regenerative hyperplasia.
KW  - Toxikologie
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60721
ER  - 
TY  - JOUR
A1  - Bommakanti, R.
A1  - Bokoch, G. M.
A1  - Tolley, J. O.
A1  - Schreiber, R. E.
A1  - Siemsen, D. W.
A1  - Klotz, Karl-Norbert
A1  - Jesaitis, A. J.
T1  - Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein: Inhibition by pertussis toxin-catalyzed ADP ribosylation
N2  - Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent Sedimentation coefficients of approximately 4 and 7 S. Tbe 7 S form can be converted to the 4 S form by guanosine 5' -0- (3-thiotriphosphate) (GTP-yS) with an EC&o of -20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. 0., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP-yS-treated neutrophil plasma membranes, was incubated with purified (>95%) G. protein from bovine brain (containing both G\(_{ia1}\) and G\(_{ia2}\)) or with neutrophil G protein (G\(_a\)), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC\(_{50}\) of 7 S complex formation induced by the two G proteins was 70 \(\pm\) 25 and 170 \(\pm\) 40 DM for G\(_a\) and G\(_1\), respectively. No complexation was measurable when bovine transducin (G\(_t\)) was used up to 30 times the EC\(_{50\) for G\(_a\). The EC\(_{50}\) for G\(_t\) was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 \(\mu\)M GTP-yS to the reconstituted 7 S complex caused a complete reversion of the receptor to the 4 S form, and anti-G\(_1\) peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gt prevented formation of the 7 S form even at 20 times the concentration of unribosylated G. normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a pbysical complex containing N-formyl chemotactic peptide receptor and G protein.
KW  - Toxikologie
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60406
ER  - 
TY  - JOUR
A1  - Cristalli, G.
A1  - Eleuteri, A.
A1  - Vittori, S.
A1  - Volpini, R.
A1  - Lohse, M. J.
A1  - Klotz, Karl-Norbert
T1  - 2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamides as selective agonists at A\(_2\) adenosine receptors
N2  - In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A\(_2\) receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cycla.se, and platelet aggregation studies. Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited by compounds 2a-d with K\(_i\) values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affmity binding at solubilized A\(_2\) receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [\(^3\)H]DPCPX and for the agonist [\(^3\)H]CCPA gave K\(_i\) values in the nanomolar range, and the compounds showed moderate A\(_2\) selectivity. In order to improve this selectivity, the correaponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. A\(_1\) expected, the 5'-N-ethyluronamide derivatives retained the A\(_2\) affinity whereas the A\(_1\) affinity was attenuated, resulting in an up to 10-fold increase in A\(_2\) selectivity. A similar patternwas observed in adenylate cyclase assays andin platelet aggregation studies. A 30- to 45-fold selectivity for platelet A\(_2\) receptors compared to A\(_1\) receptors was found for compounds 8a-c in adenylate cyclase studies.
KW  - Toxikologie
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60412
ER  - 
TY  - JOUR
A1  - Nolte, D.
A1  - Lorenzen, A.
A1  - Lehr, H.-A.
A1  - Zimmer, F.-J.
A1  - Klotz, Karl-Norbert
A1  - Messmer, K.
T1  - Reduction of postischemic leukocyte-endothelium interaction by adenosine via A\(_2\) receptor
N2  - The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. leukocytes were stained in vivo with acridine orange (0.5 mg kg\(^{-1}\) min\(^{-1}\) i.v. ). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. ·Adenosine, the adenosine A1-selective agonist 2-chloro-N\(^6\) -cyclopentyladenosine (CCPA), the Arselective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener {XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused viajugular vein starting 15 min priortorelease of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p< 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces postischemic leukocyte-endothelium interaction via A\(_2\) receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Ischemia/reperfusion
KW  - Leukocyte/endothelium interaction
KW  - Microcirculation
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60424
ER  - 
TY  - JOUR
A1  - Stopper, Helga
A1  - Metzler, M.
T1  - Carcinogenic oestrogens induce respiration deficiency mutation in yeast
N2  - 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.
KW  - Toxikologie
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63466
ER  - 
TY  - JOUR
A1  - Tas, P.
A1  - Stopper, Helga
A1  - Koschel, K.
A1  - Schiffmann, D.
T1  - Influence of the carcinogenic oestrogen diethylstilboestrol on the intracellular calcium level in C6 rat glioma cells
N2  - 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
KW  - Toxikologie
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63459
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Poetzsch, J.
A1  - Schlatter, J.
A1  - Schlatter, C.
T1  - The real role of risk assessment in cancer risk management
N2  - 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.
KW  - Toxikologie
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60730
ER  - 
TY  - JOUR
A1  - Baertsch, A.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat
N2  - 1 ,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-tenn bioassay using gavage in com oil (24 and 48 mg/kg/day), but not by inhalation (up to 150-250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Fernale F-344 rats (183-188 g) were exposed to [1,2-14C]DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l = 80 ppm for 4 h) or to a peak concentration (up to 18 mg/1 = 4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/k:g. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymaticaBy hydrolyzed to the 3' -nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density' indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The Ievel of DNA adducts was expressed in the dose-nonnalized units ofthe Covalent Binding Index, CBI = f.Lmol adduct per mol DNA nucleotide/ mmol DCE per kg body wt. In liver DNA, the different exposure regimens resulted in markedly different CBI values of 1.8 and 69, for "constant-low" and ''peak" DCE exposure Ievels. In the Jung, the respective values were 0.9 and 31. It is concluded that the DNA darnage by DCE depends upon the concentration-time profile and that the carcinogenic potency determined in the gavage study should not be used for low-Ievel inhalation exposure.
KW  - Toxikologie
KW  - 1
KW  - 2-Dichloroethane
KW  - Carcinogens
KW  - DNA
KW  - binding
KW  - Rat
KW  - Inhalation
KW  - Dose response
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60743
ER  - 
TY  - JOUR
A1  - Ohgaki, H.
A1  - Ludeke, B. I.
A1  - Meier, I.
A1  - Kleihues, P.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - DNA methylation in the digestive tract of F344 rats during chronic exposure to N-methyl-N-nitrosourea
N2  - The formation of \(O^6\)-methyldeoxyguanosine (\(O^6\)-MedGuo) was determined by an immuno-slot-blot assay in DNA of various tissues of F344 rats exposed to N-methyl-N-nitrosourea (MNU) in the drinking waterat 400 ppm for 2 weeks. Although the pyloric region of the glandular stomach is a target organ under these experimental conditions, the extent of DNA methylation was highest in the forestomach (185 \(\mu\)mol \(O^6\)-MedGuojmol guanine). Fundus (91 J.!moljmol guanine) and pylorus (105 J.!moljmol guanine) of the glandular stomach, oesophagus (124 \(\mu\)mol/mol guanine) and duodenum (109 )lmoljmol guanine) showed lower Ievels of \(O^6\) - MedGuo but differed little between each other. Thus, no correlation was observed between target organ specificity and the extent of DNA methylation. This is in contrast to the gastric carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which preferentially alkylates DNA of the pylorus, the main site of induction of gastric carcinomas by this chemical. In contrast to MNU, the nonenzymic decomposition of MNNG is accelerated by thiol compounds (reduced glutathione, L-cysteine), which are present at much higher concentrations in the glandular stomach than in the forestomach and oesophagus. During chronic exposure to MNNG (80 ppm), mucosal cells immunoreactive to 0 6-MedGuo are limited to the luminal surface [Kobori et al. (1988) Carcinogenesis 9:2271-2274]. Although MNU (400 ppm) produced similar Ievels of \(O^6\)-MedGuo in the pylorus, no cells containing methylpurines were detectable by immunohistochemistry, suggesting a more uniform methylation of mucosal cells by MNU than by MNNG. After a single oral dose of MNU (90 mg/kg) cells containing methylpurines were unequivocally identified using antibodies to \(O^6\)-MedGuo and the imidazole-ring-opened product of 7-methyldeoxyguanosine. In the gastric fundus, their distribution was similar to those methylated by exposure to MNNG, whereas the pyloric region contained immunoreactive cells also in the deeper mucosallayers. After a 2-week MNU treatment, the rate of cell proliferation, as determined by bromodeoxyuridine immunoreactivity, was only slightly enhanced in the oesophagus andin the fundus, but markedly in the forestomach and the pyloric region of the glandular stomach. lt is concluded that the overall extent of DNA methylation, the distribution of alkylated cells within the mucosa and the proliferative response all contribute to the organ-specific carcinogenicity of MNU.
KW  - Toxikologie
KW  - Gastric carcinogenesis
KW  - N-methyl-N-nitrosourea
KW  - DNA methylation
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60759
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
T1  - Dose-response relationship for chemical carcinogenesis by genotoxic agents
N2  - No abstract available
KW  - Toxikologie
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60766
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Vogt, H.
A1  - Tawfik-Schlieper, H.
T1  - Comparison of A\(_1\) adenosine receptors in brain from different species by radioligand binding and photoaffinity labelling
N2  - Radioligand binding to A\(_1\) adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1 ,3-[\(^3\)H]dipropylxanthine ([\(^3\)H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [\(^3\)H]DPCPX binding showed that the most potent compounds were DPCPX with K\(_i\) values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with K\(_i\) values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radio Iigand 2-chloro-N\(^6\) -[\(^3\)H]cyclopen tyladenosine ([\(^3\)H]CCP A) were less pronounced, rauging from a K\(_D\) value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [\(^3\)H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoadenosine (NECA) and S-N\(^6\)-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different K\(_D\) values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the "classical" A\(_1\) receptor profile (R-PIA > -NECA > S-PIA) is not sufficient to speculate that A\(_1\) receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.
KW  - Toxikologie
KW  - A1 adenosine receptors
KW  - Species differences
KW  - Radioligand binding
KW  - Photoaffinity labelling
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60388
ER  - 
TY  - JOUR
A1  - van Calker, D.
A1  - Steber, R.
A1  - Klotz, Karl-Norbert
A1  - Greil, W.
T1  - Carbamazepine distinguishes between adenosine receptors that mediate different second messenger responses
N2  - The mechanism of the therapeutic and prophylactic effects of carbamazepine (CBZ) in affective psychoses is unknown but may in part be related to the potent competitive interaction of CBZ with adenosine-binding sites in the brain. The antioonvulsant and sedative properties of CBZ are reminiscent of the effects evoked by adenosine-agonists and contrast sharply with the opposite aclions of adenosine-antagonists like caffeine. However. indirect evidence suggests an antagonist- rather than an agonist-like activity of CBZ at adenosi11e-receptors. We have used various model systems, in which adenosine receptor subtypes mediate different second messenger-responses, to investigate this apparent paradox. CBZ was found to antagonize the A\(_1\) receptor-mediated inhibition of cydic AMP accumulation in cultured astroblasts and in GH3-cells. Furthermore, CBZ also inhibits the adenosine-induced increase in the level of cyclic AMP in cultured astroblasts, which is mediated by low-affinity A\(_{2b}\)-receptors. ln contrast, CBZ does not block the inhibition elicited by adenosine-agonists of the agonist-induced increased formation of inositolphosphates in human neutrophils, which is mediated by high-affinity A\(_{2a}\)-receptors. The specific antagonism by CBZ of A\(_1\)- but not of high-affinity A\(_{2a}\)-receptors was further supported by binding experiments using rat brain membranes. These results suggest tbat the paradox of CBZ's antagonistic effects at adenosine-receptors might be at least partially reconciled by a selective antagonistic action of CBZ at A\(_1\)recertors but not at high-affinity A\(_{2a}\)-receptors.
KW  - Toxikologie
Y1  - 1991
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60392
ER  - 
TY  - JOUR
A1  - Epe, B.
A1  - Harttig, U.
A1  - Stopper, Helga
A1  - Metzler, M.
T1  - Covalent binding of reactive estrogen metabolites to microtubular protein as a possible mechanism of aneuploidy induction and neoplastic cell transformation
N2  - 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.
KW  - Toxikologie
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63478
ER  - 
TY  - JOUR
A1  - Schlatter, J.
A1  - Lutz, Werner K.
T1  - The carcinogenic potential of ethyl carbamate (urethane): risk assessment at human dietary exposure levels
N2  - Ethyl carbamate is found in fermented foods: bread contains 3-15 ng/g, stone-fruit brandies 200-20,000 ngfg, and about one-third of table-wine samples analysed contained more than 10 ng/g. In animals, ethyl carbamate is degraded to C02, H20 and NH3, with intermediate formation ofethanol. This degradation has been shown tobe inhibited (postponed) in the mouse by ethanol concentrations in the blood of about 0.15% and higher. A quantitatively minor pathway involves a two-step oxidation of the ethyl group to vinyl carbamate and epoxyethyl carbamate, the postulated electrophilic moiety that reacts with DNA. This reaction is probably the mode of the mutagenic action observed in many cellular and animal systems. The fact that only vinyl carbamate, but not ethyl carbamate, is mutagenic in a standard Ames test is probably because there is insufficient production of the intermediate oxidation product in the standard test. Consistent with this metabolism is the carcinogenic activity of ethyl carbamate in various animal species and in different organs; this activity can be seen even after a single high dose in early life. Quantitative analysis of the total tumour incidences after chronic exposure of rats and mice to 0.1-12.5 mg ethyl carbamate/kg body weightjday in the drinking-water showed a dose-related increase. The main target organs were the mammary gland (female rats and mice having similar susceptibilities) and the Jung (mice only). On the basis of sex- and organ-specific tumour data and with a linear extrapolation to a negligible increase of the lifetime tumour incidence by 0.0001% ( one additional tumour in one milüon individuals exposed for life), a "virtually safe dose .. of 20 to 80 ng/kg body weight/day was estimated. The daily burden reached under normal dietary habits without alcoholic beverages is in the range of about 20 ng/kg body weightfday. Regular table-wine consumption would increase the risk by a factor of up to five. Regular drinking of 20 to 40 ml stone-fruit brandy per day could raise the calculated lifetime tumour risk to near 0.01%.
KW  - Toxikologie
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60826
ER  - 
TY  - JOUR
A1  - Buss, P.
A1  - Caviezel, M.
A1  - Lutz, Werner K.
T1  - Linear dose-response relationship for DNA adducts in rat liver from chronic exposure to aflatoxin B1
N2  - Male F-344 rats were given eH]aßatoxin B1 (AFB1) in the drinking water at three exposure Ievels (0.02, 0.6, 20 J,Lgll, resulting in average dose Ievels of 2.2, 73, 2110 nglkg per day). After 4, 6 and 8 weeks, DNA was ~ted frorn the livers and analyzed for aßatoxin-DNA adducts. Tbe Ievel of DNA adducts did not increase significantly after 4 weeks, indicating that a steady-state for adduct formation and removal had nearly been reached. At 8 weeks, the adduct Ievels were 0.91, 32 and 850 nucleotide-aßatoxin adducts per to' nucleotides, i.e. clearly proportional to the dose. At the high dose Ievel, a near SO% tumor incidence would be expected in a 2-year bioassay with F -344 rats while the low dose used is within the range of estlmated human dietary exposures to aßatoxin in W estem countries. The proportionality seen between exposure and steady-state DNA adduct Ievel is discussed with respect to a linear extrapolation of the tumor risk to low dose.
KW  - Toxikologie
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60779
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
T1  - Dose-response relationship and low dose extrapolation in chemical carcinogenesis [commentary]
N2  - Data supporting various dose-respome relationships in chemical carcinogenesis are summarized. General principles are derived to explain the relationships between exposure dose, JI>NA adduct Ievel, induction of genetic changes, and tumor incidence. Some mechanistic aspects of epigenetic carcinogens (stimulation of ceU division and maldlfl'erentlation) are analyzed in a similar way. In a bomogeneous pnpulation, non-linearities are frequent. They are due to pbenomena of induction or saturation of enzymatic activities and to the multi-step nature of carcinog~: if a carcinogen acce1erates more than one step, the SUperposition of the dose- response curves for the indJvidual steps can result in an exponential relationship. A fourth power of the dose was the maximum seen in animals (fonnaldehyde). At the lowest dose Ievels, a proportionality between dose and tumor induction is postulated independent of the mechanism of action if the carcinogen aceeierotes the endogenous proass responsible for spootaneous tumor formation. Low-dose thresholds are expected only for situations where the carcinogen acts in a way that has no endogenous counterpart. Epidemiologfcal studies in humans show linear dose- response curves in all but two investigations. The difference from the strongly nonlinear slopes ·seen in animal studies could be due to the heterogeneity of the human population: if the individual sensitivity to a carcinogen is governed by a large number of genetic and Iife-style factors, the non-linea.rities will tend to cancel each other out and the dose- response curve becomes 'quasi-linear'.
KW  - Toxikologie
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60789
ER  - 
TY  - JOUR
A1  - Hegi, M. E.
A1  - Ulrich, D.
A1  - Sagelsdorff, P.
A1  - Richter, C.
A1  - Lutz, Werner K.
T1  - No measurable increase  in thymidine glycol or 8-hydroxydeoxyguanosine in liver DNA of rats treated with nafenopin or choline-devoid low-methionine diet
N2  - Male rats were treated for 2 months with 1000 ppm nafenopin in the diet or for 4 or 7 days with a choline-devoid low-methionine diet. DNA was isolated from the livers and analyzed for the presence of cis-thymidine glycol-3'-phosphate (cis-dTGp) by 32P-postlabeling and for the Ievel of 8-hydroxy-deoxyguanosine (8-0H-dG) by electrochemical detection (ECD). In no DNA sample was the Ievel of cis-dTGp above the Iimit of detection of 1 modified thymidine per 106 nucleotides. With 8-0H-dG, a background Ievel of this modification of 20 8-0H-dG per 106 nucleosides was found in liver DNA of control rats, which was not affected by either treatment. It is postulated for thymidine glycol that a potential increase was below the Iimit of detection or was rapidly repaired in vivo and that the steady-state Ievel of endogenous 8-hydroxydeoxyguanosine appears not tobe influenced by the treatments chosen.
KW  - Toxikologie
KW  - Oxygen radical
KW  - DNA
KW  - Genotoxicity
KW  - Rat liver peroxisome
KW  - Choline deficiency
KW  - Thymidine glycol
KW  - 8-Hydroxy-deoxyguanosine
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60790
ER  - 
TY  - JOUR
A1  - Meier, I.
A1  - Shephard, S. E.
A1  - Lutz, Werner K.
T1  - Nitrosation of aspartic acid, aspartame, and glycine ethylester. Alkylation of 4-(p-nitrobenzyl)pyridine (NBP) in vitro and binding to DNA in the rat
N2  - In a colorimetric assay using 4-( p-nitrobenzyl)pyridine (NBP) as a nucleophilic scavenger of alkylating agents, the nitrosation and alkylation reactions were investigated for a number of amino acids and derivatives. The alkylating activity increased with the square of the nitrite concentration. The nitrosation rate constants for aspartic acid, aspartame, and glycine ethylester ( = precursors C) were 0.08, 1.4 and ~ 0.2, respectively, expressed in terms of the pH-dependent \(k_2\) rate constant of the equation dNOCjdt = \(k_2\) • (C]· [nitrite]\(^2\) • The rates correlated inversely with the basicity of the amino group. The stability of the alkylating activity was astonishingly high, both in acid and at neutral pH. Half-lives of 500, 200, and 30 min were determined for aspartic acid (pH 3.5), aspartame (pH 2.5), and glycine ethylester (pH 2.5). Values of 60, 15, and 2 min; respectively, were found at pH 7. It is concluded that rearrangement of the primary N-nitroso product to the ultimate alkylating agent could be rate-limiting. The potential of nitrosated a-amino acids to bind to DN A in vivo was investigated by oral gavage of radiolabelled glycine ethylester to rats, followed irnmediately by sodium nitrite. DNA was isolated from stomach and liver and analysed for radioactivity and modified nucleotides. No indication of DNA adduct formation was obtained. Based on an estimation of the dose fraction converted from glycine ethylester to the nitroso product under the given experimental conditions, the maximum possible DNA-binding potency of nitroso glycine ethylester is about one order of magnitude below the methylating potency of N-nitrosomethylurea in rat stomach. The apparent discrepancy to the in vitro data could be due to efficient detoxification processes in mammalian cells.
KW  - Toxikologie
KW  - Nitrosation
KW  - Alkylation
KW  - Amino acids
KW  - DNA binding
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60804
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
T1  - Endogenous genotoxic agents and processes as a basis of spontaneous carcinogenesis
N2  - A list ofendogenaus DNA·damaging agents and processes is given. Endogenaus e/ectrophiles are found with the cosubstrates of physiological transfer reactions (S-adenosylrnethionine for methylation, A TP for phosphorylation, NAD\(^+\) for ADP-ribosylation, acetyl CoA for acetylation). Aldehyde groups (glyceraldehyde- 3-phosphate, formaldehyde, open forms of reducing sugars, degradation products of peroxidation) or alkylating degradation products derived from endogenaus nitrose compounds represent additional possibilities. Radical-forming reactions include leakage of the superoxide anion radical from terminal cytochromes and redox cycles, hydroxyl radical formation by the Fenton reaction from endogenaus hydrogen peroxide, and the formation of lipid peroxides. Genetic instability by spontaneaus deaminations and depurinations as well as replicative instability by tautomer errors andin the presence of mutagenic metal ions represent a third important dass of endogenaus genotoxic processes. The postulated endogenaus genotoxicity could form the mechanistic basis for what is called 'spontaneous' tumor incidence and explain the possibility of an increased tumor incidence after treatment of animals with non-genotoxic compounds exhibiting tumor-promoting activity only. Individual differences are expected to be seen also with endogenaus DNA damage. The presence of endogenaus DNA darnage implies that exogenaus DNAcarcinogen adducts give rise to an incremental darnage which is expected to be proportional to the carcinogen dose at lowest Ievels. An increased tumor risk due to exposure to exogenaus genotoxic carcinogens could therefore be assessed in terms of the background DNA damage~ for instance in multiples of the mean Ievel or of the interindividual variability in a population.
KW  - Toxikologie
KW  - Endogenous genotoxicity
KW  - Electrophiles
KW  - Radicals
KW  - Genetic instability
KW  - DNA damage
KW  - Spontaneous tumours
KW  - Carcinogen risk Individual susceptibili
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60816
ER  - 
TY  - JOUR
A1  - Gross, E.
A1  - Ruzicka, T.
A1  - Restorff, B. von
A1  - Stolz, W.
A1  - Klotz, Karl-Norbert
T1  - High-affinity binding and lack of growth-promoting activity of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) in a human epidermal cell line
N2  - No abstract available
KW  - Toxikologie
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60358
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Keil, R.
A1  - Zimmer, F. J.
A1  - Schwabe, U.
T1  - Guanine nucleotide effects on 8-cyclopentyl-1,3-[\(^3\)H]dipropylxanthine binding to membrane-bound and solubilized A\(_1\) adenosine receptors of rat brain
N2  - The effects of guanine nucleotides on binding of 8-cyclopentyl-1,3-[\(^3\)H]dipropylxanthine [\(^3\)H]DPCPX), a highly selective A\(_1\) adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A\(_1\) receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [\(^3\)H]DPCPX in a concentration-dependent manner. The rank order of potency for different guanine nucleotides for increasing [\(^3\)H]DPCPX bindingwas the same as for guanine nuc1eotide-induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., G\(_i\), in the regulation of antagonist binding is suggested. This was confirmed by inactivation ofGi by N-ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [\(^3\)H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP- or NEM-induced increase in antagonist binding was not caused by an affinity change of A\(-1\) receptors for [\(^3\)H]DPCPX but by an increased Bmu value. Guanine nucleotides had similar effects on membrane-bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when rnost receptors are in a high-affinity state for agonists, only a few receptors are labeled by [\(^3\)H]DPCPX. It is suggested that [\(^3\)H]DPCPX binding is inhibited when receptors are coupled to G\(_i\). Therefore, uncoupling of A\(_1\) receptors from G\(_i\) by guanine nucleotides or by inactivation of G\(_i\) with NEM results in an increased antagonist binding. 
Key Words: Adenosine receptors-8 -Cyclopentyl-1,3-eH]dipropylxanthine-Antagenist binding-Guanine nucleotide effects. Klotz K.-N. et al. Guanine nucleotide etfects on 8-cyclopentyl-1 ,3-eH]dipropylxanthine binding to membrane-bound and solubilized A1 adenosine receptors of rat brain. J. Neurochem. 54, 1988-1994 (1990).
KW  - Toxikologie
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60369
ER  - 
TY  - JOUR
A1  - Gimpl, G.
A1  - Gerstberger, R.
A1  - Mauss, U.
A1  - Klotz, Karl-Norbert
A1  - Lang, R. E.
T1  - Solubilization and characterization of active neuropeptide-Y receptors from rabbit kidney
N2  - Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[ (3-cholamidopropy l)dimethylammonio ]- 1-propanesulfonic acid (CHAPS). In membrane fragmentsandsoluble extracts neuropeptide Y bindingwas time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective Kn and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y bindingwas specifically inhibited by the nonhydrolyzable GTP analog guanosine 5' -0- (3-thiotripbosphate) in a concentration-dependent manner, with IC\(_{50}\) values of 28 and 0.14 \(\mu\)M for membrane- bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. CrossHoking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.
KW  - Toxikologie
Y1  - 1990
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60375
ER  -