TY  - JOUR
A1  - Kugler-Steigmeier, M. E.
A1  - Friederich, U.
A1  - Graf, U.
A1  - Lutz, Werner K.
A1  - Maier, P.
A1  - Schlatter, C.
T1  - Genotoxicity of aniline derivatives in various short-term tests
N2  - Various substituted aniline derivatives were tested for genotoxicity in several short-term tests in order to examine the hypothesis that a Substitution at both ortho positions (2,6-disubstitution) could prevent genotoxicity due to steric hindrance of an enzymatic activation to electrophilic intermediates. In the Salmonellajmicrosome assay, 2,6-dialkylsubstituted anilines and 2,4,6-trimethylaniline (2,4,6-TMA) were weakly mutagenic in strain TA100 when 20% S9 mixwas used, although effects were small compared to those of 2,4-dimethylaniline and 2,4,5-trimethylaniline (2,4,5-TMA). In Drosophila me/anogaster, however, 2,4,6-TMA and 2,4,6-trichloroaniline (TCA) were mutagenic in the wing spottestat 2-3 times lower doses than 2,4,5-TMA. In the 6-thioguanine resistance test in cultured fibroblasts, 2,4,6-TMA was again mutagenic at lower doses than 2,4,5-TMA. Two methylene-bis-aniline derivatives were also tested with the above methods: 4,4'-methylene-bis-(2-chloroaniline) (MOCA) was moderately genotoxic in al1 3 test systems whereas 4,4'-methylene-bis-(2-ethyl-6-methylaniline) (MMEA) showed no genotoxicity at all. DNA binding sturlies in rats, however, revealed that both MOCA and MMEA produced DNA adducts in the liver at Ievels typically found for moderately strong genotoxic carcinogens. These results indicate that the predictive value of the in vitro test systems and particularly the Salmonellajmicrosome assay is inadequate to detect genotoxicity in aromatic amines. Genotoxicity seems to be a general property of aniline derivatives and does not seem to be greatly influenced by substitution at both ortho positions.
KW  - Toxikologie
KW  - Aniline derivatives
KW  - Genotoxicity
KW  - Short-term tests
KW  - Covalent DNA binding
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60857
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  - 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  - THES
A1  - Duraphe, Prashant
T1  - Identification and characterization of AUM, a novel human tyrosine phosphatase
T1  - Identifizierung und Charakterisierung von AUM, einer neuen humanen Tyrosin-Phosphatase
N2  - Protein Phosphatasen werden aufgrund der Aminosäuresequenzen ihrer aktiven Zentren in drei große Familien unterteilt. In einer neu entdeckten Familie von Phosphatasen ist das aktive Zentrum durch die Sequenz DXDX(T/V) charakterisiert. Diese Aspartat-abhängigen Phosphatasen gehören zu der Superfamilie der Hydrolasen vom Haloazid Dehalogenase(HAD)-Typ, einer evolutionär konservierten und ubiquitär verbreiteten Enzymfamilie. Bislang konnten 58 menschliche HAD Enzyme durch Datenbankanalysen identifiziert werden. Ihre Funktionen sind jedoch nach wie vor nur rudimentär verstanden. Im Rahmen dieser Arbeit wurde zunächst das Komplement aller menschlichen HAD Phosphatasen durch Datenbank-Recherchen erfasst. Zusammen mit phylogenetischen Analysen gelang es, eine zum damaligen Zeitpunkt unbekannte, putative Phosphatase zu identifizieren, die eine vergleichsweise hohe Sequenz-Homologie zu der Zytoskelettregulierenden HAD Phosphatase Chronophin aufweist. Dieses neuartige Enzym wurde kloniert und mit biochemischen und zellbiologischen Methoden charakterisiert. Auf der Basis dieser Befunde bezeichnen wir dieses neuartige Protein als AUM (actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase).Mittels Northern blot, real-time PCR und Western blot Analysen konnte gezeigt werden, dass AUM in allen untersuchten menschlichen und murinen Geweben exprimiert wird. Die höchste Expression konnte in Hodengewebe nachgewiesen werden. Durch immunohistochemische Untersuchungen konnte gezeigt werden, dass AUM spezifisch in reifenden Keimzellen mit einem Expressionsmaximum zum Zeitpunkt der Spermiogenese exprimiert wird. Um die Substratpräferenz von AUM zu charakterisieren, wurde zunächst ein peptidbasierter in vitro Phosphatase-Substrat-Screen durchgeführt. Hierbei wurden 720 aus menschlichen Phosphoproteinen abgeleitete Phosphopeptide untersucht. Interessanterweise dephosphorylierte AUM ausschließlich Phosphotyrosin (pTyr)-enthaltende Peptide. Nur 17 pTyr-Peptide (~2% aller untersuchten Peptide) fungierten als AUM-Substrate. Diese Daten legen eine hohe Substratspezifität von AUM nahe. Zu den putativen AUM Substraten gehören Proteine, die in die Dynamik der Zytoskelett-Reorganisation sowie in Tyrosin Kinasevermittelte Signalwege eingebunden sind. In Übereinstimmung mit den Ergebnissen dieses Phosphopeptid-Screens konnte mittels Phosphatase overlay assays sowie in Zellextrakten aus Pervanadat-behandelten HeLa Zellen demonstriert werden, dass AUM eine begrenzte Anzahl Tyrosin-phosphorylierter Proteinen dephosphorylieren kann.In zellulären Untersuchungen wurde die mögliche Rolle von AUM im Rahmen der durch den epidermalen Wachstumsfaktor (EGF) ausgelösten Tyrosin-Phosphorylierung in einer Spermatogonien Zelllinie (GC-1 spg-Zellen) analysiert. So konnte nachgewiesen werden, dass die Überexpression von AUM zu einer moderaten Abnahme Tyrosin phosphorylierter Proteine nach EGF-Stimulation führte. Im Gegensatz dazu löste jedoch die durch RNAInterferenz vermittelte Depletion von endogenem AUM einen robusten Anstieg Tyrosinphosphorylierter Proteine aus, zu denen auch der EGF-Rezeptor selbst zählt. Zusätzlich zu dem EGF-Rezeptor wurde die Src-Kinase im Zuge des Phosphopeptid- Screens als mögliches AUM Substrat identifiziert. Daher wurden in vitro Kinase/Phosphatase-Assays mit gereinigtem Src und AUM durchgeführt. Mit diesem Ansatz konnte erstmals gezeigt werden, dass AUM in der Lage ist, die Src-Kinase zu aktivieren, während Src AUM phosphoryliert und die AUM Phosphatase-Aktivität blockiert. Diese Ergebnisse deuten auf eine gekoppelte, wechselseitige Regulation von AUM und Src hin. Obwohl die Details dieser Regulation derzeit noch unklar sind, zeigen unsere initialen Ergebnisse, dass AUM die Src-Aktivität unabhängig von seiner Phosphatase Aktivität steigert, während Src die AUM Phosphatase-Aktivität Kinase-abhängig vermindert. Auf zellulärer Ebene sind AUM-depletierte Zellen durch Veränderungen der Aktin- Zytoskelett-Dynamik und der Zelladhäsion charakterisiert. So weisen AUM-defiziente Zellen stabilisierte Aktin Streßfasern und vergrößerte fokale Adhäsionen auf. Weiterhin sind AUMdepletierte Zellen durch ein beschleunigtes spreading auf Fibronektin gekennzeichnet. Wir haben mit AUM ein bisher nicht beschriebenes Mitglied der Familie Aspartat-abhängiger Phosphatasen entdeckt. In dieser Arbeit ist es gelungen, AUM phylogenetisch, biochemisch und zellbiologisch zu charakterisieren. Unsere Ergebnisse legen nahe, dass AUM einen wichtigen, neuartigen Regulator der Src-vermittelten Zytoskelett-Dynamik im Rahmen der Zelladhäsion und Migration darstellt.
N2  - Protein phosphatases can be classified into at least three major families based on amino acid sequences at their active sites. A newly emerging phosphatase family contains the active site sequence DXDX(T/V), and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases, a ubiquitous and evolutionarily conserved enzyme family. Although the existence of 58 human HAD enzymes has been predicted by database analysis, our understanding of their biological functions remains rudimentary.By database mining amd phylogenetic analysis of human HAD phosphatases, we have found a marked increase in cell area of spreading cells, as well as accelerated cell spreading onfibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. a previously unidentified enzyme with homology to Chronophin, a cytoskeletal regulatory HAD phosphatase. We have cloned and characterized this novel enzyme and named it AUM,for actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase. By Northern blot, real-time PCR and Western blot analysis, we show that AUM is broadly expressed in all major human and mouse tissues with highest levels found in testis. Using immunohistochemistry, we can show that AUM is specifically expressed in maturing germ cells and that its expression peaks during spermiogenesis. To characterize the substrate preference of AUM, we have conducted an in vitro phosphatase substrate screen with 720 phosphopeptides derived from human phosphorylation sites. AUM exclusively dephosphorylates phosphotyrosine (pTyr)-containing peptides. Furthermore, only 17 pTyr peptides (~2% of all pTyr peptides investigated) acted as AUM substrates, indicating a high degree of substrate specificity. Putative AUM substrates include proteins involved in cytoskeletal dynamics and tyrosine kinase signaling.In accordance with the phosphopeptide screen, phosphatase overlay assays employing whole-cell extracts of pervanadate-treated HeLa cells show that AUM dephosphorylates only a limited number of tyrosyl-phosphorylated proteins.The role of AUM for cellular signaling was investigated in response to epidermal growth factor (EGF) stimulation in a spermatogonial cell line (GC-1 spg). The overexpression of AUM reduces, whereas the RNAi-mediated depletion of endogenous AUM increases EGF inducedtyrosine phosphorylation, including changes in the phosphorylation of the EGF receptor itself. Interestingly, in vitro kinase/phosphatase assays with purified Src and AUM indicate that AUM can activate Src, which in turn phosphorylates and inactivates AUM. Although it is at present unclear how Src and AUM regulate each other, our initial findings suggests that AUM enhances Src kinase activity independently of its phosphatase activity, whereas Src diminishes AUM phosphatase activity in a kinase dependent manner. On a cellular level, AUM-depleted cells are characterized by altered actin cytoskeletal dynamics and adhesion, as indicated by stabilized actin filaments, enlarged focal adhesions,a marked increase in cell area of spreading cells, as well as accelerated cell spreading on fibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration.
KW  - Tyrosin
KW  - Phosphatase
KW  - Signal transduction
KW  - Cell adhesion
KW  - Actin cytoskeleton
KW  - Src
KW  - Spermatogenesis
Y1  - 2010
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-44256
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  - 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  - THES
A1  - Vogl, Silvia
T1  - Investigation of individual differences in the metabolic elimination of drugs by the polymorphic enzymes CYP2C9, 2C19 and 2D6 based on metabolite profiling by LC-MS/MS
T1  - Untersuchung individueller Unterschiede der metabolischen Elimination von Arzneistoffen durch die polymorphen Enzyme CYP2C9, 2C19 und 2D6 basierend auf Metaboliten-Profiling mittels LC-MS/MS Analytik
N2  - Mit der vorliegenden Studie sollte zu dem wichtigen Forschungsfeld der Pharmakogenetik beigetragen werden, indem zum einen eine einfache und sichere kombinierte Phänotypisierung der drei zuvor erwähnten CYPs (CYP2D6, CYP2C9 und CYP2C19) entwickelt, und zum anderen die Vorhersagekraft des Genotyps für den gemessenen Phänotyp näher untersucht werden sollte. Es ist uns gelungen eine sichere, einfache, schnelle und kombinierte Phänotypisierung der beiden wichtigen Monooxygenasen CYP2D6 und CYP2C9 zu etablieren. Zunächst wurden dazu Wechselwirkungsstudien mit den ausgewählten Testsubstanzen Dextromethorphan (DEX, CYP2D6), Flurbiprofen (FLB, CYP2C9) und Omeprazole (OME, CYP2C19) durchgeführt. Es konnte gezeigt werden, dass DEX und FLB als Kombination verabreicht werden können. Die Gabe von OME gemeinsam mit FLB verändert jedoch das Ergebnis der CYP2C9 Phänotypisierung. Dies ist eine neue Erkenntnis, denn noch 2004 wurde ein Phänotypisierungscocktail veröffentlicht, der die Kombination von FLB und OME enthielt. Bei der genannten Studie wurden jedoch, unseres Wissens nach, keine Wechselwirkungsstudien zu den einzelnen Testsubstanz-Kombinationen durchgeführt. Die von uns entwickelte Phänotypisierungsmethode wurde durch Wechselwirkungsstudien verifiziert. Sie ist jedoch auch in anderen Bereichen den bisher veröffentlichten phänotypisierungscocktails überlegen. Zum einen wurden nur sehr kleine Dosen sicherer Testsubstanzen verwendet. Dies wurde durch Entwicklung neuer, sensitiver LC-MS/MS Methoden ermöglicht. Zum anderen ist diese neue Prozedur schnell und nicht-invasiv durchführbar. Nach Verabreichung der Testsubstanz muss der Urin nur für zwei Stunden gesammelt werden. Zudem weisen unsere Ergebnisse darauf hin, dass die normalerweise durchgeführte, aufwendige Glucuronidspaltung des CYP2D6 abhängigen DEX-Metaboliten, Dextrorphan, vermutlich vernachlässigt werden kann. Die wichtigsten Ergebnisse dieser Studie sind jedoch die Einblicke, die in die Vorhersagekraft der CYP2D6 und CYP2C9 Genotypen für die entsprechenden Phänotypen gewonnen werden konnten. Fast 300 phänotypisierte Kaukasier wurden auch in Hinsicht auf die wichtigsten varianten Allele von CYP2D6, CYP2C9 und CYP2C19 mithilfe bekannter und neu etablierter Methoden genotypisiert. Aufgrund der parallelen Phäno- und Genotypisierung konnten Geno- und Phänotyp direkt korreliert werden. Mit linearen Modellen war es möglich, allen detektierten varianten CYP2D6- und CYP2C9-Allelen Aktivitätskoeffizienten zuzuweisen. Diese können nun verwendet werden, um den Beitrag der einzelnen Allele zur resultierenden Enzymaktivität zu bestimmen, wodurch sich die Vorhersage dieser Aktivität ausgehend vom Genotyp verbessern lassen sollte. Besonders für CYP2D6 ermöglicht das neue Korrelationsmodel präzisere Vorhersagen des Phänotyps als bisher veröffentlichte Modelle. Zusammengefasst leistet diese Studie durch die Entwicklung eines sicheren und einfachen Phänotypisierungsprozesses für CYP2D6 und CYP2C9 und durch die Bestimmung von Aktivitätskoeffizienten für alle einbezogenen CYP2D6 und CYP2C9 Allele und der damit verbundenen präziseren Vorhersage des Phänotyps ausgehend vom Genotyp einen wesentlichen Beitrag zum Forschungsfeld der Pharmakogenetik.
N2  - This study should contribute to the important field of pharmacogenetics by: firstly, establishing an easy and safe phenotyping method that combines the activity determination of all three previously mentioned CYPs (CYP2D6, CYP2C9, and CYP2C19) into one phenotyping cocktail and secondly, improving the knowledge about the predictive power of the genotype for the measured phenotype. It was indeed possible to develop a save, easy-to-use, fast and simultaneous phenotyping procedure for the important genetic polymorphic enzymes CYP2D6 and CYP2C9. To accomplish that, interaction studies with the chosen probe drugs dextromethorphan (DEX, CYP2D6), flurbiprofen (FLB, CYP2C9) and omeprazole (OME, CYP2C19) were conducted. It could be proven that DEX and FLB can be administered in combination, whereas OME alters the phenotyping results of CYP2C9. This is a new finding as in 2004 a phenotyping cocktail was published that used FLB and OME in combination. However, to our knowledge, no interaction tests were carried in that study. The new phenotyping procedure is not only verified by prior probe drug interaction studies, it also has other advantages over phenotyping cocktails found in literature. Firstly, save probe drugs are used in very small doses. This is possible due to the new sensitive LC-MS/MS methods that were evaluated. Secondly, the new phenotyping procedure is very fast and on-invasive. Urine has to be collected only for 2 h and the results also suggest that the time consuming glucuronide cleavage of the CYP2D6 dependent metabolite dextrorphan, usually carried out before CYP2D6 phenotyping, may be unnecessary. Most importantly, however, new insights into the phenotype prediction from genotype for CYP2C9 and CYP2D6 could be gained within this study. Nearly 300 phenotyped Caucasian subjects were also genotyped for the most important known variant alleles for CYP2D6, CYP2C9 and CYP2C19 using several established and newly developed genoptyping methods. Therefore, a direct correlation between phenotype and genotype could be conducted for CYP2D6 and CYP2C9. Employing linear modeling, it was possible to assign activity coefficients to each of the detected CYP2D6 and CYP2C9 alleles, thereby estimating their contribution to the resulting enzyme activity. This might facilitate the prediction of the CYP2D6 and CYP2C9 metabolic status of a subject knowing only its respective genotypes. Especially the new CYP2D6 genotype phenotype correlation model might allow for more precise phenotype prediction for the included variant alleles than was possible until now. Taken together, this study substantially contributes to the important research field of pharmacogenetics by (i) developing a save and easy-to-use phenotyping combination for CYP2D6 and CYP2C9, and (ii) by establishing activity coefficients for each of the detected CYP2D6 and CYP2C9 alleles, thereby allowing for a more precise prediction of the phenotype from genotype.
KW  - Pharmakogenetik
KW  - Pharmakokinetik
KW  - Cytochrom P450
KW  - LC-MS/MS
KW  - Phänotyp
KW  - Genotyp
KW  - pharmacogenetics
KW  - pharmacokinetics
KW  - cytochrome p450
KW  - LC-MS/MS
KW  - phenotyping
KW  - genotyping
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67216
ER  - 
TY  - JOUR
A1  - Hegi, M.E.
A1  - Sagelsdorff, P.
A1  - Lutz, Werner K.
T1  - Detection by \(^{32}\)P-postlabeling of thymidine glycol in gamma-irradiated DNA
N2  - No abstract available
KW  - Toxikologie
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60863
ER  - 
TY  - JOUR
A1  - Sagelsdorff, P.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - DNA methylation in rat liver by daminozide, 1,1-dimethylhydrazine, and dimethylnitrosamine
N2  - DNA Methylation in Rat Li ver by Daminozide, 1, 1-Dimethylhydrazine, and Dimethylnitrosamine. SAGELSDORFF, P., LUTZ, W. K., AND ScHLAITER C. (1988). Fundam. Appl. Toxico/. 11, 723-730. [methyP4C]Daminozide (succinic acid 2',2'-dimethylhydrazide; 37 mgjkg), l,l( 14C]dimethylhydrazine (UDMH; 19 mgtkg), and (14C]dimethylnitrosamine (DMNA; 0.1 mg/ kg) were administered by oral gavage to male Sprague-Dawley rats. After 24 hr, the animals were killed and DNA was purified from the livers to constant specific radioactivity. After enzymatic degradation of the DNA to the 3'-deoxynucleotides the Ievel of DNA methylation was determined by HPLC analysis. Radiolabeled 7-methylguanine (7mG) was identified by cochromatography with unlabeled 7mG added as standard after acidic depurination of DNA and HPLC analysis ofpurines and apurinic acid. All three compounds were found to methylate DNA. The relative potencies were 1:47:4900 for daminozide:UDMH:DMNA. With [methyPH]UDMH, the formation of7mG was investigated as a function of dose administered, at 20, 2, and 0.2 mgj kg. The methylation ofDNA was strictly proportional to the dose. The data were used to compare the Ievel of DNA alkylation derived from residues of daminozide and UDMH in treated apple with the genotoxicity of the intake of N-nitroso compounds in Germany and Japan. It is estimated that these residues could Iead to a DNA methylation in the Ii ver of about 6% of an average exposure to DMNA
KW  - Toxikologie
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60875
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  - 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  - Jauch, A.
A1  - Lutz, Werner K.
T1  - In vivo assay for somatic point mutations induced by genotoxic carcinogens: incorporation of [\(^{35}\)S]methionine into a rat liver cytochrome b\(_5\) normally lacking sulphur-containing amino acids
N2  - The trypsin fragments of rat liver microsomal cytochron1e b\(_5\) (Tb\(_5\)) lack both methionine (met) and cysteine (cys), i.e., the sulphur-containing antino acids. Tb\(_5\) should therefore contain no 358-radioactivity after isolation from animals treated wHh [\(^{35}\)S]met or [\(^{36}\)S]cys. If, however, the nucleic acids coding for this polypeptide have been damaged by a genotoxic carcinogen, a miscoding could result in an incorporation of met or cys into the polypeptide so that Tb\(_8\) could now be \(^{36}\)S-radiolabelled. Two experiments are descrihed. the first one where a toxic regimen of N -nitrosomorpholine (NNM) to rats resulted in a significant increase of \(^{35}\)S-radioactivity in the Tbs of liver microsomes, and a second experiment with a non-toxic regimen of N,N diethylnitrosamine (DENA), where no increase was observable.
KW  - Toxikologie
KW  - MammaJian mutagenicity test
KW  - Pointmutation
KW  - Protein coding
KW  - Cytochrome b5
KW  - Amino acid composition
KW  - Rat Iiver microsomes
Y1  - 1983
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61047
ER  - 
TY  - JOUR
A1  - Meier-Bratschi, A.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Methylation of liver DNA of rat and mouse by N-nitrosodimethylamine formed in vivo from dimethylamine and nitrite
N2  - The extent of formation of N-nitrosodimethylaminc {NDMA) in the stomachs of rats and mice after sirnultancous oral administration of [\(^{14}\)C]dimethylamine and potassium nitrite was determined by measuring the methylation of liver DNA. With doses of around 1 mg dimethylamine hydrochloride/ kg body weight and 50 mg potassium nitrite/kg body weight. 0,8 % of the amine was nitrosated on average. The individual fluctuations ranged from 0.2 to 1.30% in the rat and from 0.2 to 1.9% in the mouse. Simultaneous administration of 50 mg sodium ascorbate (vitamin Cl/kg body weight inhibited the nitrosation by ahout 80% while 50 mg \(\alpha\)-tocopherol acetate [Vitamin E)/kg body weight reduced the nitrosation by about a half. Assuming similar kinctics and conditions of nitrosation in rats and man. a comparison of the formation of NDMA in vivo from dietary dimethylamine and nitrite with the estimated human uptake of preformed N DMA revealed that in vitro formation in the stomach of man is probably negligible.
KW  - Toxikologie
Y1  - 1983
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61052
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Jaggi, W.
A1  - Schlatter, C.
T1  - Covalent binding of diethylstilbestrol to DNA in rat and hamster liver and kidney [Short Communication]
N2  - No abstract available
KW  - Toxikologie
KW  - Carcinogenesis
KW  - Covalent binding index - Diethylstilbestrol
KW  - DNA binching
KW  - Estrogen
KW  - Hormone
Y1  - 1982
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61066
ER  - 
TY  - JOUR
A1  - Däniken, A. von
A1  - Friederich, U.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Tests for mutagenicity in Salmonella and covalent binding to DNA and protein in the rat of the riot control agent o-chlorobenzylidene malononitrile (CS)
N2  - The aim of this study was to determine whether o-chlorobenzylidene malononitrile ( CS) exhibits any genotoxic activity towards Salmonella or mammalian DNA in vivo. CS was synthesized with a [\(^{14}\)C]-label at the benzylic carbon atom. It was administered i. p. at a dose level of 13 mg/kg (1 mCi/kg) to young adult male rats. Liverand kidney DNA was isolated after 8, 25, and 75 h. The radioactivity was at (liver, 8 and 75 h) or below (all other samples) the limit of detection of 3 dpm. Therefore, a possible binding of CS to DNA is at least 10\(^5\) times lower than that of the strong hepatocarcinogen aflatoxin B1, and 4,000 times lower than that of vinyl chloride. In contrast to this lack of DNA binding, but in agreement with the chemical reactivity of CS, a binding to nuclear proteins could be detected with specific activities ranging between 50 and 121 dpm/mg for liver and between 3 and 41 dpm/mg for kidney. Protein binding could well be responsible for its pronounced cytotoxic effects. Cs was also tested in the Ames Salmonella/microsome assay. Strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were used with or without pre-incubation. Only with strain TA 100 and only without pre-incubation, a doubling of the number of revertants was detectable at the highest dose Ievels used, 1,000 and 2,000 !lg CS per plate. With pre-incubation of TA 100 with CS, a slight increase of the number of revertants was seen at 100 and 500 !lg per plate, and a subsequent fall below control values at 1,000 J.tg. A check for the number of surviving bacteria revealed a strong bacteriotoxicity of the higher doses of es so that the calculated mutation frequencies, i.e., the oumber of revertants per number of surviving bacteria, increased with doses up to 500 !J.g. This toxicity could be counteracted in part by the addition of increasing amounts of rat liver microsomes. In the view of these results, and taking into account the rare and low exposure of man, it is concluded that CS will not create a risk for the induction of point mutations or of carcinogenic processes mediated by DNA binding.
KW  - Toxikologie
KW  - o-Chlorobenzylidene malononitrile
KW  - Riot control agents
KW  - DNA Binding
KW  - Salmonella/microsome assay
KW  - Carcinogens
KW  - Mutagens
Y1  - 1981
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61073
ER  - 
TY  - JOUR
A1  - Däniken, A. von
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Lack of covalent binding to rat liver DNA of the hypolipidemic drugs clofibrate and fenofibrate
N2  - \(^{14}\)C-Labelled clofibric acid and fenofibric acid were administered p.o. to 200 g male and female rats. After 10 h, liver nuclear DNA and protein were isolated and the radioactivity was determined. Binding to protein was clearly measurable whereas no binding to DNA could be detected from any drug. A comparison of the Iimit of detection of such DNA binding with well-known chemical carcinogens revealed that the known hepatocarcinogenicity of clofibrate cannot be based upon an initiating, DNA damaging, mode of action but must be due to other, nongenotoxic, mechanisms such as peroxisome proliferation, hepatomegaly, or cytotoxicity due to protein binding. The risk assessment in man and the interpretation of the carcinogenicity data for rodents are discussed.
KW  - Toxikologie
Y1  - 1981
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61087
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Jaggi, W.
A1  - Lüthy, J.
A1  - Sagelsdorff, P.
A1  - Schlatter, C.
T1  - In vivo covalent binding of aflatoxin B\(_1\) and aflatoxin M\(_1\) to liver DNA of rat, mouse and pig
N2  - [\(^{14}\)C] Aflatoxin B\(_1\) (AFB\(_1\)) was isolated from cultures of Aspergillus parasiticus grown on [1-\(^{114}\)C] sodium acetate. Covalent binding of AFB1 to liver DNA of rat and mouse was determined 6-8 h afteroral administration. The effectiveness of covalent binding, expressedas DNA binding per dose in the units of a 'Covalent Binding Index' (CBI), (\(\mu\)mol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors. The corresponding values for pig liver DN A, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable. Aflatoxin M \(_1\) ( AFM\(_1\)) is a metabolite found in the milk of cows that have been fed AFB\(_1\)-contaminated diet. [\(^{14}\)C] AFM\(_1\) was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DN A revealed a CBI of 2100 shoWing that AFM\(_1\) must also be regarded as a strong hepatocarcinogen. It is concluded that AFB\(_1\) contaminations should be avoided in dairy feed.
KW  - Toxikologie
Y1  - 1980
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61097
ER  - 
TY  - JOUR
A1  - Jaggi, W.
A1  - Lutz, Werner K.
A1  - Lüthy, J.
A1  - Zweifel, U.
A1  - Schlatter, C.
T1  - In vivo covalent binding of aflatoxin metabolites isolated from animal tissue to rat-liver DNA
N2  - Ring-labelled [\(^{14}\)C)aflatoxin B\(_1\) (AFB\(_1\)), prepared by biosynthesis. or generally labelled [\(^3\)H]AFB\(_1\) was administered by oral gavage to young adult male rats. After 6 hr. the liver was removed and two fractions were isolated, namely macromolecules, which contamed about 3 % of the initial dose of AFB\(_1\) radioactivity. and water-soluble, low-molecular aftatoxin conjugates containing about0·2% of the administered radioactivity. These two fractions were administered orally to other rats in order to determine the potential of radioactive aftatoxin residues for covalent binding to DNA. Such binding can be used as an indicator for carcinogenic potency. Liver DNA was isolated 9-12 hr after admmistration of the aflatoxin derivatives and in no case was any radioactivity detected on the DNA. It can be deduced on the basis of the limit of detection of radioactivity on the DNA, that macromolecule bound AFB\(_1\) derivatives are at least 4000 times less active than AFB\(_1\) with respect to covalent binding to rat-liver DNA. and that the water-soluble conjugates are at least 100 times less potent than AFB, itself. It is concluded that the carcinogenic risk for humans who consume liver or meat. containing such aflatoxin residues is negligible when compared with the risk from intake of aftatoxins in other food items.
KW  - Toxikologie
Y1  - 1980
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61101
ER  - 
TY  - JOUR
A1  - Viviani, A.
A1  - Däniken, A. von
A1  - Schlatter, C.
A1  - Lutz, Werner K.
T1  - Effect of selected induction of microsomal and nuclear aryl hydrocarbon monooxygenase and epoxide hydrolase as well as cytoplasmic glutathione S-epoxide transferase on the covalent binding of the carcinogen benzo(a)pyrene to rat liver DNA in vivo
N2  - Groups of four adult male rats [ZUR:SIV -Z] were pretreated with corn oil (control; 2 ml/kg/day i. p. for 3 days), trans-stilbene-oxide (SO; 200 mg/kg/day i. p. for 2 days), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 \(\mu\)g/kg i. p. once, 4 days before killing), phenobarbital (PB; 1 gjliter in the drinking water for 8 days), and dieldrin (20 mg/kg/day i. p. for 3 or 9 days). They received an injection of [G-\(^3\)H]benzo(a)pyrene (BaP, 31 \(\mu\)g/kg, 7.4. 10\(^9\) dpm/kg; i. v.) 16 h before killing. In the liver of each rat, five enzymatic activities and the covalent binding of BaP to DNA have been determined. The rnicrosomal aryl hydrocarbon monooxygenase activity (AHM) ranged frorn 75% of control (SO) to 356% (TCDD), the nuclear AHM from 63% (SO) to 333% (TCDD). Microsomal epoxide hydrolase activity (EH) was induced up to 238% (PB), nuclear EH ranged from 86% (TCDD) to 218% (PB). A different extent of induction was observed in the two compartments. Highest induction of glutathione S-epoxide transferase activity (GST) was found with PB (202%). The DNA binding of BaP was modulated within 79% (dieldrin, 9 days) and 238% of control (TCDD). An enzyme digest of control DNA was analysed by Sephadex LH-20 chromatography. Multiple linear regression analysis with all data expressedas o/o of control yielded the following equation: DNA Binding = 1.49 · Microsomal AHM- 1.07 · Nuclear AHM+ 0.33 · Microsomal EH- 0.52 · N uclear EH+ 0.11 · Cytoplasmic GST + 58.2. From this analysis it is concluded that (1) AHM located in the endoplasmic reticulum is most important in the formation of DNA-binding metabolites, (2) EH in the same compar.tment is not determinative in thls respect nor has it a protective effect, (3) both membrane-bound enzyme activities located in the nucleus may inactivate potential ultimate carcinogens, and ( 4) cytoplasmic GST probably cannot reduce DNA binding due to its subcellular localization.
KW  - Toxikologie
KW  - Carcinogen
KW  - Benzo(a)pyrene-DNA binding
KW  - Enzyme induction
KW  - Aryl hydrocarbon rnonooxygenase
KW  - Epoxide hydrolase
KW  - Glutathione Stransferase
Y1  - 1980
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61114
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
T1  - In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis
N2  - The covalent binding of chemical carcinogens to DNA of mammalian organs is expressed per unit dose, and a 'Covalent-Binding Index', CBI, is defined. CBI for various carcinogens span over 6 orders of magnitude. A similar range is observed for the carcinogenic potency in long-term bioassays on carcinogenicity. For the assessment of a risk from exposure to a carcinogen, the total DN A darnage can be estimated if the actual dose is also accounted for. A detailed description is given for planning and performing a DNA-binding assay. A complete literature survey on DNA binding in vivo (83 compounds) is given with a calculation of CBI, where possible, 153 compounds are listed where a covalent binding to any biological macromolecule has been shown in vivo or in vitro. Recent, so far unpublished findings with aflatoxin Mh macromolecule- bound aflatoxin Bh ·diethylstilbestrol, and 1,2-epithiobutyronitrile are included. A comparison of CBI for rat-liver DNA with hepatocarcinogenic potency reveals a surprisingly good quantitative correlation. Refinements for a DN A-binding assay are proposed. Possibilities and Iimitations in the use of D NA binding in chemical carcinogenesis are discussed extensively.
KW  - Toxikologie
Y1  - 1979
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61122
ER  - 
TY  - JOUR
A1  - Jaggi, W.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Comparative studies on the covalent binding of the carcinogen benzo(a)pyrene to DNA in various model systems
N2  - The covalent binding of tritiated benzo(a)pyrene (BP) to DNA has been determined in rat liver in vivo, in rat liver perfused in situ, after incubation of BP with liver single cells, with liver homogenate, with liver microsomes and DNA, with fibroblasts from a rat granulorna pouch, and with · 2 cell lines. Li ver single cells were found to be a valuable compromise between the rnost sensitive system (microsomal incubation of BP with DNA) and the biologically most relevant system (in vivo ).
KW  - Toxikologie
Y1  - 1979
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61131
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Brändle, E.
A1  - Zbinden, G.
T1  - Effect of gum Arabic on aminopyrine demethylation in rats
N2  - Stimulation of aminopyrine demethylation induced in rats by oral or i.p. administration of phenobarbital was partially inhibited in animals receiving daily treatments of 2 x 200 mg/kg gum Arabic p.o.
KW  - Toxikologie
Y1  - 1978
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61146
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Böser, S.
A1  - Klotz, Karl-Norbert
A1  - Schwabe, U.
T1  - Affinities of barbiturates for the GABA-receptor complex and A\(_1\) adenosine receptors: A possible explanation of their excitatory effects
N2  - The effects of barbiturates on the GABA·receptor complex and the A\(_1\) adenosine receptor were studied. At the GABA-receptor complex the barbiturates inhibited the binding of [\(^{35}\)S]t-butylbicyclophosphorothionate [\(^{35}\)S]TBPT) and enhanced the binding of [\(^3\)H]diazepam. Kinetic and saturation experiments showed that both effects were allosteric. Whereas all barbiturates caused complete inhibition of [\(^{35}\)S]TBPT binding, they showed varying degrees of maximal enhancement of [\(^3\)H]diazepam binding; (±)methohexital was idenafied as the most efficacious compound for this enhancement. At the A\(_1\) adenosine receptor all barbiturates inhibited the binding of [\(^3\)H]N\(^6\)-phenylisopropyladenosine (\(^3\)H]PIA) in a competitive manner. The comparison of the effects on [\(^3\)H]diazepam and [\(^3\)H]PIA binding showed that excitatory barbiturates interact preferentially with the A\(_1\) adenosine receptor, and sedative/anaesthetic barbiturates with the GABA-receptor complex. It is speculated that the interaction with these two receptors might be the basis of the excitatory versus sedative/ anaesthetic properties of barbiturates.
KW  - Toxikologie
KW  - GABA-receptor complex
KW  - Adenosine receptors
KW  - Barbiturates
Y1  - 1987
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60250
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Elger, B.
A1  - Lindenborn-Fotinos, J.
A1  - Klotz, Karl-Norbert
A1  - Schwabe, U.
T1  - Separation of solubilized A\(_2\) adenosine receptors of human platelets from non-receptor [\(^3\)H]NECA binding sites by gel filtration
N2  - Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)dimethylammonio]- 1-propanesulfonate) and the solubilized extract subjected to gel ftltration. Binding of the adenosine receptor agonist [\(^3\)H]NECA (5'-N-ethylcarboxamidoadenosine) was measured to the eluted fractions. Two [\(^3\)H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [\(^3\)H]NECA binding activity eluted from the column. It bound [\(^3\)H]NECA in a reversible, saturable and GTPdependent manner with an affinity of 46 nmol/1 and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [\(^3\)H]NECA to the frrst peak with a pharmacological proftle characteristic for the A\(_2\) adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [\(^3\)H]NECA binding to the second, major peak. These results suggest that a solubilized A\(_2\) receptor-Gs protein complex of human platelets can be separated from other [\(^3\)H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A2 adenosine receptor of human plate1ets.
KW  - Toxikologie
KW  - A<sub>2</sub> Adenosine receptor
KW  - Human platelets
KW  - Radioligand binding
KW  - Adenylate cyclase
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60309
ER  - 
TY  - JOUR
A1  - Cristalli, G.
A1  - Franchetti, P.
A1  - Grifantini, M.
A1  - Vittori, S.
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
T1  - Adenosine receptor agonists: Synthesis and biological evaluation of 1-deaza analogues of adenosine
N2  - In a search for more selective A\(_1\) adenosine receptor agonists, N\(^6\)-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N\(^6\)-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N\(^6\)-cyclohexyl-l-deazaadenosine (1-deazaCHA, Sc), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-ß-D-ribofuranosyl-3Himidazo[ 4,5-b]pyridine (1). On the other band, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-ß-D-ribofuranuronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-ß-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine (4), in an attempt to find a more selective A\(_2\) agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase andin radioligand binding studies. 1-DeazaNECA (10) proved tobe a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N\(^6\)-substituted 1-deazaadenosines largely retain the A\(_1\) agonist activity of their parent compounds, but lose some of their A\(_2\) agonist activity. This results in A\(_1\)-selective compounds, of which N\(^6\)cyclopentyl- 2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A\(_1\) adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A\(_1\) receptor mediated adenosine actions.
KW  - Toxikologie
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60262
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Klotz, Karl-Norbert
A1  - Schwabe, U.
A1  - Cristalli, G.
A1  - Vittori, S.
A1  - Grifantini, M.
T1  - 2-Chloro-N\(^6\)-cyclopentyladenosine: a highly selective agonist at A\(_1\) adenosine receptors
N2  - 2-Chloro-N\(^6\)-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for At adenosine receptors. Binding of [\(^3\)H]PIA to A1 receptors of rat brain membranes was inhibited by CCP A with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N\(^6\)-cyclopentyladenosine (CPA). Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A\(_1\)-selectivity of CCPA. CCP A inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A\(_1\) receptor, with an IC\(_{50}\)-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC\(_{50}\)-value of 3500 nM. The more than 100-fold A\(_1\)-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A\(_1\) adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard At receptor agonist R-N\(^6\) -phenylisopropyladenosine (R-PIA). CCP A represents the agonist with the highest selectivity for A\(_1\) receptors reported so far.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Adenylate cyclase
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60279
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Klotz, Karl-Norbert
A1  - Diekmann, E.
A1  - Friedrich, K.
A1  - Schwabe, U.
T1  - 2',3'-Dideoxy-N\(^6\)-cyclohexyladenosine: an adenosine derivative with antagonist properties at adenosine receptors
N2  - Tbe 2',3'-dideoxy analogue of the potent A\(_1\) receptor agonist, N\(^6\)-cyclohexyladenosine (CHA), was synthesized as a potential antagonist for the A\(_1\) adenosine receptor. In sturlies on adenylate cyclase 2',3'-dideoxy-N\(^6\)-cyclohexyladenosine (ddCHA) did not show agonist properties at A\(_1\) or at A\(_2\) receptors. However, it antagonized the inhibition by R-PIA of adenylate cyclase activity of fat cell membranes via A\(_1\) receptors with a K\(_i\) value of 13 \(\mu\)M. ddCHA competed for the binding of the selective A1 receptor antagonist, [\(^3\) HJ8-cyclopentyl-1,3-dipropylxantbine ([\(^3\)H]DPCPX), to rat brain membranes with a K\(_i\) value of 4.8 \(\mu\)M; GTP did not affect the competition curve. In contrast to the marked stereoselectivity of the A\(_1\) receptor for the cx- and the natural ß-anomer of adenosine, the cx-anomer of ddCHA showed a comparable affinity for the A\(_1\) receptor (K\(_i\) value 13.9 \8\mu\)M). These data indicate that the 2'- and 3'-hydroxy groups of adenosine and its derivatives are required foragonist activity at and high affinity binding to A\(_1\) adenosine receptors and for the distinction between the cx- and ß-forms.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Adenylate cyclase
KW  - Adenosine receptor antagonists
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60282
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
A1  - Schwabe, U.
T1  - Chemical modification of A\(_1\) adenosine receptors in rat brain membranes - evidence for histidine in different domains of the ligand binding site
N2  - Chemical modification of amino acid residues was used to probe the ligand recognition site of A\(_1\) adenosine receptors from rat brain membranes. The effect of treatment with group·specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N\(^6\)-[\(^3\)H]phenylisopropyladenosine ([\(^3\)H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [\(^3\)H]8- cyclopentyl-1 ,3-dipropylxanthine ([\(^3\)H]DPCPX). Antagonists protected A\(_1\) receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [\(^3\)H]DPCPX, but reduced receptor number. From the selective protection of [\(^3\)H] PIA and [\(^3\)H]DPCPX binding from inactivation, it is concluded that agonists and antagonists oocupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of tbe inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [\(^3\)H]PIA and [\(^3\)H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the Iigand binding site. In addition, it was shown that no further amino acids witb polar side chains are present. The absence of bydrophilic amino acids frout the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A\(_1\) receptors.
KW  - Toxikologie
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60295
ER  - 
TY  - JOUR
A1  - Reddington, M.
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
A1  - Hietel, B.
T1  - Radiation inactivation analysis of the A\(_1\) adenosine receptor: decrease in radiation inactivation size in the presence of guanine nucleotide
N2  - Radiation inactivation analysis of the binding of the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes yielded a radiation inactivation size of 58 kDa. In the presence of GTPyS this was reduced to 33 kDa, in good agreement with the size of the ligand-binding subunit detected after photoaffinity labelling. The data indicate that the structural association of A\(_1\) adenosine receptors with G-protein components is altered in situ in the presence of guanine nucleotides.
KW  - Toxikologie
KW  - Adenosine receptor
KW  - A1
KW  - Radiation inactivation
KW  - Target size
KW  - G-protein
KW  - (Rat brain membrane)
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60318
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Maier, P.
T1  - Genotoxic and epigenetic chemical carcinogenesis: one process, different mechanisms
N2  - Chemieals that induce cancer in an intact organism are called carcinogens. This term does not differentiale between their various modes of action. In this review, Werner Lutz and Peter Maier make a mechanistic distinction between carcinogens that alter the genetic information and carcinogens that interfere with epigenetic processes. They considercardnogenesis tobe an ongoing, part1y unavoidable process which is based on a succession of mutations, most likely in stem cells, leading to autonomaus cellular growth regulation. Chemical carcinogens either induce such changes through mutations (genotoxic carcinogens) or they aceeierate the accumulation of critica1 spontaneaus mut11tions (epigenetic carcinogens). Examples are given for both classes of carcinogens, and for the processes that act at genoto:tic/nuclear 11nd epigenetic/mitotic Ievels.
KW  - Toxikologie
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60884
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Deuber, R.
A1  - Caviezel, M.
A1  - Sagelsdorff, P.
A1  - Friederich, U.
A1  - Schlatter, C.
T1  - Trenbolone growth promotant: covalent DNA binding in rat liver and in Salmonella typhimurium, and mutagenicity in the Ames test
N2  - DNA binding in vivo: (6,7-\(^3\)H]ß-trenbolone (ß-TBOH) was administered p.o. and i.p. to rats. After 8 or 16 h, DNA was isolated from the livers and purified to constant specific radioactivity. Enzymatic digestion to deoxyribonucleotides and separation by HPLC revealed about 90% ofthe DNA radioactivity eluting in the form of possible TBOH-nucleotide adducts. The extent of this genotoxicity, expressed in units of the Covalent Binding Index, CBI = (~mol TBOH bound per mol nucleotide)/(mmol TBOH administered per kg body weight) spanned from 8 t~ 17, i. e. was in the range found with weak genotoxic carcmogens. Ames test: low doses of ß-TBOH increased the number of revertants in Salmonella strain TAl 00 reproducibly and m a dose-dependent manner. The mutagenic potency was 0.2 revertants per nmol after preincubation of the bacteria (20 min at 37° C) with doses between 30 and 60 \(\mu\)g per plate (47 and 94 \(\mu\)g/ml preincubation mixture). Above this dose, the number of revertants decreased to control values, accompanied by a reduction in survival. The addition of rat liver S9 inhibited the mutagenicity. DNA binding in vitro: calf thymus DNA was incubated with tritiated ß-TBOH with and without rat liver S9 Highest DNA radioactivities were determined in the absence of the "activation" system. Addition of inactive S9 (without cofactors) reduced the DNA binding by a factor of up to 20. Intermediate results were found with active S9. DNA binding in Salmonella: ß-TBOH was irreversibly bound to DNA isolated from S. typhimurium TA100 after incubation of bacteria with [\(^3\)H]ß-TBOH. Conclusions: Covalent DNA binding appears to be the mechanism of an activation-independent ("direct") mutagenicity of TBOH which is not easily detected because of the bactericidal activity. The genotoxicity risk arising from exposure of humans to trenbolone residues in meat was estimated using the in vivo data and compared to that from the exposure to unavoidable genotoxins aflatoxin B1 and dimethylnitrosamine. It ts concluded that trenbolone residues represent only a low genotoxic risk.
KW  - Toxikologie
KW  - Trenbolone
KW  - Anabolieagent
KW  - DNA binding
KW  - Genotoxicity
KW  - Ames test
KW  - Salmonella typhimurium
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60897
ER  - 
TY  - JOUR
A1  - Büsser, M. T.
A1  - Lutz, Werner K.
T1  - Stimulation of DNA synthesis in rat and mouse liver by various tumor promoters
N2  - In order to investigate whether the Stimulation of liver DNA synthesis might be used to detect one class of hepatic tumor promoters, the incorporation of orally administered radiolabelled thymidine into liver DNA was detennined in rats and mice 24 h after a single oral gavage of test compounds at various dose Ievels. Three DNA-binding hepatocarcinogens, aflatoxin B1; benzidine and carbon tetrachloride, did not stimulate but rather inhibited DNA synthesis (not for CCla). Four hepatic tumor promoters, clofibrate, DDT, phenobarbital and thioacetamide, gave rise to a Stimulation in a dosedependent manner. Single oral doses between 0.02 and 0.3 mmol/kg were required to double the level of thymidine incorporation into liver DNA (= doubling dose, DD). Differentes between species or sex as obsprved in long-term carcinogenicity studies were reflected by a different stimulation of liver DNA synthesis. In agreement with the bioassay data, aldrin was positive only in male mice (DD = 0.007 mmol/kg) but not in male rats or female mice. 2,3, 7,8-TCDD was positive in male mice (DD = 10\(^{-6}\) mmol/kg) andin female rats (DD = 2 x 10\(^{-6}\) mmol/kg) but not in male rats. The assay was also able to distinguish between structural isomers with different carcinogenicities. [alpha]Hexachlorocyclohexane stimulated Iiver DNA synthesis with a doubling dose of about 0.2 mmol/kg in male rats whereas the [gamma]isomer was ineffective even at l mmol/kg. So far, only one result was inconsistent with carcinogenicity bioassay data. The different carcinogenicity of di(2-ethylhexyl)adipate (negative in rats) and di(2-ethylhe.xyl)phthalate (positive) was not detectable. 8oth plasticizers were positive in.this short-term system with DD's of 0. 7 mmol/kg for DEHA and 0.5 mmol/kg for DEHP. The proposed assay is discussed as an attempt to devise short-term assays for carcinogens not detected by the routine genotoxicity test systems.
KW  - Toxikologie
Y1  - 1987
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60908
ER  - 
TY  - JOUR
A1  - Bösch, R.
A1  - Friederich, U.
A1  - Lutz, Werner K.
A1  - Brocker, E.
A1  - Bachmann, M.
A1  - Schlatter, C.
T1  - Investigations on DNA binding in rat liver and in Salmonella and on mutagenicity in the Ames test by emodin, a natural anthraquinone
N2  - Emodin (1,6,8-trihydroxy-3-methylanthraquinone), an important aglycone found in natural anthraquinone glycosides frequently used in Iaxative drugs, was mutagenic in the Salmonellajmammalian microsome assay (Ames test) with a specificity for strain TA1537. The mutagenic activity was activationdependent with an optimal amount of S9 from Aroclor 1254-treated male Sprague-Dawley rats of 20% in the S9 mix (v jv) for 10 p.g emodin per plate. Heat inactivation of the S9 for 30 min at 60 ° C prevented mutagenicity. The addition of the cytochrome P-448 inhibitor 7,8-benzoflavone (18.5 nmoles per plate) reduced the mutagenic activity of 5.0 p.g emodin per plate to about one third, whereas the P-450 inhibitor metyrapone (up to 1850 nmoles per plate) was without effect. To test whether a metabolite" binds covalently to Salmonella DNA, [10-\(^{14}\)C]emodin was radiosynthesized, large batches of bacteria were incubated with [10-\(^{14}\)C]emodin and DNA was isolated. [G- \(^{3}\)H]Aflatoxin B1 (AFB1) was used as a positive control mutagen known to act via DNA binding. DNA obtained after aflatoxin treatment could be purified to constant specific activity. With emodin, the specific activity of DNA did not remain constant after repeated precipitations so that it is unlikely that the mutagenicity of emodin is due to covalent interaction of a metabolite with DNA. The antioxidants vitamin C and E or glutathione did not reduce the mutagenicity. Emodin was also negative with strain TA102. Thus, oxygen radicals are probably not involved. When emodin was incubated with S9 alone for up to 50 h before heat-inactivation of the enzymes and addition of bacteria, the mutagenic activity did not decrease. It is concluded that the mutagenicity of emodin is due to a chemically stable, oxidized metabolite forming physico-chemical associations with DNA, possibly of the intercalative type. In order to check whether an intact mammalian organism might be able to activate emodin to a DNA-binding metabolite, radiolabelled emodin was administered by oral gavage to male SD rats and liver DNA was isolated after 72 h. Very little radioactivity was associated with the DNA. Considering that DNA radioactivity could also be due to sources other than covalent interactions, an upper limit for the · covalent binding index, CBI = (p.moles chemical bound per moles DNA nucleotides)/(mmoles chemical administered per kg body weight) of 0.5 is deduced. This is 104 times below the CBI of AFB1. The demonstration of a lack of covalent interaction with DNA bothin Salmonellaandin rat liver is discussed in terms of a reduced hazard posed by emodin as a mutagenic drug in use in humans.
KW  - Toxikologie
KW  - DNA binding
KW  - (Rat liver)
KW  - (Salmonella)
KW  - Ames test
KW  - Emodin
KW  - Anthraquinone glycosides
KW  - natural
Y1  - 1987
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60913
ER  - 
TY  - JOUR
A1  - Shephard, S. E.
A1  - Schlatter, C.
A1  - Lutz, Werner K.
T1  - Assessment of the risk of formation of carcinogenic N-nitroso compounds from dietary precursors in the stomach
N2  - A literature review has shown that the daily intakes of various N -nitroso-precursor classes in a typical European diet span five orders of magnitude. Amides in the form of protein, and guanidines in the form of creatine and creatinine, are the nitrosatable groups found most abundantly in the diet, approaching Ievels of 100 g/day and 1 gjday, respectively. Approximately 100 mg of primary amines and amino acids are consumed daily, whereas aryl amines, secondary amines and ureas appear to lie in the 1-10 mg range. The ease of nitrosation of each precursor was estimated, the reactivities being found to span seven orders of magnitude, with ureas at the top and amines at the bottom of the scale. From this infonnation and an assessment of the carcinogenicity of the resulting N-nitroso derivatives, the potential health risk due to gastric in vivo nitrosation was calculated. The combined effects of these risk variables were analysed using a simple mathematical model: Risk = [daily intake of precursor] x [gastric concentration of nitrite]\(^n\) x [nitrosatability rate constant} x [carcinogenicity of derivative]. The risk estimates for the various dietary components spanned nine orders of magnitude. Dietary ureas and aromatic amines combined with a high nitrite burden could pose as great a risk as the intake of preformed dimethylnitrosamine in the diet. In contrast, the risk posed by the in vivo nitrosation of primary and secondary amines is probably negligib1y small. The risk contribution by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes. Thus three priorities for future work are a comprehensive study of the sources and Ievels of arylamines and ureas in the diet, determination of the carcinogenic potencies of key nitrosated products to replace the necessarily vague categories used so far, and the development of short-term in situ tests for studying the alkylating power or genotoxicity of N-nitroso compounds too unstable for inclusion in long-term studies.
KW  - Toxikologie
Y1  - 1987
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60925
ER  - 
TY  - JOUR
A1  - Grilli, S.
A1  - Lutz, Werner K.
A1  - Parodi, S.
T1  - Possible implications from results of animal studies in human risk estimations for benzene: nonlinear dose-response relationship due to saturation of metabolism
N2  - To date, all risk assessment studies on benzene have been based almost exclusively on epiderniological data. Wehave attempted a more integrated and quantitative evaluation of carcinogenic risk for hurnans, trying to utilize, in addition to the epidemiological data, all data available, specifically data on metabolism, genotoxicity, and carcinogenicity in small rodents. An integrated evaluation of the globality of the available data seems to suggest a progressive saturation of metabolic capacity both for man and rodents between 10 and 100 ppm. The most susceptible target cells seem tobe different in humans (predominant induction of myelogenous leukemia) and small rodents (induction of a wide variety of tumors). Nevertheless, both epidemiological and experimental carcinogenicity data tend to indicate a flattening ofthe response for the highest dosages, again suggesting a general Saturation of mechanisms of metabolic activation, extended to different target tissues. From a quantitative point of view, the data suggest a carcinogenic potency at 10 ppm two to three times higher than that computable by a linear extrapolation from data in the 100 ppm range. These observations are in accord with the recent proposal of the European Economic Community of reducing benzene time-weighted average occupationallevels from 10 to 5 ppm.
KW  - Toxikologie
KW  - Benzene
KW  - Risk estimation
KW  - Carcinogenicity
KW  - Genotoxicity
KW  - Metabolism saturation
KW  - Dose-response relationship
Y1  - 1987
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60936
ER  - 
TY  - JOUR
A1  - Jauch, A.
A1  - Lutz, Werner K.
T1  - Metallothionein protein variants generated in rat liver as a result of DNA and RNA ethylations by the carcinogen diethylnitrosamine
N2  - Metallothionein (MT) is a protein which contains 20 cysteine residues but no aromatic amino acids. It was tested whether treatment of male rats with the hepatocarcinogen diethylnitrosamine (DENA) could ethylate nucleic acids in such a way that protein variants containing measurable amounts of aromatic amino acid residues could be isolated from the livers of treated animals. To give a low Iimit of detection, the "wrong" amino acid precursors were administered in radiolabelled form at high Ievels of activity (7 mCi/kg each of [\(^3\)H]tyrosine and [\(^3\)H]phenylalanine). 11 \(\mu\)Ci/kg [\(^{14}\)C]cysteine was given as an intemal marker for MT biosynthesis. 6 h after amino acid administration, metallothionein (MT) was isolated from the liver and extensively purified. Afteracid hydrolysis and collection of Cys, Tyr, and Phe from an HPLC analysis of the amino acids, the \(^3\)H/\(^{14}\)C ratio was determined. The carcinogen-treated rats exhibited a significantly higher ratio than the vehicle-treated animals. This type of in vivo assay might find interesting applications in the investigation of nucleic acid alkylations as promutagenic lesions.
KW  - Toxikologie
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60946
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
T1  - Investigation of the potential for binding of di(2-ethylhexyl)phthalate (DEHP) to rat liver DNA in vivo
N2  - It was the aim of this investigation to determine whether or not covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of rodents with high doses of DEHP. DEHP radiolabeled in different positionswas administered orally to female F344 rats with or without pretreatment for 4 weeks with 1% unlabeled DEHP in the diet. Livu DNA was isolated after 16 hr and analyzed for radioattivity. Administration of [\(^{14}\)C]carboxylate unabeled DEHP resulted in no measurable DNA radioactivity. With DEHP [\(^{14}\)C]· and [\(^{3}\)H]. labeled in the alcohol moiety as well as with 2-ethyl[1-\(^{14}\)C]hexanol, radioactivity was clearly measurable in the DNA. HPLC analysis of enzyme-degraded DNA relvealed that the normal nucleosides had incorporated radiolabel whereas no radioactivity was detectable in those fractions where the carcinogen-modified nucleoside adducts are expected. A quantitative evaluation of the negative data in terms of a Iimit of detection for a covalent binding Index (CBJ) indicates that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP in rodents.
KW  - Toxikologie
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60957
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
T1  - Quantitative evaluation of DNA binding data for risk estimation and for classification of direct and indirect carcinogens
N2  - Investigation of covalent DNA binding in vivo provided evidence for whether a test substance can be activated to metabolites able to reach and react with DNA in an intact organism. Fora comparison of DNA binding potencies of various compounds tested under different conditions, a normalization of the DNA lesion with respect to the dose is useful. A covalent binding index, CBI = (\(\mu\)mol chemical bound per mol DNA nucleotide )/(mmol chemical administered per kg body weight) can be determined for each compound. Whether covalent DNA binding results in tumor formation is dependent upon additional factors specific to the cell type. Thus far, all compounds which bind covalently to liver DNA in vivo have also proven tobe carcinogenic in a long-term study, although the liver was not necessarily the target organ for tumor growth. With appropriate techniques, DNA binding can be determined in a dose range which may be many orders of magnitude below the dose Ievels required for significant tumor induction in a long-term bioassay. Rat liver DNA bindingwas proportional to the dose of aflatoxin B1 afteroral administration of a dose between 100 \(\mu\)g/kg and 1 ng/kg. The lowest dose was in the range of generat human daily exposures. Demonstration of a lack of liver DNA binding (CBI<0.1) in vivo for a carcinogenic, nonmutagenic compound is a strong indication for an indirect mechanism of carcinogenic action. Carcinogens of this class do not directly produce a change in gene structure or function but disturb a critical biochemical control mechanism, such as protection from oxygen radicals, control of cell division, etc. Ultimately, genetic changes are produced indirectly or accumulate from endogenaus genotoxic agents. The question of why compounds which act via indirect mechanisms are more likely to exhibitanonlinear rangein the dose-response curve as opposed to the directly genotoxic agents or processes is discussed.
KW  - Toxikologie
KW  - Chemical carcinogenesis
KW  - Mechanism of action
KW  - Quantitative risk assessment
KW  - Genotoxicity
KW  - Dose-response relationship
KW  - Aflatoxin B1
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60967
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
T1  - Endogenous formaldehyde does not produce detectable DNA-protein crosslinks in rat liver
N2  - Formaldehydeis an electrophilic molecule able to crosslink DNA and protein. It has been found to induce tumors in the nasal epithelium in rodents. The safety margin between the maximum tolerated FA concentration in the work place and the concentration found to be tumorigenic in animal studies is very small. Because FA is produced endogenously as a result of a variety of oxidative demethylations, the assessment of the tumor risk from exogenaus FA exposure has tobe related quantitatively to the level of DNA-protein crosslinks induced by endogenaus FA generation. It is reported here that the high level of endogenaus FA formed in the liver after a large dose of methanol or of aminopyrine did not lead to any observable increase in DNA-protein crosslinks. Using positive and negative control data from in vitro incubations of liver homogenate with FA or methanol it is estimated that the endogenous level of DNA damage in the liver must be more than three orders of magnitude below the damage observed at tumorigenic concentrations for the rat nose. The fact that FA is formed endogenously cannot, therefore, be used to claim that exogenous FA merely leads to a negligible increase in DNA damage.
KW  - Toxikologie
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60972
ER  - 
TY  - JOUR
A1  - Koch, R.
A1  - Deger, A.
A1  - Klotz, Karl-Norbert
A1  - Schenzle, D.
A1  - Krämer, H.
A1  - Kelm, S.
A1  - Müller, G.
A1  - Rapp, R.
A1  - Weber, U.
T1  - Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with  different binding properties
N2  - Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak li) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak Il). Both peaks were glycoproteins. At 4°C peak 1 showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak li bad its binding optimum at pH 7.0 and low ionic strength, where peak I bindingwas minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak 11 an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 oc the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfatejpolyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400000, 365000, 320000, 290000, and 245000 under non-reducing conditions. For peak II two major receptor bands with M\(_r\) 210000 and 115000 were found. The peak II receptor bands were also obtained aftermild reduction of peak I. After complete reduction both peaks showed one major receptor band with M\(_r\) 130000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.
KW  - Toxikologie
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60215
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
A1  - Schwabe, U.
T1  - Characterization of the solubilized A\(_1\) adenosine receptor from rat brain membranes
N2  - A\(_1\) adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-( cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A\(_1\) adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-\(N^6\)-[\(^3\)H]phenylisopropyladenosine([\(^3\)H]PJA) with K\(_D\) values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A\(_1\) adenosine receptors could be labelled not only with the agonist [\(^3\)H]PIA but also with the antagonist I ,3-diethyi-8-[\(^3\)H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein N; and that all regulatory functions are retained on solubilization. Key Words: A1 adenosine receptors - Solubilization- Rat brain membranes. Klotz K.-N. et al. Characterization of the solubilized A1 adenosine receptor from rat brain membranes. J. Neurochem. 46, 1528-1534 (1986).
KW  - Toxikologie
KW  - A1 adenosine receptors
KW  - solubilization
KW  - rat brain membranes
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60222
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
T1  - The glycoprotein nature of A\(_1\) adenosine receptors
N2  - A\(_1\) adenosine receptors from different tissues and species we~e photoaffinity labelled and then the carbohydrate content was examined by both enzymatic and chemical treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labelled membrane receptors shows that neuraminidase treatment alters the electrophoretic mobility of the receptor band indica ting the presence of terminal neurandnie acids. Neuraminidase digestion does not influence the binding characteristics of the receptor. The totally deglycosylated receptor protein obtained by chemical treatment has an apparent molecular weight Of 32,000.
KW  - Toxikologie
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60231
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Klotz, Karl-Norbert
A1  - Lindenborn Fotinos, J.
A1  - Reddington, M.
A1  - Schwabe, U.
A1  - Olsson, R. A.
T1  - 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) - a selective high affinity antagonist radioligand for A\(_1\) adenosine receptors
N2  - The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A\(_1\) adenosirre receptors were examined and conipared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A\(_1\) adenosirre receptors and the stimulationvia A\(_2\) adenosirre receptors. The K\(_i\)-values of this antagonism were 0.45 nM at the A\(_1\) receptor of rat fat cells, and 330 nM at the A\(_2\) receptor of human platelets, giving a more than 700-fold A\(_1\)-selectivity. A similar A\(_1\)-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMPphosphodiesterase activity of human platelets. [\(^3\)H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A\(_1\) receptors in membranes of bovine brain and heart, and rat brain and fat cells (K\(_D\) -values 50-190 pM). Its nonspecific binding was about 1% of total at K\(_D\) , except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [\(^3\)H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A\(_1\) receptor.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Adenylate cyclase
KW  - Phosphodiesterase
KW  - Xanthines
KW  - Radioligands
Y1  - 1987
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60246
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Winkler, F. K.
A1  - Dunitz, J. D.
T1  - Crystal structure of the antibiotic monensin similarities and differences betweeen free acid and metal complex
N2  - The structure of monensin, C36H620 11 , has been deterrnined by X-ray analysis of its crystalline monohydrate (orthorhombic, a = 15.15, b = 23.61, c = 10.65 A, Z = 4, space group P212121). Phases were assigned by direct methods, malring use of the 'tangent formula'. Although the conformation of the free acid resembles that of the silver salt in being cyclic, there are differences in the hydrogen bonding pattern. These featurcs are discussed in relation to the cornplexation of metal ions by m.onensin.
KW  - Toxikologie
Y1  - 1971
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61228
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Wipf, H. K.
A1  - Simon, W.
T1  - Alkalikationen-Spezifität und Träger-Eigenschaftender Antibiotica Nigerfein und Monensin
T1  - Alkali-cation specificity and carrier qualities of the antibiotics nigericin and monensin
N2  - It is shown by means of IR. spectroscopic methodsthat nigericin and monensin bave a cyclic conformation similar to that of their silver salts. Camplex fonnation constants with sodium and potassium ions follow the selectivity order determined by EMF. measurements on liquid membranes: nigericin: K\(^+\) >Rb\(^+\)> Na\(^+\)> Cs\(^+\) >Li\(^+\); monensin: Na\(^+\)> K\(^+\) >Li\(^+\)> Rb\(^+\)> Cs\(^+\). Transport experiments show that nigericin and monensin facilitate the diffusion of potassium ions across model membranes, although in electrolytic transport experiments the permeability is not affected.
KW  - Toxikologie
Y1  - 1970
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61233
ER  - 
TY  - JOUR
A1  - Viviani, A.
A1  - Lutz, Werner K.
T1  - Modulation of the binding of the carcinogen benzo(a)pyrene to rat liver DNA in vivo by selective induction of microsomal and nuclear aryl hydrocarbon hydroxylase activity
N2  - The lnfluence of mlcrosomal and nuclear aryl hydrocarbon hydroxylase (AHH) actlvlty on the covalent blndlng of [G·3H]benzo(a )pyrene to rat llver DNA was evaluated in viWJ. lnductlon of mlcrosomal AHH was obtalned alter phenobarbltal treatment (160% of control), whlch also lncreased DNA blndlng to 190%, but left the nuclear actlvlty unchanged. Nuclear AHH was lnduced wlth dleldrln (150%), and the blndlng was decreased to 75%, whereaa the mlcrosomal AHH was at control Ievei. The lncreaslng effect of mlcrosomal AHH lnductlon as weil as the decreaslng effect of nuclear AHH lnductlon on the blndlng was shown clearly when the data of the Individual rata were uaed to solve the equatlon Binding = e•(mlcroeomal AHH) + b•(nuclear AHH) + c Multiple linear regresslon analysls wlth the data from 10 anlmala reaulted ln positive valuea for a and c, a negative value for b, and a good multiple correlatlon coefflclent of r = 0.974. Pretreatment wlth 3-methylcholanthrene ln· duced mlcrosomal AHH to 380% of control and nuclear AHH to 590% and lncreased the blndlng' to 175,.-o. The blndlng was hlgher than predlcted by the formula found, probably because the lncreaslng lnfluence of lnduced mlcrosomal AHH overahadowed the decreaslng effect of the nuclear AHH. The study ahows clearly that the blndlng of a forelgn compound to DNA in viWJ Ia dependent not only on mlcrosomal enzyme actlvltles but also on nuclear actlvltles even lf the latter are conslderably lower than thoae of mlcrosomes.
KW  - Toxikologie
Y1  - 1978
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61150
ER  - 
TY  - JOUR
A1  - Jaggi, W.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Covalent binding of ethinylestradiol and estrone to rat liver DNA in vivo
N2  - Thecovalent bindingof [6,7-\(^3\)H]ethinylestradiol (EE)and [6,7-\(^3\)H]estrone (E) to liver DNA of 200 g female ratswas measured 8 h after the administration of 80 \(\mu\)g (9.2 mCi) estrogen by gavage. The binding is 1.5 for EE and 1.1 for E, expressedas binding to DNA/dose, in units of \(\mu\)mol hormonefmol DNA phosphate/mmole honnone/kg body wt. It is in the same order of magnitude as for benzene and about 10 000 tim es below the binding of typical liver carcinogens, such as aflatoxin B\(_1\) or N,N-dimethylnitrosamine.
KW  - Toxikologie
Y1  - 1978
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61162
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Viviant, A.
A1  - Schlatter, C.
T1  - Nonlinear dose-response relationship for the binding of the carcinogen benzo(a)pyrene to rat liver DNA in vivo
N2  - Wlth radioactive compound of high specific activity, the binding of carcinogene to DNA can be measured wlth doses that are ineffective ln long-term studies. The binding of tritiated benzo(a )pyrene to liver DNA of adult male rats has been determined 50 hr after a singie l.p. injection of doses between 40 1'9/kg and 4 mg/kg. The doseresponse relationship is linear up to 1 mg/kg, shows a step towards 2 mg/kg, and gives a shallow linear slope above that value. The observed binding ranges from 1.7 to 180 nmoles benzo(a)pyrene per mole DNA phosphate. The nonlinearity could be due to an induction of metabolizing enzymes. The microsomal aryl hydrocarbon hydroxylase activity increases significantly 24 hr after a single dose of 4 mg/kg and 48 hr after doses of 2 and 4 mg/kg, but no induction Ia found with 1 mg/kg. The binding from an equimolar dose is 35 times lower than the one found on mouse skin DNA and 300 times lower than that of N,Ndlmethylnitrosamine in rat liver. A good correlatlon exiats to the respective tumor formation in long-term studles.
KW  - Toxikologie
Y1  - 1978
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61179
ER  - 
TY  - JOUR
A1  - Viviani, A.
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclei and microsomes by phenobarbital, 3-methylcholanthrene, 2,3,7,8-tetrachloro-dibenzo-p-dioxin, dieldrin and other inducers
N2  - Aryl hydrocarbon hydroxylase (AHH) has been measured in male rat Jiver nucJei and microsomes after treatment of adult animals with various inducers for up to 14 days. After daily i.p. injections of 3-methylcholanthrene (MC, 20 mg/kg) the nuclear activity increased to a maximum of 600 per cent of the control activity after 4 days whereas the microsomal activity was 400 per cent of control at the same date. After 12 days, both activities equilibrated at 400 per cent. A similar time course was found after a single i.p. injection of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD, 0.01 mg/kg) with an induction to .500 and 300 per cent for nuclei and microsomes, respectiveJy. after 2 days, and to 400 per cent for both after 12 days. PhenobarbitaJ (PB) was given continuously in the drinking water (I g/1) and induced the microsomal activity to 200 per cent after 8 days and 170 per cent after 14 days. The nuclear activity was only slightly induced to a constant Ievei of 130 per cent between day 8 and 14. Dieldrin did not significantly increase the microsomal activity after daiJy i.p. injections (20 mg/kg), but the nuclear activity raised to 200 per cent after 3 days and levelled down tocontrol valuesafter 12 days. Other inducers tested were benz[a)anthracene (BA), hexachlorobenzene (HCB} and 1,1.1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The induction pattern with BA was similar tothat of MC, a modeJ compound for the group of cytochrome P448 inducers. The induction by HCB and DDT resembled that by PB. a typical cytochrome P450 inducer.
KW  - Toxikologie
Y1  - 1978
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61182
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Saccharin does not bind to DNA of liver or bladder in the rat [Short Communication]
N2  - No abstract available
KW  - Toxikologie
Y1  - 1977
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61194
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Schlatter, C.
T1  - Mechanism of the carcinogenic action of benzene: irreversible binding to rat liver DNA
N2  - No abstract available
KW  - Toxikologie
Y1  - 1977
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61208
ER  - 
TY  - JOUR
A1  - Lutz, Werner K.
A1  - Früh, P. U.
A1  - Simon, W.
T1  - Microcalorimetric determination of ΔH0, ΔG0 and ΔS0 for the interaction of the carrier antibiotics nigericin and monensin with sodium and potassium ions
N2  - The thermodynainic parameters ΔH0, ΔG0 and ΔS0 - and thereby the equilibrium constants - for the complexation of the carrier antibiotics nigericin and monensin with sodium and potassium ions in methanol at 25°C have been determined by microcalorimetry. Tbc results are discussed in terms of the nature of the interaction between ligands and cations.
KW  - Toxikologie
Y1  - 1971
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61218
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Maurer, K.
A1  - Klotz, Karl-Norbert
A1  - Schwabe, U.
T1  - Synergistic effects of calcium-mobilizing agents and adenosine on histamine release from rat peritoneal mast cells
N2  - 1 Adenosine and its metabolically stable analogue N.etbyl-carboxamidoadenosine (NECA) enhance histamine release from rat peritoneal mast cells when tbese are stimulated by calciummobilizing agents. NECA and adenosine shift the concentration-response curve of tbe calcium ionophore A23187 to lower concentrations. 2 The potencies of NECA or adenosinein enhancing A23187-induced histamine release are dependent on the Ievel of stimulated release in tbe absence of adenosine analogues. At high Ievels of release their potencies are up to 20 times higher than at low Ievels. Consequently, averaged concentration-response curves of adenosine and NECA for enhancing bistamine release are shallow. 3 The adenosine transport blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) has no effect by itself at low Ievels of stimulated histamine release, but abolishes the enhancing effect of adenosine. At high Ievels of release, however, NBTI alone enhances the release of histamine. 4 lt is concluded that adenosine and calcium reciprocally enhance the sensitivity of the secretory processes to the effects of the other agent. The Ievels of intracellular adenosine obtained by trapping adenosine inside stimulated mast cells are sufficient to enhance histamine release substantially, suggesting that this effect may play a physiological and pathophysiological role.
KW  - Toxikologie
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60346
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Klotz, Karl-Norbert
A1  - Jakobs, K. H.
A1  - Schwabe, U.
T1  - Barbiturates are selective antagonists at A\(_1\) adenosine receptors
N2  - Barbiturates in pharmacologically relevant . concentrations inhibit binding of (R)-\(N^6\)-phenylisopropyl[\(^3\)H]adenosine ([\(^3\)H]PIA) to solubilized A\(_1\) adenosine receptors in a concentration-dependent, stereospecific, and competitive manner. K\(_i\) values are similar to those obtained for membrane-bound receptors and are 31 \(\mu\)M for ( ± )-5-(1 ,3-dimethyl)-5-ethylbarbituric acid [( ± )DMBB] and 89 \(\mu\)M for ( ± )-pentobarbital. Kinetic experiments demoostrate that barbiturates compete directly for the binding site of the receptor. The inhibition of rat striatal adenylate cyclase by unlabelled (R)-\(N^6\)-phenylisopropyladenosine [(R)-PIA] is antagonized by barbiturates in the same concentrations that inhibit radioligand binding. The Stimulation of adenylate cyclase via A\(_2\) adenosine receptors in membranes from NIE 115 neuroblastoma cells is antagonized only by 10-30 times higher concentrations of barbiturates. lt is concluded that barbiturates are selective antagonists at the A1 receptor subtype. In analogy to the excitatory effects of methylxanthines it is suggested that A\(_1\) adenosine receptor antagonism may convey excitatory properties to barbiturates. Key Words: Adenosine receptors-Barbiturates - Adenylate cyclase-Receptor solubilization-[3H]PIA binding-N1E 115 cells. Lohse M. J. et al. Barbiturates are selective antagonists at A1 adenosine receptors.
KW  - Toxikologie
KW  - adenosine receptors
KW  - barbiturates
KW  - adenylate cyclase
KW  - receptor solubilization
KW  - N1E 115 cells
KW  - [3H]PIA binding
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60187
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Cristalli, G.
A1  - Grifantini, M.
A1  - Vittori, S.
A1  - Lohse, M. J.
T1  - Photoaffinity labeling of A\(_1\) adenosine receptors
JF  - The journal of biological chemistry
N2  - The ligand-binding subunit of the A\(_1\)-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R- \(N^6\)-phenylisopropyladenosine, R-2-azido-\(N^6\)-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific Iigand for A\(_1\)-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R·AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A\(_1\)-subtype. It competes for [\(^3\)H].\(N^6\)- phenylisopropyladenosine binding to Arreceptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [\(^3\)H)\(N^6\)-phenylisopropyladenosine binding afterextensive washing; the K; value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity Iabel of high specific radioactivity (\(^{125}\)I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for \(^{125}\)I-AHPIA binding to rat brain membranes with an order of potency characteristic for A\(_1\)-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of M\(_r\) = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A\(_1\)-subtype. The results indicate that \(^{125}\)I-AHPIA identifies the ligand-binding subunit of the A\(_1\)-adenosine receptor, which is a peptide with M\(_r\) = 35,000.
KW  - Toxikologie
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60198
VL  - 27
IS  - 260
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  - 
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  - 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  - Ukena, D.
A1  - Schirren, C. G.
A1  - Klotz, Karl-Norbert
A1  - Schwabe, U.
T1  - Evidence for an A\(_2\) adenosine receptor in guinea pig lung
N2  - Adenosine receptors in guinea pig lung were characterized by measurement of cyclic AMP formation and radioligand binding. 5'-N-Ethylcarboxamidoadenosine (NECA) increased cyclic AMP Ievels in lung slices about 4-fold over basal values with an EC\(_{50}\) of 0.32 \(\mu\)mol/l. N\(^6\) - R-(- )-Phenylisopropyladenosine (R-PIA) was 5-fold less potent than NECA. 5'-N-Methylcarboxamidoadenosine (MECA) and 2-chloroadenosine had EC\(_{50}\)-values of 0.29 and 2.6 \(\mu\)mol/l, whereas adenosine and inosine had no effect. The adenosine receptors in guinea pig Iung can therefore be classified as A\(_2\) receptors. Several xanthine derivatives antagonized the NECA-induced increase in cyclic AMP levels. 1,3-Diethyl-8-phenylxanthine (DPX; K\(_i\) 0.14 \(\mu\)mol/l) was the most potent analogue, followed by 8-phenyltheophylline (K\(_i\) 0.55 \(\mu\)mol/l), 3-isobutyl-1-methylxanthine (IBMX; K\(_i\) 2.9 \(\mu\)mol/l) and theophylline (K\(_i\) 8.1 \(\mu\)mol/l). In contrast, enprofylline (1 mmol/1) enhanced basal and NECA-stimulated cyclic AMP formation. In addition, we attempted to characterize these receptors in binding studies with [\(^3\)H]NECA. The K\(_D\) for [\(^3\)H] NECA was 0.25 \(\mu\)mol/l and the maximal number of binding sites was 12 pmol/mg protein. In competition experiments MECA (K\(_i\) 0.14 \(\mu\)mol/l) was the most potent inhibitor of [\(^3\)H] NECA binding, followed by NECA (K\(_i\) 0.19 \(\mu\)mol/l) and 2-chloroadenosine (K\(_i\) 1.4 \(\mu\)mol/l). These results correlate well with the EC\(_{50}\)- values for cyclic AMP formation in lung slices. However, the K\(_i\)-values of R-PIA and theophylline were 240 and 270 \(\mu\)mol/l, and DPX and 8-phenyltheophylline did not compete for [\(^3\)H]NECA binding sites. Therefore, a complete characterization of A\(_2\) adenosine receptors by [\(^3\)H] NECA binding was not achieved. In conclusion, our results show the presence of adenylate cyclase-coupled A\(_2\) adenosiile receptors in lung tissue which are antagonized by several xanthines.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Cyclic AMP
KW  - Lung
KW  - Theophylline
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60202
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
A1  - Schwabe, U.
A1  - Cristalli, G.
A1  - Vittori, S.
A1  - Grifantini, M.
T1  - 2-Chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) - a high affinity agonist radioligand for A\(_1\) adenosine receptors
N2  - The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10000-fold selectivity for A1 adenosine receptors, has been examined as a new agonist radioligand. [3H]CCP A was prepared with a specifi.c radioactivity of 1.58 TBqjmmol ( 43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinity K0 -value of 0.2 nmol/1. In the presence of GTP a K0 -value of 13 nmol/1 was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [3H]CCPA binding to rat brain membranes confrrmed binding to an A1 receptor. Solubilized A1 receptors bound [3H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgC12, as has been shown for the agonist [ 3H]N6-phenylisopropyladenosine ([3H]PIA). [3H]CCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A K0 -value of 0.4 nmol/1 and a Bmax-value of 16 fmol/ mg protein was determined in these membranes. In human platelet membranes no specific binding of [3H]CCPA was measured at concentrations up to 400 nmoljl, indicating that A2 receptors did not bind [3H]CCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors [ 3H]CCPA proved to be a useful agonist radioligand for characterization of A 1 adenosine receptors also in tissues with very low receptor density.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Radioligauds
KW  - agonists
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60328
ER  - 
TY  - JOUR
A1  - Tawfik-Schlieper, H.
A1  - Klotz, Karl-Norbert
A1  - Kreye, V. A. W.
A1  - Schwabe, U.
T1  - Characterization of the K\(^+\)-channel-coupled adenosine receptor in guinea pig atria
N2  - In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on 86Rb + -efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1 ,3-[\(^3\)H]dipropylxanthine ([\(^3\)H]DPCPX) to atrial membrane preparations. The rate of \8^{86}\)Rb\(^+\) -effiux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC50-values for 2-chloro-N\(^6\)-cyclopentyladenosine (CCPA), R-N\(^6\)-phenylisopropyladenosine (R-PIA), 5'-Nethylcarboxamidoadenosine (NECA), and S-N\(^6\)-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 \(\mu\)M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K\(_B\)-value of 8.1 nM, indicating competitive antagonism. [\(^3\)H]DPCPX showed a saturable binding to atrial membranes with a Bmax·value of 227 fmol/mg protein and a K\(_D\)-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K\(^+\) channel-coupled adenosine receptor in guinea pig atria is of an A\(_1\) subtype.
KW  - Toxikologie
KW  - A1 Adenosine receptors
KW  - K + -channels
KW  - Atria
KW  - Radioligand binding - 86Rb + -efflux
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60333
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  - THES
A1  - Hintzsche, Henning
T1  - Gentoxizität nichtionisierender Strahlung - Auswirkungen von Mobilfunk- und Terahertzstrahlung auf das Genom
T1  - Genotoxicity of non-ionizing radiation - Effects of mobile phone and terahertz radiation on the genome
N2  - Ziel der vorliegenden Arbeit war es, zu untersuchen, ob nichtionisierende elektromagnetische Strahlung verschiedener Frequenzbereiche Genomschaden hervorrufen kann. Im Rahmen der vorliegenden Arbeit wurde eine Biomonitoring-Studie zu dieser Thematik konzipiert und durchgeführt. Es wurden 131 Probanden detailliert zu ihrer Mobilfunknutzung befragt. Anschließend wurden Mundschleimhautzellen entnommen und für eine mikroskopische Untersuchung aufbereitet und angefärbt. In den Zellen wurden Mikrokerne und andere Kernanomalien quantifiziert. Es zeigte sich keine Erhöhung der Mikrokernfrequenz in Abhängigkeit von der Dauer der Mobiltelefonnutzung. Auch die anderen abgefragten Parameter hatten keinen Einfluss auf die Höhe des Genomschadens. Als Positivkontrollen wurden vier Patienten, die eine lokale Strahlentherapie (ionisierende Strahlung) erhielten, eingeschlossen. Hier zeigte sich eine deutliche Erhöhung der Mikrokernfrequenz. Um festzustellen, ob die Mikrokerninduktion erst bei höheren Leistungsflussdichten als denen, die beim Mobilfunk verwendet werden, auftritt, wurden in-vitro-Versuche durchgeführt, bei denen verschiedene Zelllinien einer Strahlung von 900 MHz ausgesetzt wurden. Nach Exposition und einer Postinkubationsperiode wurden die Zellen fixiert und die Mikrokernfrequenz bestimmt. Neben den Leistungen wurden hier auch die Expositionszeiten und die Postinkubationsperioden variiert. In keinem Fall konnte eine Erhöhung der Mikrokernfrequenz festgestellt werden. Insgesamt konnte ein Einfluss elektromagnetischer Strahlung auf das Genom weder am Menschen im Rahmen einer Biomonitoring-Studie noch an verschiedenen Zelllinien im Rahmen von in-vitro-Versuchen festgestellt werden. Terahertzstrahlung ist elektromagnetische Strahlung im Bereich von 0,1 bis 10 THz, d. h. sie liegt zwischen Mikrowellen und Infrarotlicht. Derzeit wird sie hauptsächlich für spektroskopische Untersuchungen und zur Qualitätskontrolle im Herstellungs-prozess verschiedener Produkte verwendet. Anwendungen in der Sicherheitstechnik (z. B. Ganzkörperscanner) und in der Medizintechnik (z. B. Bildgebung) stehen kurz vor der Markteinführung bzw. sind bereits etabliert. Diese Anwendungen bringen eine Exposition der betroffenen Menschen mit sich. Außerdem wird an weiteren Techniken wie etwa der Datenübertragung gearbeitet. Die Wirkungen auf biologische Systeme sind im Gegensatz zum Mobilfunkbereich bisher nur unzureichend untersucht. Da bisher keine vollständigen Literaturübersichten vorlagen, wurde eine umfassende Literaturrecherche durchgeführt. Ziel war es, alle bisher durchgeführten Studien zu diesem Thema aufzulisten. Um diese Datenbasis zu verbreitern wurden in-vitro-Versuche bei verschiedenen Frequenzen durchgeführt. Als Strahlungsquellen wurden eine Frequenzvervielfacherkaskade (0,106 THz), ein Rückwärtswellen-Oszillator (0,380 THz) und ein Ferninfrarot-Laser (2,520 THz) eingesetzt. Die Strahlung wurde in einen modifizierten Inkubator geführt, so dass die Expositionen bei definierter Temperatur und konstantem CO2-Gehalt durchgeführt werden konnten. Da Terahertzstrahlung durch Wasser sehr stark absorbiert wird, sind bei einer Exposition des Menschen primär die obersten Hautschichten betroffen. Aus diesem Grund wurden primäre Hautfibroblasten und HaCaT-Zellen, eine Keratinozyten-Zelllinie, als biologische Systeme verwendet. Die Zellen wurden für unterschiedliche Zeitperioden mit verschiedenen Leistungsflussdichten exponiert. Anschließend wurden die Zellen für den Comet Assay aufbereitet und analysiert. Der Comet Assay ist eine Methode zur Quantifizierung von Einzel- und Doppelstrangbrüchen der DNA. Weiterhin wurden die Zellen nach einer Postinkubationsperiode für den Mikrokerntest aufbereitet. Neben unbehandelten Kontrollen und Sham-Expositionen wurden auch Positivkontrollen durchgeführt. Es konnte keine Erhöhung der Anzahl der DNA-Strangbrüche bzw. der Mikrokernfrequenz festgestellt werden. Da bekannt war, dass im Mobilfunkbereich unter bestimmten Bedingungen Störungen der Mitose, nicht aber Erhöhungen der Mikrokernfrequenz, auftreten, wurden Mitosestörungen nach Exposition bei 0,106 THz untersucht. Hierzu wurden AL-Zellen für 30 Minuten exponiert und anschließend ohne Postinkubation direkt fixiert. Analysiert wurden Störungen in allen Phasen der Mitose. Es zeigte sich, dass die Frequenz der Störungen in der Pro- und Metaphase unverändert blieb. Die Störungen in der Ana- und Telophase nahmen dagegen mit steigender Leistungsflussdichte zu. Insgesamt konnte im Terahertzbereich unter den gewählten Expositionsbedingungen kein DNA-Schaden beobachtet werden. Bei 0,106 THz konnten Mitosestörungen als Folge der Exposition gezeigt werden. Der Zusammenhang zwischen diesen Mitosestörungen und DNA-Schäden, insbesondere der Mikrokerninduktion, konnte bisher nicht abschließend geklärt werden und bleibt Gegenstand weiterer Untersuchungen.
N2  - The aim of the present study was to investigate whether non-ionizing radiation of different frequencies can cause genomic damage. Within the course of the present work, a biomonitoring study was designed and performed. 131 participants were interviewed and asked for their mobile phone use customs. Subsequently buccal mucosa cells were retrieved, prepared and stained for microscopy analysis. Micronuclei and other nuclear anomalies indicating genomic damage were quantified. No increase in micronucleus frequency depending on mobile phone use or other retrieved parameters were observed. Four patients receiving radiation therapy (ionizing radiation) were included as positive controls. A clear increase in micronucleus frequency was observed here. To determine whether micronucleus induction only occurs at higher power densities, in vitro investigations with different cell lines exposed to 900 MHz radiation were carried out. After exposition and post-incubation, cells were fixed and the micronucleus frequency was determined. Besides power density also exposition times and post-incubation periods were varied. In no case an increase in micronucleus frequency could be observed. 7. Summary 108 All in all, no influence of electromagnetic radiation on the genome could be observed, neither on humans in the biomonitoring study nor on cell lines in the in vitro study. Terahertz radiation is electromagnetic radiation in the range from 0.1 to 10 THz, i. e. between microwaves and infrared light. Currently it is used in spectroscopy and for quality control procedures in manufacturing processes. Application in security technology (e. g. full body scanners) and medical technology (e. g. medical imaging) have recently been established or are about to enter the market. These applications also involve exposure of humans. Furthermore, other technologies such as data transfer are being developed. So far the effects on biological systems of this frequency range are investigated only insufficiently. A thorough literature search was conducted because no comprehensive literature review was available. The aim was to completely list all available studies on this topic. To enlarge this data basis, in vitro studies at different frequencies were conducted. Radiation sources were a frequency multiplier chain (0.106 THz), a backward wave oscillator (0.380 THz) and a far infrared laser (2.520 THz). The radiation beam was led into a modified incubator in order to perform expositions under controlled temperature and CO2 concentration conditions. Terahertz radiation is strongly absorbed by water, thus in case of exposure of humans the skin will be affected primarily. This is the reason why primary dermal fibroblasts and HaCaT cells, a keratinocyte cell line, were used as biological systems. The cells were exposed to different power densities for different time periods. Subsequently cells were prepared and analyzed using the comet assay which quantifies DNA single and double strand breaks. Also, after a post-incubation period cells were prepared for the micronucleus test. Besides untreated controls and sham expositions also positive controls were 7. Summary 109 performed. No induction of DNA damage, neither as strand breaks nor as micronucleus frequency elevation, was observed. Because it was known that under certain experimental conditions mitotic disturbances but not micronucleus formation occur after exposition to mobile phone radiation, these mitotic disturbances were analyzed after terahertz exposition. AL cells were exposed for 30 minutes with 0.106 THz and fixed directly after the exposition. All phases of mitosis were analyzed, but it turned out that primarily ana- and telophase were affected. These disturbances increased with increasing power density. All in all, no DNA damage could be observed under the applied experimental conditions. After exposition to 0.106 THz mitotic disturbances occurred in AL cells. The link between these mitotic disturbances and the DNA damage, particularly the micronucleus formation, could not be resolved completely and remains subject of further investigations.
KW  - Mutagenität
KW  - Nichtionisierende Strahlung
KW  - Terahertzbereich
KW  - Mobiles Endgerät
KW  - Gentoxizität
KW  - Nichtionisierende Strahlung
KW  - Terahertzstrahlung
KW  - Mobilfunkstrahlung
KW  - Angewandte Toxikologie
KW  - genotoxicity
KW  - non-ionizing radiation
KW  - terahertz radiation
KW  - mobil phone radiation
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-57684
ER  - 
TY  - THES
A1  - Bellwon, Patricia
T1  - Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing
T1  - Evaluierung der Kinetik anhand von in vitro Systemen - Ein Beitrag um die Anzahl von Tierversuchen zur Toxizitätsprüfung zu reduzieren
N2  - The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program “Predict-IV”; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of “omics” technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling. 
This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day. 
Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors. 
Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling. 
Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions. 
Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo.
Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound’s distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.
N2  - Die Erlassung von Richtlinien und Verordnungen durch die EU führte zu der Entwicklung von alternativen Testmethoden als Ersatz von Tierversuchen zur Risikobewertung von Xenobiotika. Des Weiteren weisen hohe Ausfallraten von Arzneimitteln in der späten Entwicklungsphase auf die Notwendigkeit hin, die bisher verwendeten Testmethoden der präklinischen Phase zu verbessern. Diese Punkte wurden in dem im siebten Rahmenprogramm der EU finanzierten Projekt „Predict-IV“ aufgegriffen. Ziel des Projektes war es, die Vorhersage der Arzneimittelsicherheit durch integrierte „omics“-Technologien, angewendet an etablierten in vitro Ansätzen, zu verbessern. Dabei standen drei Zielorgane bzgl. Arzneimittel-induzierter Organtoxizität im Mittelpunkt: Leber, Niere und zentrales Nervensystem, die jeweils durch Zelllinien oder primäre Zellen vertreten waren. Um die in vitro generierten Dynamik-Daten mit der in vivo Situation in Korrelation zu bringen, muss die Kinetik der Testsubstanz berücksichtigt und die Ergebnisse mit Hilfe von physiologisch-basierter pharmakokinetischer Modellierung extrapoliert werden. 
Ziel der vorliegenden Arbeit war es, Kinetik-Daten der gewählten Testsubstanzen (Cyclosporin A, Adefovir dipivoxil und Cisplatin) in vitro zu erheben und bzgl. ihrer Zuverlässigkeit sowie ihrer Relevanz verglichen mit in vivo Daten zu beurteilen. Hierfür wurden kultivierte Zellen täglich bzw. jeden zweiten Tag für zwei Wochen mit zwei verschiedenen Konzentrationen (toxisch und nicht toxisch) des Arzneimittels behandelt. Der Gehalt des applizierten Arzneimittels oder die Hauptmetaboliten wurden mittels LC MS/MS oder ICP-MS in Vehikel, Medium und Zellen sowie die vom Plastik adsorbierte Menge in Proben bestimmt, die am ersten und letzten Behandlungstag zu fünf unterschiedlichen Zeitpunkten gewonnen wurden.
Eine eindeutige Bioakkumulation von Cyclosporin A wurde in primären Rattenhepatozyten nach Behandlung mit der hohen Konzentration festgestellt. Eine effiziente CYP3A4- und CYP3A5-vermittelte Biotransformation von Cyclosporin A wurde für primäre humane Hepatozyten sowie HepaRG Zellen beobachtet. Diese Ergebnisse stimmten mit der in vivo Situation überein. Ratten sind aufgrund ihrer geringen CYP3A Aktivität schlechte Tiermodelle für CYP3A-Biotransformationsstudien. Des Weiteren wurden Interassay-Schwankungen bei primären human Hepatozyten bemerkt, die auf die biologische Variabilität der CYP3A4- sowie CYP3A5-Aktivität zwischen den menschlichen Spendern zurückzuführen sind. Rattenhepatozyten und HepaRG Zellen hingegen wiesen Interassay-Schwankungen auf, die durch unterschiedliche Cyclosporin A Behandlungskonzentrationen zwischen den Replikaten verursacht wurden. Die Cyclosporin A Biotransformation war in HepaRG Zellen am stärksten ausgeprägt, was durch stabile und wesentlich höhere CYP3A4- und CYP3A5-Aktivität in HepaRG Zellen zu erklären ist. Zusätzlich wurden die in vitro Clearance-Werte bestimmt und auf in vivo Clearance-Werte extrapoliert. Alle extrapolierten Werte waren zu hoch geschätzt (primäre Rattenhepatozyten: 10fach, primäre human Hepatpzyten: 2fach, HepaRG Zellen: 2fach). Diese Ergebnisse sollten mittels physiologisch-basierter pharmakokinetischer Modellierung sowie durch weitere Experimente überprüft werden, um zu ermitteln, ob diese hohen Schätzungen für jedes System konstant sind und somit durch die Einführung von weiteren Skalierungsfaktoren verringert werden können.
Kultivierte Gehirnzellen, primäre Nervenzellkulturen der Kortex von Mäusen und primäre Hirnzellaggregate der Ratte, zeigten schnell erreichte Cyclosporin A Gleichgewichtskonzentrationen. Diese Ergebnisse deuteten auf eine Verteilung von Cyclosporin A zwischen der wässrigen und organischen Phase hin, wobei biologische Prozesse nur eine untergeordnete Rolle spielen. Daher scheint die intrazelluläre Cyclosporin A Aufnahme Transporter-vermittelt zu sein. Ergebnisse der Transporter Experimente, die an einer künstlichen Membran und Caco-2 Zellen durchgeführt wurden, unterstützten diese Hypothese. Messungen der Plastikbindung von Cyclosporin A zeigten signifikante, aber für jedes Zellsystem unterschiedliche, Adsorptionsraten, die mittels physiologisch-basierter pharmakokinetischer Modellierung berücksichtigt werden sollten. 
Die Kinetik von Adefovir dipivoxil machte auf die Nachteile von in vitro Versuchen aufmerksam. Für die intrazelluläre Aufnahme von Adefovir sind aktive Transportproteine nötig, die jedoch in der Nierenzelllinie RPTEC/TERT1 nicht funktionell vorhanden sind. Daher war die Aufnahme von Adefovir auf die passive Diffusion von Adefovir dipivoxil beschränkt, das aber auch zeitabhängig unter den experimentellen Konditionen zerfiel. 
Die an RPTEC/TERT1 Zellen untersuchte Kinetik von Cisplatin deutete auf eine intrazelluläre Platin-Anreicherung hin, die jedoch nicht in einer signifikanten Bioakkumulation resultierte. Möglicherweise sind innerhalb von 14 Tagen die Gleichgewichtskonzentrationen von Cisplatin noch nicht erreicht. Die Kinetikprofile von Cisplatin in Medium ließen einen aktiven, von der basolateralen zur apikalen Seite gerichteten Cisplatin Transport erkennen, wie schon in vivo beschrieben, wobei die Geschwindigkeit dieser Transportprozesse in vitro langsamer zu sein scheint als in der intakte Niere. Daher müssen die generierten Daten zur Schätzung von zellulären Prozessen modelliert werden, um durch anschließende Extrapolation mit in vivo Daten verglichen werden zu können.
Abschließend bleibt zu sagen, dass das experimentelle Design vorteilhaft war. Wiederholte tägliche Administration von zwei unterschiedlichen Konzentrationen eines Medikaments ermöglichte die Erfassung von Bioakkumulation bei toxischen Konzentrationen sowie Biotransformation/Export bei nicht-toxischen Konzentrationen. Potenzielle Fehler, die zu einer Fehlinterpretation führen könnten, wurden durch die exakte Bestimmung der tatsächlich applizierten Arzneimittelmenge reduziert, da nicht immer die applizierte Konzentration mit der Nominalkonzentration übereinstimmt. Darüber hinaus erwies es sich als Vorteil, die Arzneimittelkonzentrationen in den einzelnen Kompartimenten (Medium, Zellen und Plastik) zu bestimmen. Somit konnten zum einen Erkenntnisse über die Verteilung der Substanz gewonnen werden und zum anderen Fehleinschätzungen von zellulären Prozessen, z.B. Biotransformation, verhindert werden, was letzten Endes bei die Interpretation von Kinetik-Daten behilflich ist. Jedoch, wurden auch die Grenzen von in vitro Ansätzen deutlich. Für eine korrekte Extrapolation ist es unverzichtbar, dass die untersuchten in vitro Systeme funktionierende Proteine aufweisen, die bei der untersuchten Arzneimittel-induzierten Toxizität eine Schlüsselrolle übernehmen. Abschließend kann festgehalten werden, dass es, unter Berücksichtigung der Vor- und Nachteile, von Nutzen sein kann diesen Versuchsansatz der Langzeitbehandlung zu validieren und darüber hinaus auf Co Kultursysteme sowie Organ-Chips anzuwenden hinsichtlich der Entwicklung von Alternativmethoden für Toxizitätsstudien bei wiederholter Gabe.
KW  - cell culture
KW  - pharmacokinetics
KW  - repeated dose
KW  - in vitro
KW  - toxicity testing
KW  - Zellkultur
KW  - In vitro
KW  - Pharmakokinetik
KW  - Toxizitätstest
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122693
ER  - 
TY  - JOUR
A1  - Chen, Wen
A1  - Gaßner, Birgit
A1  - Börner, Sebastian
A1  - Nikolaev, Viacheslav O.
A1  - Schlegel, Nicolas
A1  - Waschke, Jens
A1  - Steinbronn, Nadine
A1  - Strasser, Ruth
A1  - Kuhn, Michaela
T1  - Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway
JF  - Cardiovascular Research
N2  - Aims 
Cardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP.
Methods and results 
Intravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1−/− mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains.
Conclusion 
ANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.
KW  - caveolin-1
KW  - microvessel permeability
KW  - atrial natriuretic peptide
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126562
N1  - Lizenzhinweis: The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for noncommercial purposes provided that the original authorship is properly and fully attributed; the Journal, Learned Society and Oxford University Press are attributed as the original place of publication with correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated.
VL  - 93
IS  - 1
ER  - 
TY  - THES
A1  - Zink [geb. Sondergeld], Thomas Gerd
T1  - Der Cofilin-Signalweg im Glioblastoma multiforme - Ursachen für den Verlust von Chronophin und Einfluss von LIM-Kinase-Inhibitoren
T1  - The cofilin pathway in glioblastoma multiforme - Reasons for chronophin loss and effect of LIM-kinase inhibitors
N2  - Das invasive Potential maligner Gliome beeinflusst maßgeblich die schlechte Prognose dieser Tumorentität. Migration und Invasion von Tumorzellen werden entscheidend durch die Cofilin-vermittelte Umstrukturierung des Aktin-Zytoskeletts geprägt, die durch die Aktivität antagonistischer Cofilin-Kinasen und -Phosphatasen reguliert wird.

Im Rahmen der vorliegenden Arbeit konnte ein progressiver Expressionsverlust der Cofilin-Phosphatase Chronophin mit ansteigendem Malignitätsgrad astrozytärer Gliome aufgezeigt werden, der mit einer Zunahme der Phosphorylierung von Cofilin einhergeht. In den entsprechenden Gewebeproben gelang gleichzeitig der Nachweis einer gesteigerten Expression der Cofilin-Kinase LIMK-2.

Genetische und epigenetische Analysen des Chronophin-Locus konnten eine Hypermethylierung im Bereich der Promotorregion der Phosphatase identifizieren, die möglicherweise dem Verlust von Chronophin in Glioblastom-Gewebeproben zugrunde liegt. 
In Glioblastom-Zelllinien, die unterschiedliche Expressionsmuster von Chronophin aufwiesen, konnten hingegen keine molekularen Alterationen festgestellt werden. 

Untersuchungen des Einflusses von ROCK- und LIMK-Inhibitoren auf Glioblastomzellen konnten ausgeprägte Veränderungen der Zellmorphologie dokumentieren, wobei erstmals die Induktion eines stellate cell-Phänotyps unter Einfluss des LIMK-Inhibitors BMS-5 beschrieben wird. Während ROCK- und LIMK-Inhibitoren keinen Einfluss auf die 2D-Motilität der Tumorzellen hatten, wiesen die Glioblastomzellen in Abhängigkeit ihrer basalen Cofilin-Aktivität eine verstärkte bzw. verminderte 3D-Invasivität auf. 

Die Erkenntnisse dieser Arbeit unterstreichen die Bedeutung des Cofilin-Signalweges für die Migration und Invasion von Gliomzellen, zeigen neue Angriffspunkte in der Therapie maligner Gliome auf und warnen zugleich vor einem unkritischen Einsatz neuer Wirkstoffe.
N2  - The invasive potential of malignant gliomas is the main reason for the dismal prognosis of this tumor entity. Migration and invasion of tumor cells is crucially determined by the cofilin dependent reorganization of the actin cytoskeleton regulated by the activity of antagonistic cofilin kinases and phosphatases.

This study revealed a progressive loss of expression of the cofilin phosphatase chronophin with increasing malignancy grade of astrocytic glioma, accompanied by an increase of cofilin phosphorylation. Moreover, the analyzed glioma specimens were characterized by a simultaneous increase of expression of the cofilin kinase LIMK-2.

Integrated genetic and epigenetic analysis of the chronophin locus identified an aberrant promoter methylation as a possible mechanism leading to chronophin down-regulation in glioblastoma tissue samples. In contrast, molecular alterations of the chronophin locus were undetectable in glioblastoma cell lines characterized by different chronophin expression levels.       

Analysis of glioblastoma cells demonstrated striking effects of ROCK and LIMK inhibitors on cell morphology, providing first evidence of the induction of a stellate cell-phenotype by the LIMK inhibitor BMS-5. While ROCK and LIMK inhibitors had no detectable effect on the 2D motility of the tumor cells, the inhibitors increased or decreased the 3D-invasiveness of the glioma cells depending on their basal cofilin activity.

The findings of this study stress the importance of the cofilin signaling pathway as a key regulator of glioma migration and invasion, indicate novel targets for glioma therapy, but also warn against a noncritical use of new drugs.
KW  - Cofilin
KW  - Glioblastom
KW  - Chronophin
KW  - Actin
KW  - Rho-Proteine
KW  - PDXP
KW  - LIMK
KW  - BMS-5
KW  - Signalkette
KW  - Rho-GTPasen
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127065
ER  - 
TY  - THES
A1  - Makiol, Christian
T1  - Die Rolle der durch NADPH-Oxidasen produzierten reaktiven Sauerstoffspezies (ROS) in der Pathophysiologie von Hypertonie und Alterung
T1  - The role of nadph-oxidase produced reactive oxygen species (ROS) in the pathophysiology of hypertension and aging
N2  - Reaktive Sauerstoffradikale spielen eine große Rolle bei der Entstehung von Karzinomen, im Alterungsprozess und bei kardiovaskulären Erkrankungen (Valko et al., 2007). Solche Sauerstoffradikale können unter anderem durch die NADPH-Oxidase gebildet werden (Finkel and Holbrook, 2000). 
Ziel der Arbeit war es, die Rolle von ROS im Alterungsprozess und bei Bluthochdruck aufzuzeigen. Dafür wurden zwei Tiermodelle, eines mit einer geringen ROS-Konzentration und eines mit einer hohen ROS-Konzentration, verwendet. Zum einen handelt es sich um ein p47-Knockout-Mausmodell für die geringere ROS-Konzentration, im Vergleich zu alten und jungen Wildtyp-Tieren. Für die Rolle von ROS bei der Alterung wurden junge mit alten Wildtyp-Tieren verglichen. Um die Rolle der NOX2 in den ROS-Spiegeln der Organe zu ermitteln wurden gleichaltrige Wildtyp- mit p47-Knockout-Tieren verglichen. Zum anderen nutzten wir ein ren2-Tiermodell für die hohe ROS-Konzentration. Hierbei handelt es sich um ein Bluthochdruckmodell, in dem der Blutdruck mit Medikamenten normalisiert werden kann. Die Tiere wurden daher mit Ramipril (ACE-Inhibitor), Apocynin (NADPH-Oxidase-Inhibitor) und Tempol (Radikalfänger) behandelt. Als Maß für die ROS-Konzentration wurden die Superoxidionenspiegel mithilfe einer Dihydroethidium-Färbung ermittelt. Die DNA-Doppelstrangbrüche wurden mit einer γH2AX-Antikörper-Färbung nachgewiesen und die Expression von Sirtuin1 und Hsp70, welche als Anti-Aging Proteine bekannt sind, mittels Western Blot bestimmt.
N2  - Reactive oxygen species play a major role in the etiology of cancer, in the  aging process and cardiovascular diseases (Valko et al., 2007). These oxygen radicals may be formed by the NADPH oxidase enzym complex (Finkel and Holbrook, 2000). 
The aim of the study is to analyse the effect of ROS in the aging process and hypertension. Therefore two models were used. Young and old wild-type animals were used and compared with young p47 knockout mice, which have a reduced ROS level. As a model with increased  ROS levels the ren2-rat model was applied. Those animals were treated with different drugs such as ramipril (ACE inhibitor), apocynin (NADPH oxidase inhibitor) and tempol (radical scavenger). The superoxide concentration was determined in kidney and heart by using dihydroethidium-staining. The DNA double strand breaks were detected by staining with an γH2AX antibody and the expression of Sirtuin1 and Hsp70 which are known as anti-aging proteins, were determined by Western Blot.
KW  - Reaktive Sauerstoffspezies
KW  - Hypertonie
KW  - Alterung
KW  - NADPH-Oxidase
KW  - reaktive Sauerstoffspezies
KW  - Hypertonie
KW  - Alterung
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-109901
ER  - 
TY  - THES
A1  - Mandel, Philipp
T1  - Entstehung von oxidativen Stressmarkern in DNA und RNA nach der Behandlung mit den Hormonen Angiotensin II und Aldosteron in vitro und in vivo : Vergleich von drei Analysemethoden zum Nachweis von 8-Oxo-2'-desoxyguanosin in LLC-PK1-Zellen
T1  - Formation of oxidative stress markers in DNA and RNA after treatment with aldosterone and angiotensin II in vitro and in vivo
N2  - The detection of oxidative stress markers has gained increasing importancy in the early investigation of diseases like diabetes, cancer or hypertension. 8 oxo 2' deoxyguanosine (8-oxodG) is the main marker, which is used for the intracellular detection of oxidative stress levels. However, the oxidative stress markers 8 oxoguanine (8-oxoGua), a product of the DNA base excision repair and 8 oxoguanosine (8-oxoGuo), a marker for oxidative damaged RNA have received less attention up to now.
The renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation processes of the blood pressure system. During hypertension angiotensin II (Ang II) and aldosterone (Aldo) are released in high concentrations over a longer period leading to non-physiological effects of the RAAS hormones. Subsequently, an increase of the intracellular oxidative stress level in kidney cells can be measured. The aim of this thesis is the in vitro and in vivo detection of the oxidative damage in DNA and RNA by measuring oxidative stress markers, especially 8-oxodG which is triggered by Ang II and Aldo.
In vitro experiments were carried out in LLC-PK1, a cell line originated from porcine kidney cells. It could been shown that Ang II and Aldo led to a dose-dependent increase of DNA damage in the cells. A time-dependent increase was detected for the first 30 minutes of the treatment. For the rest of the experimental set up (4 h) the level of detected DNA damage remained constant. The FPG comet assay and the immunocytochemical staining showed a significant increase of 8-oxodG in the cells, whereas the HPLC-MS/MS measurement only detected a small increase of 8-oxodG in the DNA. The FPG enzyme, which recognises also other oxidized purines besides 8-oxodG, which led to an overestimation of 8-oxodG in the comet assay. Also, the 8 oxodG antibody, which was used in the immunocytochemical analysis, detected higher amounts of 8-oxodG most likely due to its side reactions with other oxidized DNA structures. One of the main advantages of the last mentioned methods is the direct measurement in damaged cells, whereas the HPLC-MS/MS requires an isolation of the DNA. During this isolation process the oxidative stress markers can be oxidized and the detection can become imprecise.
The main purpose of the in vivo experiments was the detection of the oxidative stress marker 8-oxoGua, 8-oxodG and 8-oxoGuo in the urine of test animals. The treatment of C57BL/6 mice and Sprague Dawley (SD) rats with the RAAS hormones led to an increase of the blood pressure, higher DNA damage due to oxidative stress as well as an increased excretion rate of oxidative stress markers. The inhibition of the angiotensin II type 1- or mineralocorticoid receptor and a mutation of the AT1a gene could show, that the DNA damage is independent from the hypertension. In addition, it was shown that the NOX4 is not alone responsible for the oxidative stress. Other NADPH oxidases must contribute to the induction of oxidative stress inside the cell. Moreover, the activation of the Nrf2 pathway has an influence on the effect of Aldo in SD rats.
The excretion rate of the oxidative stress markers in the 20 h urine of the treated animals showed how the equilibrium between the DNA repair and the oxidative stress level was changing over time. The measurement of 8-oxoGuo became more and more popular, because up to the fact that 80 % of the DNA is translated into RNA. Overall, the detection of 8-oxodG and 8-oxoGuo is feasible for monitoring the disease or the healing process, because the measurement is non-invasive. The detection of 8-oxodG and 8-oxoGuo in nucleic acids is a first step into the field of basic research methods, because it reveals a snapshot of the nucleic acid damage in the cell at a specific time point. Usually, there will be an overestimation of the oxidative stress marker resulting from the analytical method. Although, it is possible to detect an underestimation of oxidative stress markers in tissue samples if not all cell types are damaged equally. Therefore, a primary goal should be the detection of a stable oxidation product of guanine to insure a reliable detection strategy and for a better understanding of the equilibrium of DNA oxidation and repair.
N2  - Der Nachweis von oxidativen Stressmarkern hat bei der Untersuchung von Krankheiten wie Diabetes, Krebs und Hypertonie an großer Bedeutung gewonnen. Vor allem 8-Oxo-2’-desoxyguanosin (8-oxodG) wird gezielt mit verschiedenen Methoden gemessen und als Marker für oxidativen Stress herangezogen. Daneben haben 8 Oxoguanin (8-oxoGua), als Produkt aus der Basenexzisionsreparatur der DNA, sowie 8-Oxoguanosin (8-oxoGuo), als Biomarker für oxidativ geschädigte RNA, bisher weniger Aufmerksamkeit bekommen. Das Renin-Angiotensin Aldosteron System (RAAS) spielt eine wichtige Rolle in der Regulierung des Blutdrucks. Im Falle einer Hypertonie werden Angiotensin II (Ang II) und Aldosteron (Aldo) über einen langen Zeitraum in erhöhter Konzentration ausgeschüttet. Dieser Umstand bewirkt eine nicht physiologische Wirkung der Hormone des RAAS, welche zu einer Induktion von oxidativem Stress führt. Die Zielsetzung dieser Arbeit ist es, die oxidative Schädigung, ausgelöst durch Ang II und Aldo, in der DNA und der RNA in vitro und in vivo nachzuweisen und dabei speziell den Biomarker 8-oxodG zu untersuchen.
In-vitro-Experimente wurden mit LLC PK1-Zellen, einer Schweinenierenzelllinie, durchgeführt. Ang II und Aldo lösten einen dosisabhängigen Anstieg der DNA Schäden in LLC PK1 Zellen aus. Eine Zeitabhängigkeit wurde für die ersten 30 Minuten gezeigt. Für die restliche Zeit (4 h) blieb der nachgewiesene DNA Schaden konstant. Der FPG Comet-Assay und die immunzytochemische Färbung zeigten jeweils eine signifikante Zunahme von 8-oxodG in LLC-PK1-Zellen an, während die HPLC MS/MS Messung nur geringe Veränderungen nachwies. Das FPG Enzym erkennt neben 8-oxodG auch andere oxidierte Purine und sorgte so für eine Überbestimmung des DNA-Schadens. Bei der immunzytochemischen Färbung entsteht die Überbestimmung durch Kreuzreaktionen des 8 oxodG Antikörpers mit oxidierten Strukturen in der DNA. Der Vorteil beider Analysemethoden ist die direkte Messung von Schädigungen in der Zelle, während die HPLC-MS/MS eine Isolierung der Nukleinsäuren voraussetzt. Bei diesem Schritt kann es zur Oxidation der Marker für oxidativen Stress kommen, welche einen genauen Nachweis erschwert.
In vivo-Versuche hatten zum Ziel, die oxidativen Stressmarker 8-oxoGua, 8-oxodG und 8-oxoGuo im Urin nachzuweisen. Die Behandlung der C57BL/6-Mäuse und Sprague Dawley-Ratten (SD-Ratten) mit den Hormonen des RAAS zeigten einen Anstieg des Blutdrucks, erhöhte DNA Schäden durch oxidativen Stress sowie erhöhte Exkretionsraten der oxidativen Stressmarker. Durch eine Inhibierung des Angiotensin II-Typ1- oder Mineralkortikoidrezeptors sowie die Mutation des Gens AT1a konnte gezeigt werden, dass die Schädigungen unabhängig vom Blutdruck sind. Zudem konnte gezeigt werden, dass neben NOX4 auch andere NADPH Oxidasen für den oxidativen Stress verantwortlich sein müssen. Eine Aktivierung des Nrf2 Signalweges in den SD-Ratten hat Einfluss auf die Wirkung von Aldo.
Die Exkretionsrate der oxidativen Biomarker im 20-h-Urin der behandelten Tiere zeigen, wie sich das Gleichgewicht zwischen DNA-Reparatur und oxidativem Stress verändert. Da 80 % der DNA in RNA umgeschrieben werden, ist der Nachweis von 8 oxoGuo in den Fokus gerückt. In der praktischen Anwendung kann mit der Messung von 8 oxodG und 8-oxoGuo ein Krankheits- oder Heilungsprozess auf nicht  invasive Weise verfolgt werden. Der Nachweis von 8-oxodG und 8-oxoGuo in den Nukleinsäuren stellt einen Einstieg für die Grundlagenforschung dar, da sie nur eine Momentaufnahme der Nukleinsäureschädigung in der Zelle zeigen. Meist findet eine Überbestimmung, ausgelöst durch die Messmethode, statt. In Gewebeproben kann eine Unterbestimmung vorliegen, falls nicht alle Zelltypen vom oxidativen  Stress  betroffen sind. Daher sollte es ein vorrangiges Ziel sein,  ein stabileres  Oxidationsprodukt des Guanins nachzuweisen, um das Gleichgewicht der DNA-Oxidation und Reparatur besser zu verstehen.
KW  - Oxidativer Stress
KW  - Aldosteron
KW  - Renin-Angiotensin-Aldosteron-System
KW  - Angiotensin II
KW  - LC-MS
KW  - oxidative Stressmarker
KW  - 8-Oxo-2’-desoxyguanosin
KW  - Hydroxylradikal
KW  - DNA-Reparatur
KW  - Mineralokortikoidrezeptor
KW  - 8-oxo-2'-deoxyguanosine
KW  - DNA base excision repair
KW  - hypertension
KW  - oxidative stress marker
KW  - RNA degradation
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111190
ER  - 
TY  - JOUR
A1  - Rychlik, Michael
A1  - Humpf, Hans-Ulrich
A1  - Marko, Doris
A1  - Dänicke, Sven
A1  - Mally, Angela
A1  - Berthiller, Franz
A1  - Klaffke, Horst
A1  - Lorenz, Nicole
T1  - Proposal of a comprehensive definition of modified and other forms of mycotoxins including “masked” mycotoxins
JF  - Mycotoxin Research
N2  - As the term "masked mycotoxins" encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, "modified mycotoxins" into "biologically modified" and "chemically modified" with all variations of metabolites of the former and dividing the latter into "thermally formed" and "non-thermally formed" ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term "modified mycotoxins" should be used in the future and the term "masked mycotoxins" to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.
KW  - definition
KW  - mycotoxins
KW  - masked mycotoxins
KW  - modified mycotoxins
KW  - hidden mycotoxins
KW  - mycotoxin derivates
KW  - mycotoxin metabolites
KW  - conjugated mycotoxins
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121240
VL  - 30
IS  - 4
ER  - 
TY  - JOUR
A1  - Ott, Ilka
A1  - Lohse, Martin J.
A1  - Klotz, Karl-Norbert
A1  - Vogt-Moykopf, Ingolf
A1  - Schwabe, Ulrich
T1  - Effects of Adenosine on Histamine Release from Human Lung Fragments
JF  - International Archives of Allergy and Immunology
N2  - The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A 23187 orwith concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl] methyloxy]-phenyl]-1,3-dipropylaxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioninosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence ofXAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a si te antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.
KW  - mast cells
KW  - adenosine
KW  - histamine release
KW  - human lung
Y1  - 1982
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127877
VL  - 98
ER  - 
TY  - JOUR
A1  - Lohse, Martin J.
A1  - Klotz, Karl-Norbert
A1  - Salzer, Manfred J.
A1  - Schwabe, Ulrich
T1  - Adenosine regulates the \(Ca^{2+} \) sensitivity of mast cell mediator release : (histamine secretion/inositol phosphates/calcium)
JF  - Proceedings of the National Academy of Sciences of the United States of America
N2  - Mast cells release histamine and other mediators of allergy in response to stimulation of their IgE receptors. This release is generally thought to be mediated by an elevation of cytosolic \(Ca^{2+}\). Recent evidence suggests that there might be factors that modulate the coupling between \(Ca^{2+}\) levels and mediator release. The present report identifies adenosine as one such modulator. Adenosine and several of its metabolically stable analogues were shown to enhance histamine release from rat peritoneal mast cells in response to stimuli such as concanavalin A. Metabolizing endogenous adenosine with adenosine deaminase dampened the response to stimuli, whereas trapping endogenous adenosine inside mast cells with nucleoside-transport inhibitors markedly enhanced stimulated histamine release. The metabolically stable adenosine analogue 5' -(N-ethylcarboxamido)adenosine (NECA) did not affect the initial steps in the sequence from IgE-receptor activation to mediator release, which are generation of inositol trisphosphate and increase of cytosolic \(Ca^{2+}\). However, NECA did enhance the release induced in ATP-permeabilized cells by exogenous \(Ca^{2+}\), but it had no effect on the release induced by phorbol esters. These data suggest that adenosine sensitizes mediator release by a mechanism regulating stimulus-secretion coupling at a step distal to receptor activation and second-messenger generation.
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127883
VL  - 85
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Klotz, K.-N.
A1  - Ukena, D.
A1  - Schwabe, U.
T1  - Characterization of \([^3H]\)Phenobarbital Binding to Rat Brain Membranes
JF  - Neuroscience Letters
N2  - The binding of \([^3H]\)phenobarbital to rat brain membranes was studied in order to determine its characteristics and specificity. The binding reaction was rapid and occurred at sites of low affinity. \((K_d = 700 μM)\) and very high density \((B_{max} = 2.7 nmoll/mg protein)\). It was unaffected by temperature changes from O°C to 95°C and was maximal at pH 5. Detergents in low concentrations markedly decreased the binding, apparently without solubilizing the binding sites. It is concluded that the binding of \([^3H]\) phenobarbital is a rather non-specific interaction with the plasma membrane.
KW  - barbiturates
KW  - radioligand binding
KW  - rat brain membranes
Y1  - 1984
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127894
VL  - 52
ER  - 
TY  - JOUR
A1  - Boivin, Valérie
A1  - Beyersdorf, Niklas
A1  - Palm, Dieter
A1  - Nikolaev, Viacheslav O.
A1  - Schlipp, Angela
A1  - Müller, Justus
A1  - Schmidt, Doris
A1  - Kocoski, Vladimir
A1  - Kerkau, Thomas
A1  - Hünig, Thomas
A1  - Ertl, Georg
A1  - Lohse, Martin J.
A1  - Jahns, Roland
T1  - Novel Receptor-Derived Cyclopeptides to Treat Heart Failure Caused by \(Anti-β_1-Adrenoceptor\) Antibodies in a Human-Analogous Rat Model
JF  - PLoS One
N2  - Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the \(β_1\) adrenergic receptor \((β_1EC2)\). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195–225) every 4 weeks; n = 38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking \(β_1EC2\) (\(β_1EC2-CP\), 1.0 mg/kg every 4 weeks) or administration of the \(β_1-blocker\) bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received \(β_1EC2-CP/bisoprolol\) co-treatment. We found that \(β_1EC2-CP\) prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, \(β_1EC2-CP\) mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free \(anti-β_1EC2-antibodies\) and by targeting \(β_1EC2\)-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful \(anti-β_1EC2-antibodies\) and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to \(β_1\)-blockade represents a promising new therapeutic option in immune-mediated heart failure.
KW  - memory B cells
KW  - antibodies
KW  - T cells
KW  - B cells
KW  - heart
KW  - heart failure
KW  - kidneys
KW  - enzyme-linked immunoassays
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126028
VL  - 10
IS  - 2
ER  - 
TY  - THES
A1  - Segerer, Gabriela
T1  - Characterization of cell biological and physiological functions of the phosphoglycolate phosphatase AUM
T1  - Charakterisierung zellbiologischer und physiologischer Funktionen der Phosphoglykolat-Phosphatase AUM
N2  - Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer.
Still, most of the mammalian HAD phosphatases remain functionally uncharacterized.
This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos.
To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation.
The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells.
These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling.
Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase.
To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions.
Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under
hypoxic (~1% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3’-phosphate (GADP).
Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism.
Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.
N2  - Haloazid Dehalogenase (HAD)-Typ Phosphatasen in Säugetieren gehören zu einer großen ubiquitären Proteinfamilie, zu der auch mindestens 40 Phosphatasen, die im menschlichen Organismus vertreten sind, zählen. Eine Vielzahl dieser Phosphatasen hat wichtige physiologische Funktionen beispielsweise als regulatorische Enzyme im Metabolismus.
Gleichzeitig werden sie in Verbindung mit Erkrankungen des kardiovaskulären Systems, Stoffwechselstörungen und Krebs gebracht. Dennoch sind die Funktionen vieler Mitglieder dieser Phosphatasen Familie bis heute weitestgehend unbekannt.
In der vorliegenden Arbeit wurden die zellbiologischen und physiologischen Funktionen der Phosphoglykolat-Phosphatase PGP, auch AUM genannt, charakterisiert. Zu diesem Zweck wurde mit gereinigtem Enzym nach potenziellen Protein-Substraten von PGP gesucht.
Weiterhin wurden zellbiologische Studien mit der spermatogonialen GC1 Zelllinie sowie mit primären Endothelzellen und Lymphozyten durchgeführt. Mit biochemischen Methoden wurden zudem PGP-defiziente Mausembryonen charakterisiert.
Es wurde zunächst die Rolle von PGP für RTK- und integrin- induzierte Zellmigration untersucht. Dabei zeigte sich, dass PGP Inaktivierung die Zelladhäsion und Zellmigration steigerte. Gleichzeitig wurde eine vermehrte Bildung von RTK- und integrinvermittelten ringförmigen Plasmamembranausstülpungen, sogenannten Circular Dorsal Ruffles (CDR) auf der dorsalen Zelloberfläche beobachtet. PGP wurde zudem als negativer Regulator integrinund GPCR-induzierter gerichteter Lymphozytenmigration identifiziert. Der zugrundeliegende molekulare Mechanismus wurde näher untersucht. Es konnte gezeigt werden, dass PGP die Bildung von CDRs und die gerichtetete Zellmigration in Abhängigkeit der Phospholipase C- (PLC-), Proteinkinase C- (PKC-) sowie Src Kinase-Aktivität steuert. Nach Integrin- und RTKAktivierung waren die Tyrosinreste 1068 und 1173 des EGF-Rezeptors in PGP-depletierten Zellen vermehrt phosphoryliert und PLCγ1 in diesen Zellen hyperaktiviert. Interessanterweise wurde zudem eine beschleunigte PKC-vermittelte Reorganisation des Zytoskeletts beobachtet. In stimulierten Lymphozyten führte PGP-Inaktivierung zu einer erhöhten PKCAktivität.
Durch massenspektrometrische Analysen konnten erhöhte Spiegel des Membranlipids Phosphatidylserin (PS) in PGP-defizienten Zellen nachgewiesen werden.
Diese Ergebnisse sind konsistent mit der Hypothese, dass die Anreicherung von PS in der Plasmamembran PGP-defizienter Zellen zu einer Vor-Rekrutierung von Signalproteinen führt, die die Aktivierung von Integrinen, EGF-Rezeptoren und GPCRs mit einer beschleunigten Zytoskelett-Reorganisation verbindet. Hierdurch konnte gezeigt werden, dass PGP durch die Dephosphorylierung von Phosphoglykolat die Zelladhäsion und Zellmigration reguliert.
Um die physiologischen Funktionen von PGP zu verstehen, wurden konditional PGPinaktivierte Mäuse untersucht. Die Inaktivierung von PGP im gesamten Organismus führte zu einem Wachstumsdefekt ab Tag E8.5 und dem Tod der Embryonen im Uterus zwischen Tag E9.5 und E11.5. Die beobachtete embryonale Letalität war nicht durch einen Defekt des (kardio-)vaskulären Systems zu erklären.
PGP-inaktivierte Embryonen starben zu einem Zeitpunkt, an dem der intrauterine Übergang von einem hypoxischen zu einem normoxischen Millieu stattfindet. Der Einfluss von Sauerstoff wurde deshalb weiter untersucht. Zellwachstumsanalysen unter normoxischen und hypoxischen Bedingungen mit GC1 Zellen und embryonalen Maus-Fibroblasten, die aus E8.5 Embryonen gewonnen wurden zeigten, dass normoxische Bedingungen (~20% O2) einen Wachstumsdefekt PGP-inaktivierter Zellen verursacht, wohingegen dies unter hypoxischen Bedingungen (~1% O2) nicht der Fall war. Mechanistisch konnte gezeigt werden, dass die Aktivität der Triosephosphatisomerase (TPI), ein durch PG in vitro gehemmtes Enzym, in PGP inaktivierten Zellen und Embryonen vermindert war. TPI stellt einen entscheidenden Verzweigungspunkt des Glukose- und Lipidstoffwechsels dar. TPI katalysiert die Isomerisierung der aus der Glykolyse stammenden Intermediate Dihydroxyacetonphosphat (DHAP, eine Vorstufe des für die Triglycerid-Biosynthese benötigten Glycerol-Grundgerüsts) und Glyceraldehyd-3’-phosphat (GADP). Eine Verringerung der TPI-Aktivität in PGPinaktivierten Zellen resultierte in erhöhten Glycerol-3-phosphat Spiegeln und einer gesteigerten Triglycerid-Biosynthese. Die Analyse des zellulären ATP Gehalts und des Sauerstoffverbrauchs bei der mitochondrialen Atmung zeigte, dass sowohl die ATP Produktion als auch die mitochondriale Atmung in Abhängikeit der Lipolyse in PGP-defizienten Zellen erhöht waren. Unter hypoxischen Bedingungen, die zu einer Normalisierung der Zellproliferation führten, wiesen PGP-profiziente und -defiziente Zellen keinen Unterschied bezüglich ATP Produktion und mitochondrialer Atmung auf.
Wir vermuten deswegen, dass die Inhibierung der TPI-Aktivität durch PG-Anreicherung aufgrund ausbleibender Hydrolyse durch PGP zu einer Verschiebung des zellulären Energiehaushaltes von Seiten eines pro-proliferativ glykolytischen auf die Seite eines lipogenetisch/lipolytischen Metabolismus führt.
Zusammenfassend konnte gezeigt werden, dass PGP als eine metabolische Phosphatase Zellmigration, Zellproliferation wie auch den zellulären Energiehaushalt reguliert. Die vorliegende Arbeit stellt somit die Grundlage für weitere Untersuchungen an der Schnittschnelle dieser zellulären Prozesse dar und lässt auf eine wichtige Rolle von PGP im Glukose- und Lipidstoffwechsel im adulten Organismus schließen.
KW  - Phosphoglykolatphosphatase
KW  - Phosphoglykolat-Phosphatase
KW  - Maus
KW  - Cytologie
KW  - Physiologie
KW  - Phosphatasen
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123847
ER  - 
TY  - THES
A1  - Stumpf, Anette D.
T1  - Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors
T1  - Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren
N2  - Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors
A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements.
For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement
of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can
lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future.
N2  - Adenosin Rezeptoren, die zur Gruppe der Rhodopsin-ähnlichen G Protein-gekoppelten Rezeptoren (GPCRs) gehören, sind in eine Vielzahl regulatorischer Prozesse eingebunden und weit im Körper verbreitet. Das macht sie zu einer interessanten Zielstruktur für Arzneistoffe. Das Wissen über die Struktur der Adenosin Rezeptoren ist jedoch noch begrenzt. Ein großer Fortschritt zu mehr strukturellem Wissen war die Entwicklung der ersten Kristallstruktur des Adenosin A2A Rezeptors im Jahr 2008. Mit der Kristallstruktur wurden die Aminosäuren bekannt, die die Ligandenbindetasche dieses Rezeptors formen. Zudem gab die Kristallstruktur Einblick in den Endpunkt der dynamischen Rezeptorbewegung nach Ligandenbindung. Im Rahmen der hier vorgestellten Arbeit wurden zwei Mitglieder der Adenosin Rezeptor Familie, der Adenosin A1 Rezeptor und der Adenosin A2A Rezeptor (A1R, A2AR), genauer untersucht. Der A1R wurde auf Basis des vor kurzem veröffentlichten A2AR entwickelt. Die Rezeptoren wurden mit Fluorophoren versehen, zum einen mit dem cyan fluoreszierenden Protein (CFP) am C-Terminus des Rezeptors und zum anderen mit der Bindesequenz des kleinen Fluorophors "Fluorescein Arsenical Hairpin binder" (FlAsH) in der dritten intrazellulären Schleife des Rezeptors. Die daraus resultierenden Rezeptorsensoren A1 Fl3 CFP und A2A Fl3 CFP wurden mit Hilfe des Fluoreszenz Resonanz Energie Transfers (FRET) in lebenden Zellen erforscht. FRET Messungen ermöglichen es, eine Änderung der Distanz zwischen
den beiden Fluorophoren und damit Rezeptorbewegungen zu untersuchen. Um A1R und A2AR bezüglich dynamischer Rezeptorbewegungen nach Ligandenbindung vergleichen zu können, wurden die fluoreszierenden Rezeptorsensoren A1 Fl3 CFP und A2A Fl3 CFP mit verschiedenen Liganden umspült. Die Ergebnisse der FRET Messungen bezüglich ihrer Höhe des FRET Ratio wurden verglichen, welche
mit der Konformationsänderung des Rezeptors nach Ligandenbindung zusammenhängt. Neben der unterschiedlichen Richtung des FRET Ratio nach Ligandenbindung am A1R und A2AR Sensor waren Unterschiede bezüglich der Signalhöhe und der Bindungs- und Dissoziationskinetiken feststellbar, wenn die verschiedenen Liganden miteinander verglichen wurden. Unterschiede zwischen den Adenosin Rezeptor Subtypen waren speziell für den A1R subtypselektiven Agonist CPA und für den
A2AR subtypselektiven Agonist CGS 21680 feststellbar. Einen weiteren Punkt in diesem Projekt stellte die Erforschung des Einflusses, den einzelne Aminosäuren im fluoreszierenden A1R Sensor auf den Prozess der Ligandenbindung haben, dar. Die Position der Aminosäuren wurde der Kristallstruktur des A2AR entnommen und entsprechende Aminosäuren im A1R mutiert. Die konfokalmikroskopische Analyse des A1R Sensors und seiner Mutanten ergab, dass einige Aminosäuren direkt an der zellulären Expression des Rezeptors beteiligt waren. Wurden diese Aminosäuren mutiert, wurde der Rezeptor nicht in der Plasmamembran der Zellen exprimiert. Einige Aminosäuren die untersucht wurden,hatten einen generellen Einfluss auf die Bindung der Liganden, andere Aminosäuren hatten mehr Einfluss auf die Bindung bestimmter struktureller Gruppen der untersuchten Liganden. In einem weiteren Schritt wurden A1R und A2AR am N-terminalen Rezeptorende
mit SNAP oder CLIP versehen, was eine Markierung der Rezeptoren mit einer Vielzahl an Fluorophoren erlaubt. Mit Hilfe dieser Technik konnte die Verteilung der Rezeptoren in der Zelle mit konfokaler Mikroskopie untersucht werden. Des Weiteren wurde die Bindung von fluoreszierenden Adenosin Rezeptor Liganden und deren Verdrängung mit einem nicht-fluoreszierenden Adenosin Rezeptor Antagonist erforscht. Am Ende des Projekts wurden die zuvor beschriebenen fluoreszierenden Rezeptorsensoren zu dreifach fluorophormarkierten Rezeptorsensoren kombiniert, was zu den Sensoren SNAP A1 Fl3 CFP und SNAP A2A Fl3 CFP führte. Beide Rezeptorsensoren waren funktionell bezüglich FRET Experimenten und der Expression in der
Plasmamembran der Zellen. In Zukunft können mit dieser Methode gleichzeitig die Bindung von fluoreszierenden Liganden am SNAP-markierten Rezeptor, so wie die Rezeptorbewegung beobachtet
werden, die durch eine Distanzänderung zwischen CFP und FlAsH angezeigt wird. Das kann zu einem besseren Verständnis der Rezeptorfunktion und der dynamischen Rezeptorbewegung nach Ligandenbindung führen, die in Zukunft zur Entwicklung spezifischerer Wirkstoffe am A1R und A2AR beitragen könnte.
KW  - Adenosinrezeptor
KW  - Fluoreszenz-Resonanz-Energie-Transfer
KW  - G-Protein gekoppelte Rezeptoren
KW  - Adenosine receptors
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125469
ER  - 
TY  - JOUR
A1  - Calebiro, Davide
A1  - Maiellaro, Isabella
T1  - cAMP signaling microdomains and their observation by optical methods
JF  - Frontiers in Cellular Neuroscience
N2  - The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains.
KW  - G protein-coupled receptor
KW  - cyclic AMP
KW  - signaling microdomain
KW  - fluorescence resonance energy transfer
KW  - neurons
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118252
SN  - 1662-5102
VL  - 8
ER  - 
TY  - THES
A1  - Kopp, Eva Katharina
T1  - Biotransformation and Toxicokinetics of Acrylamide in Humans
T1  - Biotransformation und Toxikokinetik von Acrylamid im Menschen
N2  - The widely used chemical acrylamide (AA) has been classified as a probable human carcinogen. This classification was based on positive results in rodent carcinogenicity studies as well as on a number of in vitro mutagenicity assays. In 2002, AA was discovered to be formed during the preparation of starch-containing foods. According to the latest FDA exposure assessment (2006), the average daily intake has been estimated from AA levels in foodstuffs and from nutritional habits to be around 0.4 µg/kg b.w. with a 90th percentile of 0.95 µg/kg b.w.. In children and adolescents however, the daily AA intake is about 1.5 times higher, due to lower body weight and differing consumption patterns. Apart from the diet, humans may be exposed to AA during the production or handling of monomeric AA, from AA residues in polyacrylamides, and from cigarette smoke. After oral administration, AA is readily absorbed and distributed throughout the organism. AA is metabolized to the reactive epoxide glycidamide (GA) via the CYP 450 isoenzyme CYP 2E1. Both, AA and GA are conjugated with glutathione. After enzymatic processing, the mercapturic acids N-Acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) as well as the regioisomers N-Acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) and N-Acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-L-cysteine (iso-GAMA) are excreted with urine. An additional pathway for the metabolic conversion of GA is the epoxide hydrolase mediated hydrolysis to the diol compound glyceramide. Following administration of AA at doses exceeding the daily dietary intake by a factor of 1000 - 6000 to human subjects, a new urinary metabolite was found, which could be identified as the S-oxide of AAMA (AAMA-sulfoxide). In general, data from animal studies are used for risk assessment of (potential) human carcinogens. However, inter-species differences in toxicodynamics or toxicokinetics, e.g. in biotransformation may lead to under- or overestimation of human risk. The objective of this work was to establish a highly specific and sensitive analytical method to quantify the major urinary metabolites of AA. Other aims apart from measurements concerning the human background exposure were the evaluation of biotransformation and toxicokinetics of AA in humans and rats after oral administration of 13C3-AA. The obtained data was intended to help avoid linear extrapolation from animal models for future risk assessments of AA carcinogenicity.
N2  - Die weitverbreitete Chemikalie Acrylamid (AA) wurde als möglicherweise krebserregend im Menschen eingestuft. Diese Klassifizierung beruhte auf positiven Ergebnissen aus Kanzerogenitätsstudien in Nagern sowie einer Reihe von in vitro Mutagenitätstests. Im Jahr 2002 wurde entdeckt, dass AA während der Zubereitung von stärkehaltigen Nahrungsmitteln entsteht. Nach den neuesten Expositionsabschätzungen der FDA (2006) wurde auf der Basis von AA-Gehalten in Lebensmitteln und Ernährungsgewohnheiten eine tägliche Aufnahme von etwa 0.4 µg/kg KG (Körpergewicht) errechnet. Die 90. Percentile lag bei 0.95 µg/kg KG. In Kindern und Heranwachsenden ist die tägliche AA Aufnahme jedoch um etwa Faktor 1.5 höher. Dies beruht auf einem vergleichsweise geringeren Körpergewicht und anderen Ernährungsvorlieben (175). Außer über die Nahrung können Menschen während der Produktion oder Verarbeitung von monomerem AA, über Rückstände in Polyacrylamiden und über Zigarettenrauch gegenüber AA exponiert sein. Nach oraler Gabe wird AA schnell resorbiert und im ganzen Organismus verteilt . AA wird über das Cytochrom P450 Isoenzym CYP 2E1 in das reaktive Epoxid Glycidamid (GA) umgewandelt. Sowohl AA als auch GA binden an Glutathion. Nach enzymatischer Verstoffwechslung werden die Mercaptursäuren N-Acetyl-S-(2-carbamoylethyl)-L-cystein (AAMA) sowie die Regioisomere N-Acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cystein (GAMA) und N-Acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cystein (iso-GAMA) mit dem Urin ausgeschieden. Ein weiterer Weg für die metabolische Umwandlung von GA ist die durch Epoxidhydrolase katalysierte Hydrolyse zum Diol Glyceramid. Nach der Verabreichung von AA an Menschen in Dosen, die die tägliche Aufnahme über die Nahrung um das 1000 bis 6000-fache überschritten, wurde ein neuer Metabolit im Urin entdeckt, der als das S-oxid von AAMA identifiziert werden konnte. Im Allgemeinen werden Daten aus Tierstudien für die Risikobewertung von (möglichen) Kanzerogenen im Menschen verwendet. Spezies-Unterschiede bezüglich der Biotransformation können demzufolge zu Unter- oder Überbewertung des Risikos für den Menschen führen. Das Ziel dieser Arbeit war es, eine hochspezifische und empfindliche analytische Methode für die Quantifizierung der wichtigsten Metaboliten von AA im Urin zu entwickeln. Neben Messungen bezüglich der Hintergrundbelastung im Menschen sollten Biotransformation und Toxikokinetik von AA in Menschen und Ratten nach oraler Gabe von 13C3-AA bestimmt werden. Die gewonnenen Daten sollten dazu beitragen, lineare Extrapolation ausgehend von Tiermodellen für zukünftige Risikoabschätzungen zu vermeiden.
KW  - Acrylamid
KW  - Metabolismus
KW  - Toxikokinetik
KW  - Risikobewertung
KW  - Speziesunterschiede
KW  - Acrylamide
KW  - Metabolism
KW  - Toxicokinetics
KW  - Risk Assessment
KW  - Species Differences
Y1  - 2009
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-37273
ER  - 
TY  - THES
A1  - Merkle, Sabine
T1  - Kardiale Caspase-1 ist ein proapoptotischer Induktor für Herzinsuffizienz
T1  - A role for Caspase-1 in heart failure
N2  - Herzmuskelschwäche (Herzinsuffizienz) ist in industralisierten Ländern neben malignen Erkrankungen noch immer die zweithäufigste Todesursache. Das Zusammenspiel der zu einer Herzmuskelschwäche führenden, molekularen Signalwege und die zugrunde liegenden Mechanismen sind trotz intensiver Forschung bisher noch weitgehend unverstanden. Ein detailierteres Verständnis der Entstehung einer Herzinsuffizienz könnte somit entscheidend zur Entwicklung innovativer Therapiestrategien beitragen. Mit Hilfe eines cDNA Expressions Arrays wurde die differentielle Genexpression in einem murinen Herzinsuffizienzmodell untersucht. Die Cysteinprotease Caspase-1 ist eines der in diesem Array identifizierten Kandidatengene. Die verstärkte Expression der Caspase-1 konnte zunächst sowohl für die murine als auch die humane Herzinsuffizienz verifiziert werden. Caspasen werden klassischerweise, ihrer Funktion entsprechend, in proinflammatorische und proapoptotische Caspasen unterteilt. In der Literatur ist Caspase-1 als ein über die Generierung von aktivem IL-1? und IL-18 wirkender, proinflammatorischer Mediator beschrieben. IL-? und IL-18 sind proinflammatorische Zytokine und als solche zentrale Mediatoren bei entzündlichen Prozessen. Während die Funktion der proapoptotischen Caspasen bei Herzmuskelschwäche weitgehend bekannt ist, ist unklar, welche Bedeutung der verstärkten kardialen Expression der Caspase-1 bei der Entstehung und Progression der Herzmuskelschwäche zukommt. Zur funktionellen Analyse der verstärkten Expression der Caspase-1 in vivo wurde die Caspase-1 in einem transgenen Mausmodell kardial überexprimiert. Herzen mit verstärkter Expression der Caspase-1 entwickelten ab dem vierten Lebensmonat morphologische und funktionelle Veränderungen, die charakteristisch für eine angehende Herzinsuffizienz sind. Dennoch war, trotz progressiver Kardiomyozytenhypertrophie und Myokardfibrose, zu keinem Zeitpunkt des untersuchten Zeitraumes (ein bis vierzehn Monate alt) eine Zunahme des Ventrikelgewichtes evident. Die funktionelle Analyse der Herzfunktion von neun Monate alten Caspase-1-transgenen Mäusen zeigte eine signifikante Einschränkung der Linksherzkontraktilität. Diese Herzinsuffizienz war schließlich auch makroskopisch an einer starken Linksherzdilatation als auch an einer Reduktion der linksventrikulären Wandstärke erkennbar. Ist die Induktion einer Herzinsuffizienz durch Caspase-1 als proinflammatorischer Mediator im Herzen zu erklären? In einer Reihe von Versuchen konnten keine Hinweise auf eine Induktion inflammatorischer Prozesse durch kardiale Caspase-1 festgestellt werden. Weder eine verstärkte Bildung der proinflammatorischen Zytokine IL-1? und IL-18, noch eine vermehrte Infiltration von Entzündungszellen oder eine verstärkte Proteinexpression weiterer Zytokine waren detektierbar. Ferner waren der oxidative Stresstatus und der nekrotische Gewebeuntergang im linksventrikulären Myokard Caspase-1-transgener Mäuse unverändert. Doch besonders im Herzmuskelgewebe junger Caspase-1-transgener Mäuse zeigte sich eine deutliche, mit fortschreitendem Alter bestehende Zunahme des apoptotischen Zelltods von Herzmuskelzellen. Diese direkte, proapoptotische Wirkung der kardialen Caspase-1 konnte ebenfalls in isolierten Kardiomyozyten von Caspase-1-transgenen Mäusen ex vivo und in vitro, nach adenoviraler Infektion von neonatalen Rattenkardiomyozyten (NRCM) mit Caspase-1, demonstriert werden. Durch Infektion von NRCM mit dem Caspase-1-exprimierenden Adenovirus (Adv-Casp-1) konnte eine selektive Aktivierung des intrinsischen apoptotischen Signaltransduktionsweges durch Caspase-1 nachgewiesen werden. Ist die endogen exprimierte Caspase-1 ein westenlicher Regulator kardialer Apoptose? In dem Kardiomyozytenapoptose-induzierenden Schädigungsmodell der Ischämie/Reperfusion waren die Caspase-1-defizienten Mäuse durch eine Reduktion der Kardiomyozytenapoptose um nahezu 75% gegenüber der Kontrollgruppe deutlich begünstigt. Des Weiteren zeigte die Ausschaltung der endogenen Caspase-1 in dem Herzinsuffizienzmodell des operativen Herzinfarkts eine deutliche Verminderung der reaktiven Kardiomyozytenhypertrophie und eine geringere Einschränkung der Herzkontraktilität. Diese Verbesserung des kardialen Phänotyps spiegelte sich ferner in einer reduzierten Sterblichkeit gegenüber der Kontrollgruppe wieder. Eine Inhibition der Caspase-1 stellt somit ein interessantes therapeutisches Target zur Prävention der Entwicklung einer Herzinsuffizienz dar.
N2  - To date, heart failure is still, besides malignant diseases, one of the most frequent causes of death in industrialized countries. Despite intensive research progress, the interplay of molecular signaling pathways and the underlying mechanisms leading to heart failure are still poorly understood. However, more detailed insights in the development of heart failure are essential to develope novel therapeutic strategies. A cDNA-array was performed to identify differentially expressed genes in a murine heart failure model. Thereby, the cystein protease caspase-1 was identified as a novel potential target gene for heart failure development. First, the increased expression of caspase-1 was confirmed for murine and human heart failure. Accordingly to their function, caspases are traditionally divided into proinflammatory and proapoptotic caspases. Due to the generation of active IL-1? and IL-18, caspase-1 has been described as a potent proinflammatory mediator. The proinflammatory cytokines IL-1? and IL-18 are central mediators during inflammatory processes. While the function of the proapoptotic caspases during heart failure has been intensively studied, the role of caspase-1 during the development and progession of heart failure is unknown. To study the functional effect of an increased caspase-1 expression in vivo, transgenic mice with cardiac specific expression of caspase-1 were generated. From four month onwards, hearts with increased transgenic expression of caspase-1 displayed morphological and functional changes characteristic for heart failure. Despite progressive cardiomyocyte hypertrophy and myocardial fibrosis, the ventricular weight was not increased at any age studied (one to fourteen months old). The analysis of cardiac function revealed a significant impairment of left ventricular contractility at nine months of age. Finally, the manifest heart failure was also seen in a massive dilatation of the left ventricle and a reduced left ventricular wall thickness. Is the induction of heart failure originating from caspase-1 being a proinflammatory mediator in the heart? In a set of experiments, no indices for a induction of proinflammatory processes via cardiac caspase-1 were seen. Neither increased formation of the proinflammatory cytokines IL-1? and IL-18 nor enhanced infiltration of inflammatory cells nor increased production of other cytokines was detectable. Moreover, there were no signs for a raised oxidative stress and necrotic cell death in left ventricular tissue of caspase-1-transgenic mice. Heart sections of young caspase-1-transgenic mice in particular, displayed a pronounced and robust increase in cardiomyocyte apoptosis that was persistent at all ages studied. The direct proapoptotic effect of cardiac caspase-1 was corroborated by assessing apoptosis in isolated caspase-1-transgenic cardiomyocytes ex vivo and by further in vitro experiments with neonatal rat cardiomyocytes (NRCM). Moreover, adenovirally expressed caspase-1 in NRCM induced a selective activation of the intrinsic apoptotic signaling pathway. Can the proapoptotic function of overexpressed caspase-1 also be seen for the endogenous caspase-1? Caspase-1-deficient mice were protected from ischemia-reperfusion induced cardiomyocyte apoptosis by almost 75%, as compared to the corresponding wild-type control group. In the setting of heart failure after myocardial infarction, the deletion of endogenous caspase-1 significantly ameliorated reactive hypertrophic growth of cardiac myocytes and partly preserved cardiac contractility. Furthermore, the improved cardiac phenotype was reflected in a reduced mortality of caspase-1-deficient mice after myocardial infarction. To conclude, the inhibition of caspase-1 represents a potential therapeutic strategy to inhibit development and progression of heart failure.
KW  - Herzinsuffizienz
KW  - Apoptosis
KW  - Caspase-1
KW  - Transgenes Mausmodell
KW  - Heart failure
KW  - Apoptosis
KW  - Caspase-1
KW  - Transgenic mice
Y1  - 2007
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-25938
ER  - 
TY  - THES
A1  - Kampfinger, Katja
T1  - Nachweis einer Mismatch-Reparatur-Defizienz in L5178Y Tk+/--3.7.2C-Mauslymphomzellen
T1  - Evidence of a mismatch repair deficiency in L5178Y Tk+/--3.7.2C mouse lymphoma cells
N2  - Die Entwicklung und Zulassung von Arzneimitteln sowie die Bewertung von Xenobio-tika erfordern eine Reihe von Testsystemen zur Toxizitätsermittlung. Für die Überprüfung der Gentoxizität stehen eine Vielzahl etablierter Testsysteme zur Verfügung, die oft auf Krebszelllinien basieren. Krebszelllinien haben jedoch die Eigenschaft, neben den für die Testung notwendigen Veränderungen weitere Veränderungen zu tragen, die zu Reaktionen führen können, wie sie in den Primärzellen des Organismus nicht auftreten. Daher ist die Kenntnis des genetischen Hintergrunds der verwendeten Krebszelllinien wertvoll, um Testergebnisse bewerten und gentoxische Risikopotentiale abschätzen zu können. Die Mauslymphomzelllinie L5178Y nimmt unter den auf Krebszellen basierenden Testsystemen eine besondere Stellung ein, da sie die weltweit in der Gentoxizi-tätsprüfung am häufigsten eingesetzte Zelllinie ist. In der vorliegenden Arbeit wurde in dieser Zellllinie eine Veränderung nachgewiesen, die das Mismatch-Reparatur-System (MMR-System) betrifft. Bei der MMR handelt es sich um einen Mechanismus, der daran beteiligt ist, die Integrität des Genoms zu gewährleisten. In MMR-profizienten Zellen werden Fehler in der DNA, die bei der Replikation, der homologen Rekombination oder durch äußere gentoxische Einwirkungen entstehen, entweder erkannt und repariert, oder die geschädigten Zellen werden durch die Induktion von Apoptosen eliminiert. Im Gegensatz dazu überleben MMR-defiziente Zellen trotz gravierender DNA-Schäden und akkumulieren diese. In der vorliegenden Arbeit wurde die Akkumulierung von Genomschäden bei L5178Y-Zellen als Reaktion auf Behandlung mit alkylierenden Agenzien beobachtet, während andere Vergleichszelllinien Apoptosen induzierten. Dieses Verhalten der L5178Y-Zellen, das in der Literatur bei MMR-defizienten Zellen für alkylierende Agenzien beschrieben ist, führte zu der Vermutung, dass die L5178Y-Zellen einen MMR-defizienten Phänotyp aufweisen. Dieser MMR-defiziente Phänotyp wurde durch gezielte Behandlung von L5178Y-Zellen und Zellen mit bekanntem MMR-Status mit dem alkylierenden Agenz MNNG und dem anschließenden Vergleich der Reaktionen geprüft und bestätigt. Der Ver-gleich erfolgte durch den Nachweis gentoxischer Effekte im Mikrokern-Test und im Comet Assay. Auf Proteinebene konnte für den gezeigten MMR-defizienten Phänotyp bei den drei wichtigsten, in die MMR involvierten Proteine, MLH1, MSH2 und MSH6 keine Ursa-che gefunden werden: Alle untersuchten Proteine zeigten eine Expression, die mit denen der MMR-profizienten Kontrollzelllinien vergleichbar war. Auf DNA-Ebene wurde durch die Analyse aller bekannter, in die MMR involvierter Gene durch die Sequenzierung der kodierenden Bereiche als wichtigste Verände-rung eine Insertions-Mutation (964(insC)) in pms2 gefunden. Diese führt nach 260 Aminosäuren zu einer Leserasterverschiebung und nach 313 Aminosäuren zu einem Abbruch der Aminosäuresequenz aufgrund eines Stop-Codons. Zwar ist somit die Information für den N-terminalen Bereich von PMS2, der die DNA-Bindedomäne und die ATP-ase aktiven Stellen beinhaltet, vorhanden, die für den C-Terminus hingegen, der für die Dimerisierung mit dem MMR-Protein MLH1 und damit für die Funktion essentiell ist, fehlt. Insgesamt wurde in dieser Arbeit gezeigt, dass die L5178Y-Zelllinie MMR-defizient ist. Mit der Insertions-Mutation (964(insC)) in pms2 wurde eine molekulare Ursache gefunden, die diese Defizienz erklären kann. Daraus folgt für den Einsatz der L5178Y-Zelllinie in Gentoxizitätstests, dass die Berücksichtigung ihrer MMR-Defizienz die Möglichkeit der Bewertung von Testergebnissen erheblich erweitern kann.
N2  - The development and approval of pharmaceuticals as well as the evaluation of xenobiotics require several test systems for the detection of genotoxicity. There is a number of established genotoxicity test systems, which are often based on cancer cell lines. In addition to mutations that are essential for genotoxicity testing, cancer cell lines may also carry mutations that might cause reactions not occurring in the primary cells of the organism. Therefore the knowledge of the genetic background of the cell line used is important for the evaluation of test results and the subsequent genotoxicity risk assessment. Among test systems that are based on cancer cells the mouse lymphoma cell line L5178Y adopts a very prominent position due to its worldwide application for genotoxicity testing. The dissertation on hand provides evidence that there are mutations in the L5178Y cell line that are related to the mismatch-repair system (MMR system). MMR participates in safeguarding the genomic integrity. In MMR-proficient cells, DNA defects that arise during replication, homologous recombination or as a result of genotoxic effects are either recognized and repaired or the genetically altered cells are eliminated by induction of apoptosis. MMR-deficient cells, however, survive despite serious DNA defects and accumulate them. The accumulation of DNA damage as result of treatment with alkylating agents had been observed in L5178Y cells while other cell lines had reacted with an induction of apoptosis. The induction of apoptosis after treatment with alkylating agents is described in the literature as a typical behaviour for MMR-deficient cells. From this the hypothesis was established, that L5178Y-cells might exhibit a MMR-deficient phenotype. This MMR-deficient phenotype was proven by selective treatment of L5178Y cells and cells with known MMR status with the alkylating agent MNNG followed by the subsequent comparison of the different reactions. The comparison was carried out by the detection of genotoxic effects using the micronucleus test and the comet assay. On the protein level there was not an indication that the observed MMR-deficiency was related to the the three most important MMR-proteins MSH2, MLH1 and MSH6: All proteins demonstrated expression levels that were comparable to the levels of the MMR-proficient control cells. On the DNA level, however, several mutations were detected by sequence analysis of the coding regions of all genes known to be involved in MMR. The most important among these mutations was an insertion mutation (964(insC)) in pms2, that caused a frameshift after 260 amino acids. By this frameshift, a stop-codon was introduced, leading to an interruption of the sequence after 313 amino acids. While the information of the N-terminal region of pms2 containing the DNA-binding domain as well as the ATPase active sites is still present, the information of the C-terminus is lost. This region is responsible for the dimerisation with the MMR-protein MLH1. Therefore, the MMR-function that is due to this complex, is missing. In conclusion, a MMR-deficiency of L5178Y cells was demonstrated. This MMR-deficiency is explained by an insertion-mutation in pms2 (964(insC)). Consideration of this MMR-deficiency enhances the meaningfulness of the evaluation of test results with L5178Y mouse lymphoma cells in risk assessment.
KW  - Maus
KW  - Zelle
KW  - L5178Y-Zellen
KW  - MMR-Reparatur
KW  - pms2
KW  - Genotoxizität
KW  - Alkylantien
KW  - L5178Y cells
KW  - mismatch repair
KW  - pms2
KW  - genotoxicity
KW  - alkylating agent
Y1  - 2007
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-26023
ER  - 
TY  - THES
A1  - Klenk, Johann Christoph
T1  - Effekte von Parathormon auf die Struktur und Komplexierung des Parathormonrezeptors 1
T1  - Effects of parathyroid hormone on the structure and complexation of parathyroid hormone receptor 1
N2  - Der Parathormonrezeptor Typ 1 (PTHR) ist ein G-Protein-gekoppelter Rezeptor der Gruppe 2 und wichtigster Regulator des Kalziumstoffwechsels. Im ersten Teil der Arbeit wurde eine neuartige posttranslationale Modifikation des PTHR in Form einer proteolytischen Spaltung der Ektodomäne identifiziert, charakterisiert und deren Regulation beschrieben. Nach langanhaltender Stimulation des Rezeptors mit Agonisten – aber nicht mit Antagonisten – wurde eine Massen- und Mengenzunahme des Rezeptorproteins beobachtet. Es konnte gezeigt werden, dass der Rezeptor unter basalen Bedingungen einer Spaltung unterliegt. Der Massenunterschied entsteht durch die proteolytische Spaltung der Ektodomäne des PTHR, was nachfolgend die Stabilität des Rezeptors beeinträchtigt. Die Spaltung erfolgte innerhalb einer unstrukturierten Schleife der Ektodomäne, welche die Bereiche für die Ligandenbindung miteinander verbindet. Hierbei handelt es sich um eine Region, die im Vergleich zu anderen Gruppe 2-Rezeptoren spezifisch für den PTHR ist. Das durch die Spaltung entstandene N-terminale Fragment bleibt durch eine Disulfidbrücke mit dem Transmembranteil des Rezeptors verbunden. Durch Versuche mit verschiedenen Proteaseinhibitoren konnte die verantwortliche Protease der Familie der zinkabhängigen extrazellulären Proteasen zugeordnet werden. Diese Ergebnisse beschreiben einen Mechanismus wie die Homoöstase des PTHR reguliert sein könnte. In einem zweiten Abschnitt wurde die Interaktion der Adapterproteine NHERF1 und beta-Arrestin2 mit dem PTHR untersucht. Beide Proteine interagierten unabhängig mit dem Rezeptor, wobei NHERF1 über eine PDZ-Domäne konstitutiv an den C-Terminus des Rezeptors bindet. beta-Arrestin2 hingegen bindet nach Aktivierung des Rezeptors und führt zur Desensitisierung des Rezeptors. Mittels biochemischer und mikroskopischer Methoden konnte gezeigt werden, dass beide Proteine gemeinsam einen ternären Komplex mit dem PTHR bilden, welcher durch die direkte Interaktion zwischen NHERF1 und beta-Arrestin2 vermittelt wird. Dies hat zur Folge, dass beta-Arrestin im basalen Zustand durch NHERF1 an den Rezeptor gekoppelt wird. Durch Analyse der Assoziationskinetik mittels Fluoreszenz-Resonanz-Energietransfer-Messungen zeigte sich, dass diese Kopplung zu einer zweifach erhöhten Rekrutierungsgeschwindigkeit von beta-Arrestin2 an den PTHR führt. Somit stellt unterstützt NHERF1 die beta-Arrestin2-vermittelte Desensitisierung des PTHR.
N2  - The Parathyroid hormone receptor type 1 (PTHR) belongs to the class 2 of G-protein coupled receptors (GPCRs) and is the main regulator of calcium homeostasis of the body. The first part of the dissertation describes a novel mechanism of receptor regulation based on a proteolytic cleavage of the receptor’s extracellular domain. Prolonged stimulation with PTH led to an apparent increase in molecular mass and in stability of the PTHR. Biochemical analysis of the receptor protein revealed that the PTHR undergoes posttranslational cleavage. Agonistic but not antagonistic PTH-peptides prevented this cleavage, thereby stabilizing the molecular mass and also increasing the half life of the receptor. The cleavage was shown to occur within an unstructured stretch of the extracellular domain of the receptor, which connects two parts required for ligand binding and which is unique in the PTHR amongst all class 2 GPCRs. The cleaved N-terminal fragment was further connected by a disulfide bridge and could only be released by reducing agents. By testing a panel of different protease inhibitors, a protease belonging to the family of zinc-dependent metalloproteases could be identified to be responsible for the PTHR cleavage. Thus, these findings describe a new mechanism how PTHR homeostasis may be regulated. In the second part, the interaction between the adaptor proteins NHERF1 and beta-arrestin2 with the PTHR was assessed. Both proteins interacted independently with the receptor. While NHERF1 formed a constitutive interaction with the PTHR C-terminus, beta-arrestin2-binding required activation of the receptor. Using biochemical and microscopic methods it was shown that both proteins formed a ternary complex with the receptor. This complex was mediated by a direct interaction between NHERF1 and beta-arrestin2 which has been identified in this work. As a consequence, NHERF1 leads to a coupling of beta-arrestin2 close to the PTHR. Association kinetics of beta-arrestin2 with the PTHR measured by fluorescent resonance energy transfer were two-fold increased in the presence of NHERF, suggesting that the ternary complex facilitates the desensitization of the PTHR by beta-arrestin2.
KW  - Parathormon
KW  - Proteolyse
KW  - Endokrinologie
KW  - Calcium
KW  - Rezeptor
KW  - Retinales S-Antigen
KW  - GPCR
KW  - Matrix-Metalloprotease
KW  - NHERF
KW  - Signaltransduktion
KW  - G-Protein
KW  - GPCR
KW  - Matrix-Metalloproteinase
KW  - NHERF
KW  - signal transduction
KW  - G-protein
Y1  - 2009
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47288
ER  - 
TY  - THES
A1  - Schewe, Victoria Kristina
T1  - Mikrokernbildung in Mundschleimhautzellen von Patienten mit Kopf-Hals-Tumoren während Radio-/Radiochemotherapie
T1  - Micronucleus formation kinetics in buccal mucosa cells of head and neck cancer patients undergoing radio-/radiochemotherapy
N2  - Das Ziel der vorliegenden Arbeit war die Mikrokernbildung in Mundschleimhautzellen von 35 Patienten mit Kopf-Hals-Tumoren während einer sechswöchigen Radio-/Radiochemotherapie und sechs Wochen danach darzustellen. Die Ergebnisse der vorliegenden Arbeit zeigten, dass Patienten mit Kopf-Hals-Tumoren im Vergleich zu gesunden Probanden erhöhte Mikrokernraten aufwiesen. Ebenfalls konnte gezeigt werden, dass es zu einer vermehrten Bildung von Mikrokernen während einer sechswöchigen Radio-/Radiochemotherapie kam. Nach Therapiebeendigung sanken die Werte nach drei bis sechs Wochen und lagen unter dem Ausgangswert, in dem Bereich von spontan entstehenden Mikrokernen. In Bezug auf die Tumorgröße konnte nur in der zweiten Woche ein signifikanter Unterschied in der Mikrokernrate zwischen T1- und T4-Stadium beobachtet werden. Es konnte keine Korrelation zwischen einer zusätzlich verabreichten Chemotherapie, Grading des Tumors, Alter sowie Geschlecht der Patienten und einem Anstieg der Mikrokernrate festgestellt werden.
N2  - In the present Study, normal tissue buccal mucosa cells from 35 patients with head and neck cancer were analyzed for formation of micronuclei during a 6-week radio-/radiochemotherapy and 6 weeks afterwards. The results showed that patients with head and neck cancer had increased micronucleus rates compared to healthy test persons. It could also be observed that there was an increased formation of micronuclei during a 6-week radio-/radiochemotherapy. After the end of therapy, the values decreased after three to six weeks. The tumor size showed in the second week a significant difference in the micronucleus rate between T1 and T4 stage. Age, gender, and tumor stage did not have an influence on the micronuclei formation kinetics.
KW  - Kleinkern
KW  - Mundschleimhaut
KW  - Strahlentherapie
KW  - Chemotherapie
KW  - Hals-Nasen-Ohren-Tumor
KW  - Mikrokern
KW  - micronucleus
KW  - Kopf-Hals-Tumor
KW  - head and neck cancer
KW  - Mundschleimhaut
KW  - buccal mucosa
KW  - Bestrahlung
KW  - radiation
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215354
ER  - 
TY  - THES
A1  - Witzinger, Linda
T1  - Rolle der Pyridoxal 5´-Phosphat Phosphatase PDXP im Vitamin B6-Metabolismus muriner Erythrozyten und Hippocampi
T1  - Role of the pyridoxal 5´-phosphate phosphatase PDXP in the vitamin B6 metabolism of murine red blood cells and hippocampi
N2  - Die Phosphatase PDXP (auch bekannt als Chronophin) gehört zur Familie der HAD Phosphatasen, einer ubiquitär exprimierten Enzymklasse mit wichtigen physiologischen Funktionen. PDXP zeigt Phosphatase-Aktivität gegenüber seinem Substrat Pyridoxal 5´-Phosphat (PLP), der aktivierten Form von Vitamin B6. PDXP-defiziente Mäuse (Knockout-Mäuse) weisen im Vergleich zu Wildtypen verdoppelte PLP-Konzentrationen in Erythrozyten sowie im Gesamthirn auf. Vermutlich kommt PDXP daher eine wichtige Funktion in Erythrozyten und im Hirn zu. Ziel dieser Arbeit war es, erste Einblicke in diese Funktion(en) von PDXP zu erlangen. 
Hierzu wurden HPLC-basierte Analysen der erythrozytären PLP-Konzentrationen in Wildtyp- sowie PDXP-defizienten Mäusen durchgeführt. Dabei ließen sich die rund doppelt so hohen erythrozytären PLP-Level in den KO-Mäusen bestätigen. Zudem ist es gelungen, eine Methode zur Messung der endogenen Phosphatase-Aktivität von PDXP in Erythrozytenlysaten zu etablieren. So konnte im Wildtyp anhand der Verringerung der PLP-Konzentrationen pro Zeiteinheit eine erythrozytäre PDXP-Aktivität nachgewiesen werden. Dazu waren die Inkubation mit Pyridoxin, sowie die Anwendung eines Inhibitors der PDXK notwendig. Eine bis dato vermutete Funktion der PDXP, zur Mobilisation von erythrozytärem PLP während Fastenzeiten, konnte ausgeschlossen werden. So zeigte der Vergleich der erythrozytären PLP-Konzentrationen aus gefasteten mit normal gefütterten Tieren in beiden Genotypen exakt dieselbe prozentuale PLP-Verringerung. Während Nahrungszufuhr ließ sich jedoch eine Funktion der Phosphatase PDXP als „Converter“ von Pyridoxin zu Pyridoxal erkennen. Ausgehend von PN konnte im Wildtyp (über die Zwischenprodukte PNP und PLP) eine PDXP-abhängige Dephosphorylierung von PLP zu PL erfolgen. So wies der Wildtyp eine rund vierfach höhere PL-Produktion auf, verglichen mit der PDXP-defizienten Maus. Die Phosphatase PDXP erwies sich als essenziell für die erythrozytäre Konversion von Pyridoxin zu Pyridoxal. Dadurch erreicht der Organismus eine metabolische Flexibilität, die ihn bis zu einem gewissen Grad unabhängig von der Nahrungsauswahl macht. Zudem können Zellen oder Organe, denen durch das Fehlen der PNPO, die Konversion zu PLP nicht möglich ist, mit PL versorgt werden. 
Aus der hohen Reaktivität von PLP mit umliegenden Nucleophilen ergibt sich eine gewisse Problematik für die Zelle im Umgang mit freiem PLP. So liegt der Großteil des erythrozytären PLPs gebunden an Proteine (vor allem Hämoglobin) vor. Anhand von Filtern (MWCO, 3000) ließ sich zwischen der hier definiert als „freien“ und der „gebundenen“ Form von PLP differenzieren. So konnten erste Erkenntnisse zur Rolle von PDXP als Determinator freier PLP-Konzentrationen in Erythrozyten und insbesondere im Hippocampus erlangt werden. Im Hippocampus ergaben sich insgesamt deutlich höhere Konzentrationen an freiem PLP als in den Erythrozyten und es bestand zudem ein Unterschied zwischen den Genotypen. So wiesen die KO-Mäuse ~1/3 höhere freie PLP-Konzentrationen im Vergleich zu den Wildtypen auf. Schließlich konnte ein Effekt des Tieralters auf den PLP-Metabolismus festgestellt werden. Sowohl in den Erythrozyten als auch im Hippocampus ergaben sich alterskorrelierte Änderungen ihrer PLP-Konzentrationen. Zudem zeigten Western Blot Analysen altersbedingte Unterschiede ihrer Vitamin B6-Enzymexpressionen. So wiesen ältere Wildtypen im Hippocampus eine fünffach erhöhte PDXP-Expression verglichen mit jüngeren Tieren auf. In den Erythrozytenlysaten hingegen zeigten ältere Tiere beider Genotypen eine rund vierfach geringere PNPO-Expression gegenüber jüngeren Tieren. Die mit dem Alter eintretende physiologische Verringerung der erythrozytären PNPO-Expression würde somit für den Organismus einen Verlust seiner metabolischen Flexibilität bedeuten, die mit der Konversion von PN zu PL einhergeht.
N2  - The phosphatase PDXP, also called Chronophin, is a member of the ubiquitously expressed HAD-phosphatases, which have some important physiological functions in the organism. Its substrate pyridoxal 5´-phosphate (PLP) is the active form of vita-min B6, an important cofactor of several reactions. PDXP-deficient mice (KO-mice) have PLP-concentrations in erythrocytes and in the whole brain twice as high as wildtype mice. It is assumed that PDXP therefore has an important function in erythrocytes and in the brain. The aim of the study was to gain initial insights into these functions of PDXP. 
For this purpose, HPLC-based analyses of the PLP-concentrations in erythrocytes from WT- and KO-mice were carried out. The doubled PLP-levels in the RBCs of KO-mice could be confirmed. In addition, a method for measuring the endogenous phosphatase activity of PDXP in red cell lysates was established. The activity of PDXP could be measured by the reduction of its substrate PLP over time. This required the incubation with pyridoxine and the inhibition of PDXK by ginkgotoxine. An assumed function of PDXP in mobilization of PL(P) from the erythrocytes in fasting conditions could be ruled out. Therefore, a comparison between the PLP-concentrations in RBCs of fasted mice with normal fed ones was done. Surprisingly the fasted KO-mice showed the same percentaged decrease of cellular PLP-level as the fasted WT-mice. During vitamin B6 intake however, a function of PDXP as being a “converter” of pyridoxine to pyridoxal was found. Starting with PN, a PDXP-mediated dephosphorylation from PLP to PL could take place in the wildtype mice (via the intermediate steps PNP and PLP). Consequently, the WT´s production of PL quadrupled compared to the KO´s. PDXP turned out to be essential for the conversion of pyridoxine to pyridoxal in erythrocytes. This conversion confers some metabolic flexibility to the organism and to a certain extent makes it independent of the choice of food. Moreover, cells and organs, that due to the absence of PNPO cannot produce PL(P) themselves, can be provided via erythrocytes. 
The high reactivity of PLP with surrounding nucleophiles poses a certain problem for the cell in dealing with free PLP. The majority of the PLP in RBCs is bound to proteins (primarily hemoglobin). It was distinguished between the here termed “free” PLP and the bound PLP by using filter devices with a MWCO at 3 kDa. First insights could be gained about PDXP as a determinant of free PLP-levels in erythrocytes and hippocampus. The amount of free PLP in the hippocampus was significantly higher than in the RBCs. Additionally, the hippocampus showed some differences in the con¬centration of free PLP between WT- and KO-mice. The level of free PLP in PDXP deficient mice was one third higher than in wildtype mice. Finally, some correlation between the age of the mice and their PLP-metabolism was found. The results revealed changes of the PLP-concentrations with age in the RBCs and the hippocampus. Moreover, western blot analyses showed some age-related differences in the expression of vitamin B6 enzymes. In the hippocampus older wildtype mice showed a quintupled expression of PDXP compared to younger ones. However, western blot analyses of red blood cell lysates from older animals revealed a lower expression of PNPO by a factor of four. For the organism this physiological reduction of its PNPO expression with age would mean a loss of metabolic flexibility, that is accompanied by the conversion from PN to PL.
KW  - Vitamin B6
KW  - Vitamin-B6-Stoffwechsel
KW  - Pyridoxalphosphat
KW  - Erythrozyt
KW  - Phosphatasen
KW  - PDXP
KW  - PLP
KW  - HAD-Phosphatasen
KW  - Vitamin B6 Metabolismus
KW  - pyridoxal phosphate
KW  - red blood cells
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216546
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  - Werthmann, Ruth
T1  - Echtzeit-Untersuchungen zur Thrombin-abhängigen Änderung der cAMP-Konzentration in lebenden Endothelzellen
T1  - Real-time monitoring of thrombin-dependent changes of the cAMP concentration in living endothelial cells
N2  - Das Endothel bildet eine einschichtige Zellbarriere zwischen Blut und interstitiellem Gewebe, deren Durchlässigkeit entscheidend durch die sekundären Botenstoffe Ca2+ und cAMP reguliert wird. Während Ca2+ durch eine verstärkte Kontraktion der Endothelzellen die Permeabilität erhöht, fördert cAMP die Adhäsion der Zellen und unterstützt somit die Barrierefunktion. Es ist bekannt, dass Thrombin durch einen Anstieg der intrazellulären Ca2+-Konzentration und vermutlich auch durch eine Hemmung der cAMP-Konzentration zu einer Permeabilitätserhöhung führt. Ziel dieser Arbeit war es, Thrombin-induzierte Änderungen der cAMP-Konzentration in Echtzeit in lebenden Endothelzellen mittels Fluorescence-Resonance-Energy-Transfer (FRET) zu untersuchen. Hierfür wurden Human-Umbilical-Vein-Endothelial-Cells (HUVECs) mit dem FRET-basierten cAMP-Sensor Epac1-camps transfiziert. Die Bindung von cAMP an Epac1-camps führt zu einer Konformationsänderung des Sensors und damit zu einer Abschwächung des FRET. Mit Hilfe dieses Sensors kann die cAMP-Konzentration mit hoher zeitlicher Auflösung in einzelnen lebenden Zellen gemessen werden. Untersucht wurde der Effekt von Thrombin auf die cAMP-Konzentration in Endothelzellen, deren cAMP-Konzentration durch Stimulierung endogener &#946;-Rezeptoren erhöht war. Thrombin erniedrigte Ca2+-abhängig die cAMP-Konzentration um ca. 30 %. Dieser Abfall der cAMP-Konzentration folgte zeitlich verzögert dem Thrombin-induzierten Ca2+-Signal. Die cAMP-Konzentration erreichte ca. 30 s nach der Thrombinzugabe ein Minimum und stieg danach wieder an. Durch die Herunterregulierung der durch Ca2+ direkt inhibierten Adenylatzyklase 6 (AC6) mittels siRNA wurde die Thrombin-induzierte Abnahme der cAMP-Konzentration vollständig aufgehoben. Dies bestätigte, dass Thrombin durch die Ca2+-vermittelte Inhibierung der AC6 eine Abnahme der cAMP-Konzentration verursacht. Ohne &#946;-adrenerge Stimulation führte die Applikation von Thrombin zu einem langsamen Anstieg der cAMP-Konzentration, der mehrere Minuten anhielt. Dieser cAMP-Konzentrationsanstieg beruhte auf der Ca2+-abhängigen Aktivierung der Phospholipase A2 (PLA2). Diese setzt Arachidonsäure aus Membranphospholipiden frei, die als Substrat für die Synthese verschiedener Prostaglandine dient. Durch die pharmakologische Beeinflussung von Zyklooxygenasen und Prostazyklinrezeptoren konnte gezeigt werden, dass die Synthese von Prostazyklin und die anschließende Stimulation Gs-gekoppelter Prostazyklinrezeptoren zum Thrombin-induzierten Anstieg der cAMP-Konzentration führte. Da die Physiologie der Endothelzellen im Gefäß stark von Faktoren aus der unmittelbaren Umgebung beeinflusst wird, ist die Messung der Änderungen der cAMP-Konzentration in Endothelzellen, die sich innerhalb eines Gewebes befinden, von sehr großer Bedeutung. Deshalb war die Generierung transgener Mäuse mit einer gewebespezifischen Expression des FRET-Sensors Epac1-camps in Endothelzellen ein weiteres Ziel dieser Arbeit. Durch Anwendung eines Cre-Rekombinase/loxP-Ansatzes konnten transgene Mäuse generiert werden, die Epac1-camps spezifisch in Endothelzellen exprimierten. An isolierten pulmonären Endothelzellen konnte die Funktionalität des transgen exprimierten Sensors Epac1-camps nachgewiesen werden. Die Echtzeitmessung der Thrombin-induzierten Änderungen der cAMP-Konzentration verdeutlichte ein zeitlich sehr komplexes Wechselspiel zwischen Ca2+- und cAMP-Signalen, das die Barrierefunktion des Endothels maßgeblich beeinflussen wird. Die transgene Expression von Epac1-camps in Endothelzellen ermöglicht in Zukunft die Untersuchung der Thrombin-verursachten Änderungen der cAMP-Konzentration und der Permeabilität innerhalb eines intakten Gefäßes.
N2  - Endothelial cells form a semi permeable barrier between blood and interstitial tissues. The permeability of this barrier is mainly regulated by the second messengers Ca2+ and cAMP. While Ca2+ increases the permeability by inducing cell contraction, cAMP increases the adherence of the cells and, thereby, supports the barrier function. The Ca2+-elevating agent thrombin was demonstrated to increase endothelial permeability and to decrease cAMP levels. The aim of this thesis was to investigate thrombin-induced changes of the cAMP concentration in real time in living endothelial cells via fluorescence resonance energy transfer (FRET). Therefore, human umbilical vein endothelial cells (HUVECs) were transfected with the FRET-based cAMP sensor Epac1-camps. Binding of cAMP to the binding domain of Epac1-camps induces a conformational change of the sensor that results in a decrease of FRET. With help of this sensor, changes in cAMP concentration can be monitored with high temporal resolution. First, the influence of thrombin on cAMP levels was investigated after elevating cAMP levels by stimulation of &#946;-adrenergic receptors. Thrombin led to a Ca2+-dependent decrease of cAMP levels by approximately 30 %. The decrease of cAMP levels was delayed compared to the thrombin-induced Ca2+ signal. This decrease was also transient and reached a minimum value 30 s after thrombin stimulation. A siRNA-mediated downregulation of the Ca2+-inhibited adenylyl cyclase 6 (AC6) completely abolished the thrombin-induced decrease of cAMP concentration. This provided the first direct evidence that the Ca2+-mediated inhibition of AC6 accounts for the thrombin-induced decrease in cAMP levels. In the absence of a &#946;-adrenergic-mediated increase of cAMP concentration, thrombin led to a slow increase in cAMP concentration that lasted for several minutes. This increase in cAMP concentration was caused by the Ca2+-dependent activation of phospholipase A2 (PLA2). PLA2 releases arachidonic acid, which represents the substrate for prostaglandin synthesis. It was confirmed by pharmacological interference of cyclooxygenases and prostacyclin receptors that the synthesis of prostacyclin and subsequent stimulation of Gs-protein-coupled prostacyclin receptors caused the thrombin-induced increase in cAMP concentration. The real time monitoring of changes in cAMP concentration in endothelial cells within the vascular system is highly important as the physiology of endothelial cells in vivo is strongly influenced by factors contained in the surrounding blood or tissue. Therefore, a further aim of this thesis was the generation of transgenic mice expressing the FRET-based sensor Epac1-camps specifically in endothelial cells. Using a Cre-recombinase/loxP-approach transgenic mice were generated that specifically expressed Epac1-camps in endothelial cells, and the functionality of the transgenic sensor was proven in isolated pulmonary endothelial cells. Real time monitoring of thrombin-induced changes of cAMP concentration in endothelial cells revealed a temporally complex crosstalk between Ca2+ and cAMP signals that will affect endothelial barrier function. The transgenic expression of Epac1-camps opens the door for the investigation of thrombin-induced changes of cAMP levels and of endothelial permeability within intact vessels.
KW  - Cyclo-AMP
KW  - Endothelzelle
KW  - Fluoreszenz-Resonanz-Energie-Transfer
KW  - Thrombin
KW  - cyclo-AMP
KW  - endothelial cells
KW  - fluorescence resonance energy transfer
KW  - thrombin
Y1  - 2009
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46066
ER  - 
TY  - THES
A1  - Zürn, Alexander
T1  - Spezifische Markierungsverfahren von Rezeptoren mit kleinen Fluorophoren zur Analyse der Rezeptoraktivierung mittels FRET
T1  - Specific labelling techniques with small fluorophores for visualzing ligandselective conformations on receptors with FRET
N2  - Es gibt viele Hinweise, dass G-Protein-gekoppelte Rezeptoren bei ihrer Aktivierung durch einen Agonisten ligandenselektive Konformationen eingehen. Ein tatsächlichen Beleg hierfür konnte bisher in lebenden Zellen noch nicht erbracht werden. Zu diesem Zweck wurde in dieser Arbeit ein Fluoreszenz-Resonanz-Energie-Transfer (FRET)-basierter Ansatz gewählt, um ligandenselektive Konformationen in der dritten intrazellulären Schleife des &#945;2a-adrenergen Rezeptors (&#945;2a-AR) in lebenden Zellen darzustellen. Dazu wurden Rezeptorsensoren erstellt, welche jeweils ein CFP am Ende des C-Terminus trugen und in der dritten intrazellulären Schleife an verschiedenen Stellen mit einem Tetracysteinmotiv versehen wurden. Drei Konstrukte wurden verglichen, die das Tetracysteinmotiv N-terminal in der Nähe der Transmembrandomäne V (I3-N), in der Mitte der dritten intrazellulären Schleife (I3-M) beziehungsweise C-terminal in der Nähe der Transmembrandomäne VI (I3-C) trugen. Die drei Rezeptorsensoren unterschieden sich hinsichtlich ihrer Ligandenbindung sowie ihrer G-Proteinaktivierung nicht vom Wildtyp &#945;2a-AR. Durch das Tetracysteinmotiv ist es möglich, den Rezeptor spezifisch mit dem niedermolekularen Fluorophor FlAsH (fluorescein arsenical hairpin binder) zu markieren, welcher als Akzeptor für den Donor CFP in FRET-Experimenten dient. Die Änderung des FRET-Signals zwischen den beiden Fluorophoren, das durch den vollen Agonist Norepinephrin ausgelöst wurde, war bei allen drei Rezeptorsensoren vergleichbar. Der starke partielle Agonist Clonidin war ebenfalls in der Lage, in allen drei Konstrukten ein ähnliches FRET-Signal hervorzurufen. Dagegen zeigte der partielle Agonist Dopamin an dem Konstrukt I3-N ein signifikant schwächeres Signal, als an I3-C. Die schwachen partiellen Agonisten Octopamin und Norphenephrin konnten an den Konstrukten I3-N und I3-M keine Änderung des FRET-Signals bewirken, wobei an I3-C eine deutliche Signaländerung detektiert wurde. Dies legt nahe, dass die Transmembrandomäne V bei der Aktivierung des Rezeptors eine kleinere Bewegung eingeht als die Transmembrandomäne VI, und bestätigt damit ein auf Röntgenstrukturanlysen basierendes Modell der Rezeptorbewegung. Außerdem wurden die Aktivierungskinetiken für die Agonisten Norepinephrin und Dopamin verglichen. Hierbei konnte gezeigt werden, dass die durch Norepinephrin ausgelöste Bewegung an allen beobachteten Punkten gleich schnell war. Im Gegensatz dazu aktivierte Dopamin I3-C und I3-M ca. 1,5-mal langsamer, als Norepinephrin. Für das I3-N Konstrukt wurde sogar eine 3-mal langsamere Aktivierung gemessen. Diese Daten zeigen, dass unterschiedliche Agonisten in der dritten intrazellulären Schleife spezifische Konformationen auslösen können. Die Untersuchungen zur Rezeptorbewegung im ersten Teil dieser Arbeit wurde mit dem kleinen Fluorophor FlAsH in Kombination mit einer großen GFP-Variante durchgeführt. Im zweiten Teil dieser Arbeit wurde eine Methode entwickelt, bei der es möglich ist Proteine spezifisch mit beiden kleinen Fluorophoren FlAsH und ReAsH in einer lebenden Zelle zu markieren. Hierfür wurden zwei Tetracysteinmotive, CCPGCC und FLNCCPGCCMEP, gewählt, an die beide kleine Fluorophore kovalent binden. Durch Verdrängungsexperimente mit BAL konnte gezeigt werden, dass FlAsH für beide Motive eine dreifach höhere Affinität besitzt, als ReAsH. Dabei besitzt das FLNCCPGCCMEP-Motiv jedoch eine dreifach höhere Affinität zu dem jeweiligen Fluorophor besitzt als CCPGCC. Durch Ausnutzung dieser Affinitätsunterschiede konnte ein Protokoll entwickelt werden, mit dem es möglich ist, beide Motive in einer Zelle zu markieren. Dabei werden zunächst beide Motive mit ReAsH markiert. Durch anschließendes Waschen mit einer geeigneten Konzentration von BAL wird das ReAsH ausschließlich von der CCPGCC-Sequenz verdrängt, wohingegen die FLNCCPGCCMEP-Sequenz mit ReAsH markiert bleibt. Die nun unbesetzte CCPGCC-Sequenz kann dann anschließend mit FlAsH markiert werden, ohne dabei die Bindung des ReAsH an die FLNCCPGCCMEP-Sequenz zu beeinflussen. Um die Funktionalität dieses Protokolls zu überprüfen, sollten zwei verschiedene Proteine mit unterschiedlicher subzellulärer Lokalisation in einer lebenden Zelle spezifisch mit jeweils einem kleinen Fluorophor markiert werden. Hierzu wurden ein PTH-Rezeptor, in dem im C-Terminus die FLNCCPGCCMEP-Sequenz eingebracht wurde, mit ReAsH und ein &#946;-Arrestin-2, dem die CCPGCC-Sequenz eingebracht wurde, in Zellen co-exprimiert und gemäß dem Protokoll mit FlAsH und ReAsH markiert. Beide Proteine konnten spezifisch markiert werden, wobei der mit ReAsH markierte PTH-Rezeptor eine deutliche Lokalisation in der Zellmembran zeigte. Durch sequentielle Exzitation konnte in der gleichen Zelle das zytosolisch lokalisierte, mit FlAsH markierte &#946;-Arrestin-2 detektiert werden. Wurden die so markierten Zellen mir 1 µM PTH stimuliert, wurde das FlAsH-markierte &#946;-Arrestin-2 an die Zellmembran rekrutiert. Somit konnte durch die Entwicklung dieses Protokolls eine duale spezifische Markierung von Proteinen mit zwei kleinen Fluorophoren zu innerhalb einer Zelle erreicht werden.
N2  - Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in &#61537;2A-adrenergic receptors (&#61537;2A-AR) in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein (CFP) and with fluorescein arsenical hairpin binder (FlAsH) bound at a tetracysteine-motif at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties compared to the wildtype-&#61537;2A-AR. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET-changes for all three constructs. The strong partial agonist clonidine induced partial FRET-changes for all constructs. The partial agonist dopamine envoked a significantly weaker FRET-signal in I3-N than in I3-C. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C, but no change in I3-M and I3-N. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor. The kinetics of the receptor activation was compared between dopamine and norepinephrine. The kinetics for norepinephrine were similar for all three constructs. Dopamine-induced FRET-signals were &#8776;1.5-fold slower than those for norepinephrine in I3-C and I3-M, but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. Specific labeling of proteins in living cells with two different molecular probes would be an important further development for multiparameter imaging of cellular functions. Here we report a strategy to selectively label two different proteins in living cells with two different fluorophores, FlAsH and ReAsH. Recently improved tetracysteine binding motifs have been described to selectively bind FlAsH or ReAsH. We compared the six amino acid motif CCPGCC and the twelve amino acid motif FLNCCPGCCMEP with respect to their affinity for FlAsH and ReAsH. For both fluorophores, we observed a 3-fold higher affinity for the FLNCCPGCCMEP motif compared to CCPGCC, when washed off with BAL (british anit lewisite; 2,3-Dimercaptopropanol). For both target sequences, FlAsH showed more stable interactions than ReAsH. Based on these observations, we developed a protocol to demonstrate selective labeling of different proteins in the same cell. We used two target proteins that are localized in different cellular compartments. As model proteins we chose a plasmamembrane localized G protein-coupled receptor for PTH (PTH-receptor) which was C-terminally modified with the FLNCCPGCCMEP motif for labeling with ReAsH, and the cytosolic &#61538;-arrestin-2 protein which was C-terminally modified with the CCPGCC motif for labeling with FlAsH. Both proteins were specifically labelled with the respective Fluorophores and &#61538;-arrestin-2 will translocate to the plasmamembrane upon agonist stimulation of the PTH receptor. Taken together our data demonstrate that FlAsH and ReAsH can be used for orthogonal labeling to different binding motifs fused to different target proteins in living cells.
KW  - Fluoreszenz-Resonanz-Energie-Transfer
KW  - Fluoreszenz
KW  - Fluoreszenz <Motiv>
KW  - Wirkstoff-Rezeptor-Bindung
KW  - GPCR
KW  - Konformationsänderung
KW  - ligandenselektive Konformationen
KW  - Mikroskopie
KW  - FlAsH
KW  - ReAsH
KW  - Tetracystein-Motivee
KW  - GPCR
KW  - lignaselective conformations
KW  - FlAsH
KW  - ReAsH
KW  - Tetracystein-Motive
Y1  - 2009
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-35961
ER  - 
TY  - THES
A1  - Rached, Eva Katharina
T1  - Neue Ansätze zur Entwicklung von Alternativmethoden zur Prüfung auf chronische Nierentoxizität
N2  - Die Niere ist eines der wichtigsten Zielorgane für Toxizität, allerdings stellt die frühzeitige Erkennung einer Nierenschädigung und/oder kanzerogenen Wirkung infolge einer wiederholten Exposition gegenüber toxischen Verbindungen ein großes Problem dar, da traditionelle Marker für Nierenfunktionsstörungen wenig empfindlich sind. Daher ist es notwendig, verbesserte Testmethoden (Alternativmethoden) zur Prüfung auf chronische Nierentoxizität zu entwickeln. Ziel dieser Arbeit war es daher, mögliche Alternativmethoden zur Prüfung auf Nephrotoxizität nach wiederholter Exposition zu untersuchen. Zum einen wurden dazu in einem in vivo-Modell für chronische Nierentoxizität neue Biomarker für Stress und Gewebeschädigung untersucht, deren erhöhte Genexpression in mehreren Modellen für akute Schädigung des Nierengewebes gezeigt wurde, einschließlich kidney injury molecule-1 (KIM-1), Lipocalin-2 (LCN2), Clusterin (CLU), Osteopontin (OPN), tissue inhibitor of metalloproteinases-1 (TIMP-1), Vimentin (VIM) und Hämoxygenase-1 (HO-1). Diese Marker wurden nachfolgend auch in einem zellkulturbasierten in vitro-Modell untersucht. Ein weiterer Teil der Arbeit befasste sich mit Veränderungen der Zellteilung als möglicher Marker für die Früherkennung kanzerogener Effekte. Das in vivo-Modell bestand in einer Studie in männlichen F344/N-Ratten, die 14, 28 oder 90 Tage oral mit 0, 21, 70 oder 210 µg/kg Körpergewicht (KG) Ochratoxin A (OTA) behandelt wurden. OTA ist ein Mykotoxin, das in Ratten bei wiederholter Gabe eine Nierenschädigung und Nierenkrebs verursacht. Die Analyse der mRNA-Expression der neuen Biomarker in Nierengewebe zeigte bei Tieren, die mit 70 oder 210 µg/kg KG behandelt wurden, eine frühzeitige, zeit- und dosisabhängige Induktion von KIM-1, LCN2, TIMP-1, OPN und CLU, die mit histopathologischen Veränderungen in Form von Zelldegeneration und Regeneration einherging und das Fortschreiten der Schädigung gut widerspiegelte. Auch die mRNA-Expression von HO 1 und VIM wurde durch OTA moduliert, allerdings war eine Erhöhung nicht zu allen Zeitpunkten zu messen bzw. trat nicht so früh auf wie bei den anderen Markern. Effekte auf traditionelle Marker für Nephrotoxizität (Serum-Kreatinin, N-Acetyl-&#946;-D-glucosaminidase und &#947;-Glutamyltransferase im Urin) wurden im Vergleich zu den neuen Markern zu einem späteren Zeitpunkt und zumeist nur in der Hochdosisgruppe festgestellt. Zusätzlich zu den Effekten auf die Genexpression konnte in den Zielzellen von OTA im proximalen Tubulusepithel eine erhöhte Proteinexpression von KIM-1, CLU, OPN und VIM gezeigt werden; nur für KIM-1 wurde allerdings auch im Urin eine erhöhte Konzentration nachgewiesen, die mit den Effekten auf die mRNA- und Proteinkonzentration im Gewebe korrelierte. Damit stellt KIM-1 in dieser Studie hinsichtlich Empfindlichkeit und Messbarkeit den empfindlichsten Biomarker für Nephrotoxizität dar. Die Untersuchung der Zellteilung nach wiederholter Gabe von OTA zeigte einen dramatischen, zeit- und dosisabhängigen Anstieg der Proliferation von proximalen Tubulusepithelzellen in Nieren von Tieren, die mit 70 oder 210 µg/kg KG behandelt wurden. Dagegen wurden nach wiederholter Exposition gegenüber 21 µg/kg KG über 90 Tage keine OTA-abhängigen Effekte auf die renale Zellproliferation festgestellt. Somit korrelieren die Veränderungen der Zellteilung in der Niere in der 90-Tages-Studie sehr gut mit dem Ergebnis der 2-Jahres-Kanzerogenitätsstudie mit OTA, in der Nierentumoren nur nach Behandlung mit 70 oder 210 µg/kg KG auftraten. Ausgehend von den verschiedenen Endpunkten für Toxizität, die in der Studie untersucht wurden, liegt der no-observed-adverse-effect-level (NOAEL) bei 21 µg/kg KG OTA. Dies entspricht dem NOAEL der 2-Jahres-Kanzerogenitätsstudie. In einem weiteren Teil der Arbeit wurden die neuen in vivo-Biomarker für Nephrotoxizität in NRK 52E-Zellen als in vitro-Modell ausgetestet. Allerdings konnte eine erhöhte mRNA-Expression von KIM-1, einem sensitiven Marker in vivo, nach 24 oder 48 Stunden Behandlung mit verschiedenen nephrotoxischen Modellverbindungen (OTA, Kaliumbromat (KBrO3), Cisplatin oder Cadmiumchlorid (CdCl2)) in den Zellen nicht nachgewiesen werden. Die mRNA-Expression anderer Marker (VIM, CLU, TIMP-1, LCN2, OPN) war dagegen in unbehandelten Zellen bereits so hoch, dass die Behandlung mit Nephrotoxinen zu keiner weiteren Induktion führte. Allein die Gen- und Proteinexpression von HO-1 wurde durch CdCl2, KBrO3 und OTA induziert und könnte daher einen potentiellen Marker für screening-Studien in vitro darstellen. Insgesamt war der Nachweis zytotoxischer Wirkungen jedoch der empfindlichste Endpunkt in der Zellkultur. Die Ergebnisse stützen somit die Verwendung der neuen in vivo-Biomarker als gewebespezifische Marker für Nephrotoxizität in vitro nicht.
N2  - The kidney is a main target organ of toxicity, but early detection of kidney damage and/or carcinogenic effects following the repeated exposure to toxic substances presents a major problem, since traditional markers of renal malfunction suffer from lack of sensitivity. Therefore, nephrotoxic effects are often detected only in long-term experiments in animals. Ethical reasons as well as the immense time and costs required for these animal studies have prompted the search for alternative methods by which animal numbers and duration of studies can be reduced. A further, albeit challenging, attempt is to replace experiments in animals by studies in vitro. The aim of this work was to test possible alternative methods for the detection of nephrotoxicity after repeated exposure. One the one hand, the expression of new biomarkers of stress and tissue damage was studied in an in vivo-model of chronic nephrotoxicity; enhanced gene expression of these biomarkers, including kidney injury molecule-1 (KIM-1), lipocalin-2 (LCN2), clusterin (CLU), osteopontin (OPN), tissue inhibitor of metalloproteinases-1 (TIMP-1), vimentin (VIM), and heme oxygenase-1 (HO-1), had been demonstrated before in several models of acute kidney damage. In addition to the experiments in vivo, the markers were also studied in a cell culture-based in vitro-model to assess their use as sensitive endpoints of toxicity in vitro. A further part of this work included the determination of cell proliferation as potential early marker of toxin-induced carcinogenic effects. As an in vivo-model, a toxicity study in male F344/N rats was performed. Rats were treated 14, 28 or 90 days with 0, 21, 70 or 210 µg/kg body weight (bw) ochratoxin A (OTA) by oral administration. OTA is a mycotoxin that is known to cause kidney damage and renal tumors in rats after repeated exposure. Analysis of mRNA expression of the new biomarkers showed early, time- and dose-dependent induction of KIM-1, LCN2, TIMP-1, OPN and CLU in kidney tissue of animals treated with 70 or 210 µg/kg bw. The induction of these biomarkers accompanied histopathological changes like cell degeneration and regeneration and mirrored well the progression of tissue damage. mRNA expression of HO-1 and VIM was also modulated by OTA, but overexpression was not evident at all time points or occurred later than for the other markers. Compared with the new biomarkers, effects on traditional markers of nephrotoxicity (serum creatinine, urinary N-acetyl-&#946;-D-glucosaminidase and &#947;-glutamyltransferase) were restricted to later time points and the high dose group. In addition to the effects on gene expression, enhanced protein expression of KIM-1, CLU, OPN and VIM was observed in target cells of OTA in the proximal tubule epithelium; however, only for KIM-1, increased protein levels were also measured in urine, which correlated with the effects on the gene and protein expression in kidney tissue. Therefore, KIM-1 appeared to be the most sensitive biomarker of nephrotoxicity in this study. The study of the renal cell division after repeated administration of OTA demonstrated a dramatic, time- and dose-dependent increase in the proliferation of proximal tubule epithelial cells in kidneys of rats exposed to 70 or 210 µg/kg bw. In contrast, no OTA-dependent effects on renal cell proliferation were observed after repeated administration of 21 µg/kg bw. Thus, changes of renal cell proliferation in this 90-day-study correlate well with the results of the 2-year-carcinogenicity study with OTA, where renal tumors were only detected at 70 or 210 µg/kg bw. Based upon the different endpoints of toxicity determined in this study, the no-observed-adverse-effect-level (NOAEL) is 21 µg/kg KG OTA. This is consistent with the result of the 2-year-carcinogenicity study. In another part of this work, the new in vivo biomarkers of nephrotoxicity were studied in NRK-52E cells as in vitro-model. However, mRNA expression of KIM-1, one of the best biomarkers in vivo, was not detected in the cells after treatment for 24 or 48 hours with several nephrotoxic model substances (OTA, potassium bromate (KBrO3), cisplatin or cadmium chloride (CdCl2)). In contrast, high basal mRNA expression of other markers (VIM, CLU, TIMP-1, LCN2, OPN) was evident even in untreated cells and treatment with nephrotoxins did not further enhance marker gene expression. Only in the case of HO-1, both gene and protein expression were induced by CdCl2, KBrO3 and OTA, and could therefore represent potential markers in screening studies in vitro. In summary, measurement of cytotoxicity was still the most sensitive endpoint of toxicity in vitro. Thus, results from this study do not support the use of the new in vivo-biomarkers as tissue-specific markers of nephrotoxicity in vitro.
KW  - Niere
KW  - Toxizität
KW  - Biomarker
KW  - Carcinogenese
KW  - Niere
KW  - Toxizität
KW  - Biomarker
KW  - Carcinogenese
KW  - nephrotoxicity
KW  - biomarker
KW  - carcinogenesis
Y1  - 2009
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47812
ER  -