Naunyn-Schmiedeberg´s Arch Pharmacol (2012) 38 5 (Suppl 1):S1–S116 D OI 10.1007/s00210-012-0736-0
Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie e.V.
Abstracts of the 78 th Annual Meeting March 19 – 22 , 2012 , Dresden , Germany
This supplement was not sponsored by outside commercial interests. It was funded entirely by the publisher.
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Dear Colleagues,
The underlying abstract-issue of the 78th Annual Congress of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT) contains all abstracts submitted for poster presentation, for free oral communication and those of the invited speakers. The abstracts are printed in alphabetical order according to the main author (in bold), the presenting author is underlined.
Responsible for the congress programme are three scientific societies: Deutsche Gesellschaft für Pharmakologie (DGP), Gesellschaft für Toxikologie (GT) and Deutsche Gesellschaft für Klinische Pharmakologie und Therapie (DGKliPha) which belong to the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT).
Thus, the 78th Annual Congress in 2012 should contribute to the further development of experimental pharmacology, clinical pharmacology and toxicology. Combined sessions of the three mentioned societies demonstrate the common aims of DGPT.
Prof. Dr. Dr. W. Kirch Congress President 78th Annual Meeting DGPT
Dresden, in March 2012
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001 regulates cAMP levels and cardiac contractility through a receptor-independent G- protein activation involving direct G protein β subunit phosphorylation. The precise role of NDPK C in the heart is unknown and was the object of this study. NDPK C function Transcriptional regulation of human three prime exonuclease I (TREX1) upon was assessed in neonatal (NRCM) and adult (ARCM) rat cardiomyocytes with real-time exposure to UV light and anticancer drugs is mediated by AP-1 PCR, immunoblotting, and quantification of cAMP content. Heart failure was induced by chronic treatment with isoproterenol (ISO, 2.4 mg/kg/d 4 days) via minipumps. Chronic Aasland D., Tomicic M., Kaina B., Christmann M. ISO increased mRNA levels of NDPK C by 9.4±1.9-fold and its protein levels by Universitätsmedizin Mainz Toxikologie, Obere Zahlbacher Straße 67, 55131 Mainz, 2.1±0.11-fold. Immunoprecipitation of the G protein β subunit resulted in co- Germany immunoprecipitation of NDPK C and the stimulatory Gαs subunit: ISO enhanced this interaction. Upon ISO stimulation, NDPK C translocated from the cytosol to the plasma Cells respond to genotoxic stress with the induction of DNA damage defense functions. membrane within 3 hours in both NRCMs and ARCMs. Adenoviral overexpression of Previously we showed that mouse fibroblasts respond to UV light and benzo(a)pyrene NDPK C in NRCMs caused a 1.5-fold increase in basal and ISO induced cAMP diol epoxide (BPDE) with the induction of the three prime exonuclease I (trex1) on synthesis, whereas siRNA mediated knockdown of endogenous NDPK C decreased mRNA and protein level. This induction was dependent on AP-1 mediated promoter cAMP levels by ~50%. Our results establish NDPK C as a novel and critical regulator of activation, which was mediated by c-Fos and led to enhanced protection against UV and cAMP synthesis and Gs signaling in the heart. The up-regulation of NDPK C and the BPDE. Here we show that TREX1 is also regulated in human cells by AP-1. In human increased responsiveness to ISO in failing hearts point to NDPK C as a potential fibroblasts exposed to UV, the induction is mainly mediated via c-Jun and not c-Fos. counterregulatory factor in the onset of heart failure. Transcriptional upregulation of TREX1 is observed already after 8h and precedes the induction of c-Fos, which is not present in untreated normal fibroblasts and synthesized 16 h following UV treatment. However, upon its transgenic expression via stable transfection, c-Fos was found to be involved in the UV-mediated induction of TREX1. Also in glioma and melanoma cell lines, induction of TREX1 was observed upon 004 exposure to the anti-cancer drugs CCNU, fotemustine and topotecan. In these tumor cell lines, which express c-Fos protein at high basal level, both c-Fos and c-Jun are involved in the regulation of TREX1 as shown by electromobility shift assays and siRNA Uptake and metabolism of methylated myricetin derivatives: Studies in cell culture experiments. Our data indicate that the dimeric transcription factor AP-1, consisting of c- and C. elegans 1,2 1 1 2 1 Jun and c-Fos, is not only involved in the regulation of TREX1 in normal fibroblasts upon Ackermann D. , Büchter C. , Chovolou Y. , Proksch P. , Wätjen W. UV exposure, but also mediates up-regulation of this protein in tumor cells, which might 1Heinrich-Heine-Universitaet Duesseldorf Institut fuer Toxikologie, P.O. Box 101007, impact their sensitivity to anti-cancer drugs. 40001 Duesseldorf, Germany 2Heinrich-Heine-Universitaet Duesseldorf Institut fuer Pharmazeutische Biologie und Biotechnologie, Universitaetsstrasse 1, 40225 Duesseldorf, Germany
002 Secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. Besides Evidence for the potential usefulness of STW 5 in an experimental model of reflux their antioxidative effects, flavonoids may also modulate cell signaling pathways and esophagitis thereby performing their disease-protective actions. Though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is Abdel-Aziz H.1,2, Kelber O.2, Weiser D.2, Khayyal M. T.3 1 known about their metabolic fate in vivo. Therefore, we investigated the absorption and Institute of Pharmaceutical Chemistry Pharmacology, Münster, Germany metabolism of several flavonoids such as myricetin and its methylated derivatives 2Steigerwald Arzneimittelwerk GmbH Scientific Department, Darmstadt, Germany 3 laricitrin, syringetin, and myricetin-3’,4’,5’-trimethylether in the human colon carcinoma Faculty of Pharmacy, Cairo University Pharmacology, Cairo Egypt cell line Hct116 and the human hepatoma cell line HepG2 as well as the model organism Caenorhabditis elegans. Proton pump inhibitor (PPI) therapy is the most effective medical treatment for symptom All flavonoids were rapidly taken up by both cell lines as shown by HPLC analyses. The relief in gastro-esophageal reflux disease (GERD). However, up to 40% of patients do intracellular amount of myricetin and laricitrin did not increase with time and was only not achieve adequate symptom control with PPIs. Most of these patients originate from half of that of syringetin and myricetin-3’,4’,5’-trimethylether. Interestingly, no metabolites the group with non-erosive reflux disease, for which the symptomatic response rate to of these flavonoids could be detected which might at least in part be due to their low PPIs was shown to be only 37%. Therefore, the search continues for alternative intracellular amounts. Absorption as well as intracellular distribution was also evidenced treatments. by using the fluorescent dye “Naturstoff reagent A” (2-aminoethyl-diphenylborinate; STW 5, a multi-component herbal preparation, was shown to relieve heartburn and NSRA). Fluorescence microscopy indicated a predominant cytosolic distribution of the concomitant reflux symptoms in patients with functional dyspepsia and to prevent employed flavonoids. In the model organism Caenorhabditis elegans, the flavonoids inflammation in an acute model of reflux esophagitis (RE) in rats, without affecting the were exclusively distributed in the intestine as visualized by NSRA. pH of the refluxate. In the present study, the efficacy of STW 5 was assessed in a more The antioxidative capacity of the four flavonoids (measured by using the cell free TEAC chronic model of RE, and the underlying mechanisms of action were investigated. Rats assay and the H2DCF-DA assay in Hct116 cells) decreased with increasing methyl were pretreated for 7 d with STW 5 (0.5 or 2 ml/kg) or omeprazole (as reference drug). groups in the B-ring. Myricetin-3’,4’,5’-trimethylether (three methyl groups) was the least Esophagitis was induced surgically by ligation of the forestomach and corpus and by effective radical scavenging flavonoid in both the cell free system and in Hct116 cells covering the duodenum near the pylorus with a piece of Nelaton catheter. Animals were compared to myricetin (no methyl groups). treated for a further 10 d with the drugs and then sacrificed. The esophagi were excised, In conclusion, for exerting their biological effects, uptake and distribution as well as weighed and evaluated macroscopically. Esophagus samples were fixed in formalin and metabolism of flavonoids in certain organs such as liver and gut are important and more examined histologically. Tissue homogenates were used for the semi-quantitative research in that field is warranted. determination of cytokines using a proteome profiler cytokine array and for measuring NF-κB p65 DNA binding. Nf-kß levels were not affected by surgery or any of the treatments. Both, STW 5 and omeprazole, improved body weight, tissue damage score and esophageal weight index to similar extents. However, STW 5 had a much more pronounced anti-inflammatory effect. This was evidenced by the cytokine array, which 005 showed a marked increase in the number as well as the amount of pro-inflammatory cytokines released in the esophagitis group as compared to sham operated animals. STW 5 inhibited the vast majority of these changes in a dose dependent manner. The Phospho p38 MAPK dependent Upregulation of vascular AT2 receptor expression effect was much more prominent than that of omeprazole, suggesting a direct anti- by nitric oxide inflammatory and/or mucosa protecting action. Agouri S., Bisha M., Dao V. T. - V., Suvorava T., Kojda G. The present findings suggest that multi-target anti-inflammatory drugs like STW 5 might institute of pharmacology and clinical pharmacology Heinriche Heine Universty, present an alternative/additional treatment option for GERD patients not responding Moorenestr. 5, 40225 düsseldorf, Germany adequately to PPIs. Further elucidation of its exact mechanism of action might pave the way for a new class of anti-reflux agents. Purpose: The aim of this study was to investigate whether endothelial nitric oxide (NO) has any influence on the expression on angiotensin (AT) type 1 (AT-1) and (AT-2) receptors and P38 dependent protein kinase G . Methods:Primary porcine aortic endothelial cells (PAEC) were routinely cultured in 003 growth medium which was supplemented with 10% fetal serum, and 1% penicillin- streptomycin in an atmosphere of 10% CO2. Cells were grown until passage 2 in 75- cm² flasks to 80% confluency. Cells were incubated for 1 and 3 h with the NO- donor Nucleoside Diphosphate Kinase C is an Essential Regulator of G Protein Content DEA / NO (Diethylamine NONate 10mM). Cells were incubated for 3 h with 1µM , 5µM and Cyclic AMP Production in Cardiomyocytes and 10µM with 8-pcpT-cGMP( 8- para-chlorophenyl thioguanosine ) which is an activator 1 2 3 3 1 4 of cGMP dependent kinase. Cells were incubated for 3 h with the NO donor DEA /NO in Abu-Taha I. , Wolf N. M. , Lutz S. , El-Armouche A. , Dobrev D. , Hippe H. - J. , the presence of 10µM of the p38 MAPK inhibitor (SB 203580) and in the presence of Wieland T.2 1 10µM of protein kinase G inhibitor RP-8pcpT-cGMP(RP-8-4-chlorophenylthioguanosine- Medical Faculty Mannheim, University of Heidelberg Division of Experimental 3,5-cycl monophosphothioate ). Brain endothelial cells (bENDs) were incubated with NO Cardiology, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany 2 donor DETA/NO for 3, 6, 12 and 24 hours. P38 MAPK phosphorylation and AT2 protein Medical Faculty Mannheim, University of Heidelberg Institute for Experimental and level was measured by western blot. Results: Incubation of PAEC with DEA/NO Clinical Pharmacology and Toxicology, Maybachstrasse 14, 68169 Mannheim, Germany 3 resulted in significant higher expression levels of AT2 receptors (216.8±19.80, p<0.01, University Hospital Göttingen, Georg-August University Göttingen Department of n= 7 ) after 3 h. At the same time p38-MAPK phosphorylation was increased to Pharmacology, Robert-Koch-Str. 40, 37075 Göttingen, Germany 4 (163.5±22.21, p=0.0288) as a related to total p38-MAPK. In striking contrast the University Hospital Münster Department of Cardiology and Angiology, Albert- expression on the AT1 receptors did not change (p>0.05, n= 4). SB203580 completly Schweitzer-Campus 1, Gebäude A1, 48149 Münster, Germany inhibited the effect of DEA/NO on AT2 receptor expression ( p<0.001) and p38MAPK phosphorylation (p<0.05), and likewise PKG inhibitor blocked the effect of DEA/NO on Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved in a AT2 receptor expression (p<0.01) and p38MAPK phosphorylation (p<0.05), while variety of cellular processes including cancer metastasis and heart diseases. The incubation with 8-pcpT-cGMP resulted in upregulation of AT2 receptor protein levels plasma membrane content of the three major NDPK isoforms NDPK A, B and C is (186.6±59.3, p=0.2183, n=5) and P38 MAPK phosphorylation. Incubation bENDs cells increased in human heart failure. We have previously shown that the NDPK B isoform S4
with DETA/NO for 3, 6, 12 and 24 h resulted of in continuous upregulation of AT2 008 receptor protein levels (p<0.05, n= 6). Conclusion: These results suggest that NO can upregulate vascular AT-2 receptors in PAEC and bENDs. This newly discovered regulation is most likely mediated by cGMP-dependent protein kinase: induced Lack of gene mutagenic potential of MD 288, a hybrid of chloroquine and phosphorylation of p38-MAPK. primaquine
Albrecht A. E.1, Diegel M.2, Esch H.1, Bringmann G.2, Lehmann L.1 1University of Wuerzburg, Institute of Pharmacy and Food Chemistry Section of Food Chemistry, Am Hubland, 97074 Wuerzburg, Germany 006 2University of Wuerzburg, Institute of Organic Chemistry, Am Hubland, 97074 Wuerzburg, Germany
The Gly389Arg polymorphism determines the activation kinetics of the human β1- MD 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious adrenergic receptor diseases such as malaria. Since one moiety of the hybrid, the known antimalarial drug 1 2,3 1 4 1,5 Ahles A. , Rochais F. , Rodewald F. , Bünemann M. , Engelhardt S. chloroquine, is a known intercalator, the potential of MD 288 to intercalate into DNA was 1TU München Institut für Pharmakologie und Toxikologie, Biedersteiner Straße 29, determined. Due to the ability of intercalators to cause frame shift mutations, the 80802 München, Germany mutagenic potential of MD 288 was also investigated. 2Université de la Méditerranée Developmental Biology Institute, Marseille, France The potential of MD 288 to intercalate into DNA was investigated by means of 3Universität Würzburg Rudolf-Virchow-Zentrum, DFG-Forschungszentrum für fluorescence based micro plate assay using ethidium bromide (EB) and isolated calf Experimentelle Biomedizin, Josef-Schneider-Straße 2, 97080 Würzburg, Germany thymus double stranded DNA. As a positive control chloroquine was used. The potential 4Universitaet Marburg Institut für Pharmakologie und Klinische Pharmazie, Karl-von- of MD 288 to cause gene mutations was determined using the hypoxanthine-guanine Frisch-Straße 1, 35043 Marburg, Germany phosphoribosyltransferase (HPRT) test in Chinese hamster V79 lung fibroblasts (V79 5Munich Heart Alliance, München, Germany cells). V79 cells were treated with 0.4 µM, 0.8 µM and 1.5 µM MD 288 or the positive control, the direct mutagen 4-nitroquinoline-N-oxide (NQO, 1 µM) for 24 h. On day 6, Signaling through G protein-coupled receptors is affected by receptor polymorphisms, mutants exhibiting loss of hprt function were selected with 6-thioguanine (6-TG). yet the molecular basis for the functional differences of individual receptor variants is Whereas at 1 µM chloroquine, a 38% decrease in fluorescence intensity of EB unclear. (indicating DNA intercalation) was observed, 10 µM MD 288 were needed to observe a To investigate the impact of the frequent Gly389Arg variant of the β1-adrenergic receptor similar decrease (28%) in fluorescence intensity of EB. Therefore, MD 288 is 10fold less (β1AR) on receptor conformation we used β1AR-sensors capable of fluorescence potent to intercalate into DNA than its moiety chloroquine. The frequency of resonance energy transfer (FRET). These sensors retained the pharmacological and spontaneous 6-TG resistant mutants per 106 colony-forming cells was 9 ± 2. As functional characteristics of the native receptors. expected, 1 µM NQO caused a significant increase in the mutant frequency (MF, 168 ± Upon stimulation of the sensors we determined the activation characteristics of the 9). In contrast, MF was not significantly affected by treatment with MD 288 at both non- polymorphic receptors in real time and in living cells. We found the β1AR variants to cytotoxic (0.4 µM: 5 ± 4) and cytotoxic (0.8 µM: 9 ± 7) concentrations. behave similar upon a single stimulation with an agonist, but to differentially respond In conclusion, MD 288 is a less potent intercalator than the known antimalarial drug with a change of their activation kinetics during subsequent stimulations. While the chloroquine. Furthermore, MD 288 does not cause gene mutations in the HPRT test. Arg389-β1AR did not show altered activation kinetics after prestimulation, the Gly389- Since current studies show that various metabolites of MD 288 are formed in vitro, their β1AR became slower compared to the initial stimulation suggesting that β1ARs possess mutagenic potential is currently under investigation as well. a memory of previous activation. We then permeabilized β1AR-sensor-expressing cells with saponin to remove soluble cytosolic factors. Upon permeabilization the β1AR variants did not display receptor memory, suggesting that the β1AR memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial 009 activation including the phosphorylation of agonist-bound receptors by protein kinase A or G protein-coupled receptor kinases. Our findings suggest an intrinsic, polymorphism-specific property of βARs that alters Regulation of mast cell activation by TRP protein-mediated Ca2+ entry activation kinetics upon continued stimulation and that might account for individual drug Almering J.1, Tsvilovskyy V.1, Mannebach S.2, Kriebs U.1, Weißgerber P.2, Flockerzi responses. 2 3 1 V. , Birnbaumer L. , Freichel M. 1Universität Heidelberg Pharmakologisches Institut, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany 2Universität des Saarlandes Experimentelle und Klinische Pharmakologie und 007 Toxikologie, Gebäude 46, 66421 Homburg, Germany 3NIEHS Transmembrane Signaling, 111 TW Alexander Drive, Research Triangle Park, North Carolina 27709, United States Micro-RNA replacement therapy: Nanoparticle-mediated in vivo delivery of miRNA-145 or miRNA-33a exerts antitumor effects in colon carcinoma xenograft Ca2+ influx is essential for mast cell activation induced by various ligands. It does not mouse models only rely on the opening of Ca2+-conducting channels but depends critically on the 1 1 2 2 2 Weirauch U. , Ibrahim A. F. , Thomas M. , Lange-Grünweller K. , Grünweller A. , membrane potential. TRP channels form cation entry channels thereby either 2 1 Hartmann R. K. , Aigner A. contributing to Ca2+ entry or depolarisation. Recently, we showed that TRPM4 acts as a 1Universiität Leipzig, Rudolf-Boehm-Institut für Pharmakologie und Toxikologie Klinische Ca2+-activated non-selective cation channel and critically determines the driving force for Pharmakologie, Härtelstrasse 16-18, 04107 Leipzig, Germany Ca2+ influx in mast cells following FcεRI-stimulation (1). In addition to TRPM4 we also 2Philipps-Universität Marburg Pharmazeutische Chemie, Ketzerbach, 35032 Marburg, identified the expression of other TRP transcripts in bone marrow derived mast cells Germany (BMMC) including those encoding TRPC2, TRPC3, TRPC5, TRPC6 and TRPM7. In peritoneal mast cells (PMC), RT-PCR indicated expression of TRPC1, TRPC4, TRPC5, Micro-RNAs (miRNAs) control the expression of various genes, and under pathological TRPC6, TRPM2, TRPM4 and TRPM5. conditions several miRNAs are up- or downregulated. Previous in vitro studies have To identify the functional role of those TRP channel proteins for mast cell activation we established a pro-apoptotic and anti-proliferative role of miR-145, which shows analysed Ca2+ signaling using microfluorimetry in BMMCs and PMCs after stimulation decreased levels in colon carcinoma. In contrast, while miR-33a is only weakly with substances known to activate TRPC6 channels in other cell systems such as the expressed in several tumors as well, its role in cancer has not been analysed so far. In diacylglycerol analogue OAG, the hyperforin analogue HYP-9 and flufenamic acid (FFA), 2+ this study, we demonstrate the tumor-relevance of miR-33a and identify the proto- but could not evoke a rise in the [Ca ]i in both PMC and BMMC. Sphingosine 1- oncogenic kinase Pim-1 as a target of miR-33a. Pim-1 harbours a highly conserved miR- phosphate and lysophosphatidylcholine, which were reported to activate TRPC5 2+ 33a binding site within its 3'-UTR, and seed mutagenesis of this target sequence channels, induced only minor rise in [Ca ]i in BMMCs, respectively. Here, we will abolishes the miR-33a-mediated downregulation of Pim-1. The knockdown of Pim-1 by present our analysis of Ca2+ signaling following stimulation of the FcεRI receptor and RNAi or miRNA transfection inhibits proliferation in leukemia and in colon carcinoma application of secretagogues that are supposed to affect Ca2+-dependent mast cell cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function activation such as adenosine, endothelin-1, substance P and compound 48/80 in of miR-33a. We furthermore introduce polyethylenimines (PEIs) for the therapeutic BMMCs and PMCs derived from mouse lines with inactivation of TRPC1, TRPC3, application of miRNAs in vivo, which is critically dependent on the development of TRPC4, TRPC5 or TRPC6 since specific antagonists are still lacking for these TRP appropriate delivery tools. PEIs are able to form non-covalent complexes with miRNAs, channels. leading to miRNA protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. Therapeutic effects References of PEI-mediated miRNA delivery were demonstrated in subcutaneous colon carcinoma (1) Increased IgE-dependent mast cell activation and anaphylactic responses in mice xenograft mouse models. The in vivo application of miRNA-145 through systemic or lacking the calcium-activated nonselective cation channel TRPM4. Vennekens et al., Nat local injection of PEI/miRNA complexes resulted in efficient miRNA delivery and in Immunol. 8: 312-20 (2007) antitumor effects, based on the concomitant repression of ERK5. Likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with PEI-complexed miR- 33a. This is due to the miR-33a-mediated downregulation of Pim-1 expression and resembles the Pim-1 knockdown through RNAi / Pim-1 siRNAs. Taken together, in tumor xenograft mouse models we establish miRNA replacement therapy through the PEI-complexation of miRNAs as a novel therapeutic strategy and demonstrate that miR- 145 and miR-33a may be promising miRNAs in colon carcinoma therapy.
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Inflammatory and cytotoxic effects of amorphous SiO2 nanoparticles in Real-time monitoring of norepinephrine-mediated α2A-adrenergic receptor and Gi- mammalian cells protein activation Al-Rawi M.1, Marquardt C.1, Panas A.1, Ruh H.2, Diabaté S.1, Weiss C.1 Ambrosio M., Lohse M. J. 1Karlsruhe Institut für Technologie (KIT) Institut für Toxikologie und Genetik (ITG), Universität Würzburg Institut für Pharmakologie und Toxikologie, Versbacher Strasse 9, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany 97078 Würzburg, Germany 2Universität Heidelberg / Hochschule Mannheim Institut für Medizintechnologie, Paul- Wittsack-Straße 10, 68163 Mannheim, Germany The α2A-AR is the main AR in the central nervous system and it plays a crucial role in regulating norepinephrine (NE) release from nerve terminals via presynaptic feedback As the understanding of materials at the nanoscale and the ability to control their inhibition. It is also associated with a number of physiological effects, including structure improves, nanotechnology manufactures a wide range of materials with novel hypotension, pain perception, sedation and modulation of mood. NE, once released in characteristics and applications in, e.g., electronics, engineering and more recently in the synaptic space, binds to α2A-ARs and induces a rearrangement of the receptors from biomedical research. Although the technological and economic benefits of engineered the inactive state into an active conformation. This allows the binding and activation of nanomaterials (ENMs) are obvious, concerns have been raised about adverse effects if the cognate Gi-protein and, hence, the transduction of the transmembrane signal to the such material is inhaled, ingested, applied to the skin or even released into the downstream effectors. Generally, α2A-AR activation has been deduced from the environment. Besides the physicochemical characterization of pristine ENMs also the stimulation of a receptor-mediated biological response that could be easily followed interaction of proteins with ENMs, the so-called protein corona, becomes more and more experimentally. However, most of these approaches do not employ living cells and are important to understand potential adverse health effects normally applied under equilibrium conditions that need prolonged incubation periods Here we investigated metal oxide nanoparticles (NPs) as those are produced in large incompatible with the physiological temporal dynamics of NE. scale and are therefore included in the OECD list of manufactured nanomaterials for Here, we monitored the NE-mediated α2A-AR and Gi-protein activation by using a future safety assessments. As the lung is the primary portal of entry for airborne NPs we fluorescence resonance energy transfer (FRET)-based approach in living cells. To exposed human lung alveolar epithelial cells A549 and murine RAW264.7 macrophages examine the effects of increasing concentrations of NE on the speed and extent of α2A- to various NPs in the presence or absence of fetal calf serum (FCS). As read-outs AR activation with very high temporal resolution, we took advantage of the previously FlAsH/CFP uptake of NPs, cytotoxicity, formation of reactive oxygen species (ROS) and the anti- described α2A-AR sensor [1]. The results indicate that in our system the efficacy FlAsH/CFP oxidative and pro-inflammatory response were analysed. Surprisingly, in medium without of NE in eliciting α2A-AR activation increases in a time-dependent way and FCS amorphous SiO2-NPs were the most cytotoxic NPs and induced a significant pro- reaches the maximum with a half-life of ~ 70 ms. The EC50 values decrease in an inflammatory response in both cell types. Furthermore, pre-coating of SiO2-NPs with exponential manner and arrive at ~ 2 µM with a half-life of ~ 330 ms. Next, we analyzed FCS proteins or simply bovine serum albumin abrogated responses in A549 lung the ability of increasing concentrations of NE to trigger a downstream intracellular epithelial cells. Also in the presence of human serum SiO2-NPs adsorb albumin and response after α2A-AR stimulation by monitoring the kinetics and amplitude of Gi CFP/YFP several other proteins as revealed by mass spectrometry (MALDI-TOF). Thus, the activation in living cells. We applied the previously well characterized Gi sensor protein corona bound to the surface of SiO2-NPs suppresses their biological effects, an [2]. The results show that both the efficacy and the potency of NE in inducing Gi issue which needs to be more carefully considered for in vitro – in vivo extrapolations (1, activation reach the steady state slower compared to receptor activation (half-life ~ 700 2). Ongoing experiments address the toxicity pathways triggered by SiO2-NPs. Assays ms and ~ 3,000 ms respectively). were developed to monitor cellular toxicity by high-content and high-throughput In conclusion, we were able to monitor NE-mediated events occurring in the millisecond microscopy which are superior to conventional toxicity assays as they monitor more time scale and reaching the equilibrium in a time interval compatible with physiological accurately adverse effects even at the single cell level and will allow in the future to conditions. screen different libraries (siRNA, cDNA, chemicals) for their effects on NP- mediated toxicity. References 1. Nikolaev, V.O., et al., J Biol Chem, 2006. 281(34): p. 24506-11. References 2. Bunemann, M., M. Frank, and M.J. Lohse. Proc Natl Acad Sci U S A, 2003. 100(26): 1 Ruh et al., Toxicology Letters, 2012 p. 16077-82. 2 Panas et al., Nanotoxicology, in revision
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Interaction of Opioids and Trastuzumab in Human Breast Cancer Cells Antagonistic regulation of smooth muscle differentiation through Gq-G11– and G12- Speckmaier E., Ammer H. G13–mediated signaling pathways in vascular remodeling Institute of Pharmacology, Toxicology and Pharmacy, Ludwig Maximilians University of 1,2 1 1 1 1 Althoff T. , Albarrán-Juárez J. , Takefuji M. , Wirth A. , Wettschureck N. , Offermanns Munich, Koeniginstrasse 16, 80539 Muenchen, Germany 1,3 S. 1Max-Planck-Institut für Herz- und Lungenforschung Abteilung Pharmakologie, Recent data indicate that opioids may interfere with several aspects of tumor growth, like Ludwigstr. 43, 61231 Bad Nauheim, Germany proliferation, metastasis and survival. The human SKBR-3 cell line serves as a model 2Charité - Universitätsmedizin Berlin Medizinische Klinik für Kardiologie u. Angiologie, system for antibody based tumor therapy strategies targeting HER2, a receptor tyrosine Charitéplatz 1, 10117 Berlin, Germany kinase over-expressed in about 30% of human breast cancer patients. Here we report 3Goethe-Universität Frankfurt Medizinische Fakultät, Theodor-Stern-Kai 7, 60590 that HER2 positive SKBR-3 cells carry a single population of functional active kappa- Frankfurt, Germany opioid receptors, as revealed by RT-PCR, radioligand binding and intracellular cAMP accumulation studies. Chronic exposure of the cells to the selective kappa agonist Smooth muscle cells (SMCs) are highly plastic, and their dedifferentiation underlies U69,593 only marginally attenuated cell growth when measured by crystal violet or MTT many vascular remodeling processes. We found a reduced expression of SMC marker assays. However, when given in combination with Trastuzumab it strongly enhanced the genes in vessels from mice lacking the G-protein subunits Gα12/13 specifically in SMCs. inhibitory effect of the anti-HER2 monoclonal antibody. Investigations into the underlying In addition, we observed an exaggerated downregulation of SMC marker genes in regulatory mechanisms revealed that in naïve SKBR-3 cells activation of kappa-opioid Gα12/13–deficient SMCs, in response to carotid artery ligation. This was accompanied by receptors stimulate ERK1/2 signaling by transactivation of HER1 receptors. Chronic a dramatically enhanced vascular remodeling, resulting in a several–fold increased Trastuzumab treatment desensitizes this pathway and switches kappa-opioid receptors intima-media thickness compared with WT mice. Moreover, SMC–specific Gα12/13– to activation of the PI3K/AKT pathway. Under these conditions, U69,593 enhances deficiency promoted SMC dedifferentiation, as well as plaque–progression and – Trastuzumab-stimulated procaspase-3 and PARP cleavage. A similar interaction of vulnerability in the Apo-/-–model of atherosclerosis. In contrast, vessels from mice opioids with Trastuzumab is absent in HER2 negative MCF-7 cells that also lacking Gαq/11 specifically in SMCs, displayed a normal basal expression of SMC marker endogenously express kappa-opioid receptors. These results demonstrate that SKBR-3 genes and an attenuated downregulation in response to carotid artery ligation. cells carry functional kappa-opioid receptors that are able to enhance the cytotoxic Moreover, SMC-specific Gαq/11-deficiency protected mice from neointimal hyperplasia. efficiency of anti-HER2 directed antineoplastic therapy.
We then investigated which downstream effectors mediate the opposing effects of Gq- G11– and G12-G13–initiated signaling on SMC differentiation. We found that G12-G13 but not Gq-G11 were required for the activation of RhoA in VSMCs in response to carotid 014 artery ligation. Since the RhoGEF LARG is a major mediator of G12-G13–induced activation of RhoA, we generated mice with smooth muscle–specific LARG-deficiency. As in Gα12/13–deficient mice, carotid artery ligation in mice lacking LARG, resulted in an The role of heterotrimeric G-proteins in age-related hypertension enhanced SMC dedifferentiation and excessive vascular remodeling. Using primary 1,2 2 2 3 4 2,5 SMCs, isolated from the respective KO-mice, we revealed that G -G - and LARG–, but Angela W. , Mikito T. , Shengpeng W. , Bolz S. - S. , Schweda F. , Offermanns S. 12 13 1 not G -G –mediated signaling promotes nuclear translocation of the myocardin related Universität Heidelberg; Pharmakologie, INF 366, 69120 Heidelberg, Germany q 11 2 transcription factor A, that induces SMC marker gene expression. On the other hand, we MPI Bad Nauheim Pharmakologie, Ludwigstr. 43, 61231 Bad Nauheim, Germany 3 found that, upon carotid artery ligation, G -G –, but not G -G –mediated signaling Universität Toronto, Dept. of Physiology, 1 Kings college Circle, Toronto Canada q 11 12 13 4 induces phosphorylation of ERK1/2 and transcription of the ERK1/ 2–regulated early Universität Regensburg, Physiologie, Universitätsstr. 31, 93053 Regensburg, Germany 5 response genes c-fos, ets-1 and egr-1, thereby counteracting SMC differentiation. Universität Frankfurt,, Theodor Stern Kai 7, 60590 Frankfurt, Germany
Our results demonstrate that G12-G13– and Gq-G11–mediated signaling pathways The age-dependent progression of blood pressure is a well-known phenomenon which antagonistically regulate SMC differentiation in pathological remodeling processes, contributes to the increased risk of cardiovascular diseases in the aged population. In defining these pathways as potential targets for pharmacological interventions. contrast to its eminent importance, the underlying causes of age-related hypertension are still rather unclear. We found that also in aged mice (1 year), basal blood pressure shows a significant increase compared to young animals. A detailed characterisation of the kidney and heart function of aged and young mice showed no difference that could explain the increase in blood pressure. Also, vessel stiffness appeared to be still normal in 1-year-old mice. S6
When young and old mice were exposed to various pressor substances and receptor only 20% of LPS-induced IL-12p70 levels and was less sensitive to TNF-alpha co- antagonists, we observed that in particular in vivo pressor responses to ET-1 were stimulation. However, SphK1-deficiency strongly augmented CpG-dependent IL-12p70 significantly reduced in aged mice whereas other constricting agonists like angiotensin II production. or phenylephrine elicit similar increases in blood pressure in aged and young mice. In ongoing experiments we started to analyze the details of TRAF2/RIP1 and TRAF6/TAK1 Furthermore, in aged WT mice the blockade of ETA receptors by darusentan led to a activation by ubiquitination blots in WT, SphK1- and S1Plyase-deficient DCs. In conclusion, strong drop in blood pressure while young WT animals reacted only mildly to ETA- we hope to unravel possible mechanisms of the observed differential effects of S1P and its receptor inhibition. enzymes on inflammation and cancer-relevant cytokines. Consistent with a role of the vascular endothelin-system including Gq/11- as well as G12/13-coupled ETA-receptors in age-dependent hypertension, we saw a normalization of blood pressure in old animals after induction of Gαq/Gα11 or Gα12/Gα13 deficiency in smooth muscle cells. Thus, elevated blood pressure in aged mice results from an 017 increased activation of vascular smooth muscle cells by procontractile stimuli resulting in an elevation of total peripheral resistance. In addition, our data indicate that age- dependent elevation of vascular tone centrally involves the endothelin-system. Identification of the KH type splicing regulatory protein (KSRP) as a new important mediator of the anti-inflammatory effects of resveratrol Art J.1, Besche V.2, Bros M.2, Li H.1, Handler N.3, Bauer F.3, Erker T.3, Behnke F.4, Mönch B.5, Förstermann U.1, Dirsch V. M.6, Werz O.5, Kleinert H.1, Pautz A.1 015 1University Medical Centre Johannes Gutenberg University Mainz Department of Pharmacology, Obere Zahlbacher Str. 67, 55131 Mainz, Germany 2University Medical Centre Johannes Gutenberg University Mainz Department of Prediction of metabolites using computational approaches Dermatology, Obere Zahlbacher Str. 63, 55131 Mainz, Germany 1,2 2 2 1 Anger L. T. , Brigo A. , Kansy M. , Gundert-Remy U. 3University Vienna Department of Pharmaceutical and Medicinal Chemistry, Althanstr. 1Charité University Medical School Berlin Institute of Clinical Pharmacology and 14, 1090 Wien, Austria Toxicology, Luisenstr. 7, 10117 Berlin, Germany 4Eberhard Karls University Tübingen Pharmaceutical Institute, Auf der Morgenstelle 8, 2F. Hoffmann-La Roche Ltd. Structure/Property Effect Relationships, Non-Clinical 72076 Tübingen, Germany Safety, 4070 Basel, Switzerland 5Friedrich Schiller University Jena Institute of Pharmacy, Philosophenweg 14, 07743 Jena, Germany An increasing number of computational tools are nowadays available, which are claimed 6University of Vienna Department of Pharmacognosy, Althanstr. 14, 1090 Wien, Austria to predict drug metabolism. Those tools could most probably be used in an extended way to consider potential drug metabolites earlier in toxicity prediction of for Resveratrol, a polyphenol derived from different plants, possesses multiple example.genotoxicity or carcinogenicity. Therefore it’s important to understand the pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anti- quality and limitations of metabolism prediction tools if applied in a broader context. cancer, neuroprotective and anti-inflammatory properties. Many of these effects have However, to date, the software packages used in metabolism prediction have only been been attributed to its anti-oxidative activity but resveratrol also modulates signal validated with relative small datasets containing limited number of compounds. Hence, transduction pathways like the p38 MAPK pathway or the activity of different they require a more comprehensive and explorative evaluation. transcription factors like NF-κB redox-independently. Moreover, the histone deacetylase Aim: Meteor (Lhasa Ltd., Leeds, UK), an available prediction tool, has been used to sirtuin 1 (SIRT1) is an important mediator of resveratrol effects. Nevertheless the direct predict the metabolites of 325 drugs in therapeutic use. The results of the prediction molecular target of resveratrol remains unclear. were compared to the known human metabolites. In target fishing experiments we identified the RNA-binding protein KSRP as direct Methods: The WHO ATC index was used to extract drugs for the therapeutic classes of resveratrol binding partner. KSRP is an RNA-binding protein that controls pro- antiinfectives (A07E, M01A, S01BA, G02CC, M02AA, S01BC), central nervous system inflammatory gene expression on the post-transcriptional level by modulation of mRNA drugs and muscle relaxants (N, M03), cardiovascular drugs (C), oncologic substances stability. Moreover, it is involved in the biogenesis of miRNAs. Resveratrol treatment of (L01) and virustatics (J05, D06BB, S01AD). Additional information was received by human DLD-1 cells resulted in a decreased mRNA expression of a number of well queries in the DrugBank database. PharmaPendium and DrugBank were the sources to known KSRP target mRNAs (IL-8, iNOS and TNF-α) and enhanced miRNA-155 obtain information on the human metabolites of the therapeutic drugs observed in function. Downregulation of KSRP expression by siRNA prevented the mRNA clinical studies. destabilizing effect of resveratrol. As the activity of KSRP is mainly regulated on the Meteor (version 13.0.0, Lhasa Ltd.) is a so called knowledge-based expert system. As post-translational level by phosphorylation of different serine and threonine residues we such it uses structured expert knowledge and its output offers the structures of analyzed whether resveratrol changes KSRP activity by altering the phosphorylation of metabolites with a guess on the probability of the occurrence of the metabolite. the protein. Indeed, our immunoprecipitation experiments demonstrated that resveratrol Results: 325 drug substances were included in the study from which the occurrence of reduces the p38 MAPK-mediated phosphorylation of threonine residues in the KSRP 876 human metabolites was confirmed in experimental studies. Under the selected protein and thus leads to an increase of KSRP activity. Interestingly, resveratrol does not program settings 398 of the 876 metabolites were correctly predicted by Meteor (true block p38 MAPK activation or activity. In addition we have evidence that SIRT1 is not positives), 478 metabolites could not be predicted (false negatives) and 9272 involved in the resveratrol mediated activation of KSRP. So we believe that activation of metabolites were predicted which however are not known to occur in vivo (unconfirmed KSRP by resveratrol is the major mechanism mediating the anti-inflammatory effects of positives). resveratrol. The sensitivity of the prediction in this study is 45.4%, the positive predictive value is 4.1%. This evaluation study shows that the reliability of the prediction of human metabolites is presently limited. Hence, further development is necessary to improve the prediction 018 potential.
This study was performed as part of a Master thesis in the Master program Toxicology at In vitro testing of OECD reference nanomaterials (NM-series) in rat precision cut Charité University Medical School Berlin, Germany. lung slices
Aumann A.1, Vogel S.1, Kolle S.1, Schulz M.1, Wohlleben W.1,2, van Ravenzwaay B.1, Landsiedel R.1 1BASF SE Experimental Toxicology and Ecology, Carl-Bosch-Str. 38, 67056 016 Ludwigshafen, Germany 2BASF SE Polymer Physics, Carl-Bosch-Str. 38, 67056 Ludwigshafen, Germany
Modulation of dendritic cell cytokine profiles via extra- and intracellular targets of The OECD has defined reference nanomaterials (NM) to be tested in different endpoints sphingolipids concerning human health and environmental safety (1) in order to evaluate if the toxicity 1 1 1 1 1 2 Arlt O. , Giegold O. , Neske C. , Schröder M. , Pfeilschifter J. , Huwiler A. , Radeke H. of nanomaterials can be linked to their physico-chemical properties. 1 H. For nanomaterials, inhalation presents the major exposure route of concern and can be 1Uniklinikum der Goethe-Universität pharmazentrum / ZAFES, Theodor-Stern-Kai 7, assessed using acute inhalation toxicity studies in rodents. However, these in vivo 60590 Frankfurt am Main, Germany studies are resource intensive and animal consuming. The OECD working party on 2Universität Bern Institut für Pharmakologie, Bern, Switzerland nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (PCLS) to Sphingosine-1-phosphate (S1P) is an immune modulator produced by sphingosine estimate respiratory toxicity. kinase 1 (SphK1) and sphingosine kinase 2 (SphK2) and de-phosphorylated or We have tested all 16 NM in PCLS measuring cytotoxicity, apoptosis, oxidative stress degraded irreversibly by S1P phosphatases and a lyase, respectively. We recently and inflammatory response of the tissues as well as observing them histological. For in showed that TLR4-induced IL-12p70 is selectively counter regulated by SphK1, S1PR1 vitro exposure of PCLS the test material was dispersed in medium. Since it is the nature and its extracellular ligand S1P. On the other hand, Spiegel et al. have demonstrated of these materials to change their surface characteristics and agglomeration state in that specific, SphK1-dependent, binding of S1P to TRAF2 enhances the TNF-alpha different environments, a standardized dispersion method (nanoCare) using bovine signaling. Therefore we were interested whether the TLR/TIR and TNF-alpha-signaling serum albumin as a stabilizing agent, was used. Particle size-distributions of the pathways are interfered with each other and are modulated by S1P. In a first approach nanomaterial dispersions were characterized via analytical ultracentrifugation and found we focused our investigations on SphK1 effects on both TRAF2 and TRAF6 stimulatory the nanomaterials well dispersed. signals and cytokines produced downstream. Silver and Zinc oxide but none of the other NM showed cytotoxicity to the lung tissue in Experimentally, with GM-CSF expanded, bone marrow-derived DCs we first the tested concentrations. However, differences in cytokine profiles among the NM were desensitized the LPS-TLR4 or CpG-TLR9 signal by a defined time period of co- observed and showed several correlations to the results obtained in in vivo inhalation or stimulation with TNF-alpha. The initial results showed a partial decrease of IL-12p70 instillation studies. secretion in TNF-alpha-co-stimulated DCs in contrast to LPS stimulation alone. This might indicate that TRAF2 activated via TNF-alpha interacted with the TRAF6 pathway References to reduce IL-12p70. Further series with DCs derived from SphK1-deficient mice 1 GUIDANCE MANUAL FOR THE TESTING OF MANUFACTURED confirmed our former results that IL-12p70 in contrast to other cytokines is specifically NANOMATERIALS: OECD’s SPONSORSHIP PROGRAMME., available at: sensitive to SphK1-S1P feedback, but did not change the effects of TNF-alpha on WT http://www.olis.oecd.org/olis/2009doc.nsf/linkto/env-jm-mono(2009)20 DCs IL-12p70 release. In comparison, CpG-TLR9-induced IL-12p70 release reached S7
019 Circulating sulfated steroid hormones like estrone sulfate (E1S) or dehydroepiandrosterone sulfate (DHEAS) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (SOAT). Inside TMEM2 proteins are localized in lysosomes and other acidic compartments the cell these sulfated steroids can be metabolized to active steroid hormones by the 1 1 1 2 2 3 catalytic activity of the steroid sulfatase (StS), which shows high enzymatic activity in the Bach A. , Kriebs U. , Tsvilovskyy V. , Wissenbach U. , Mannebach S. , Scholz A. , testis (“sulfatase pathway”). In addition to SOAT, other candidate carriers like the Flockerzi V.2, Weißgerber P.2, Lipp P.3, Freichel M.1 1 organic solute carrier protein 1 (OSCP1) and the organic anion transporting polypeptides Universität Heidelberg Pharmakologisches Institut, Im Neuenheimer Feld 366, 69120 OATP6A1 and OATP1C1 are predominantly expressed in the human testis and Heidelberg, Germany 2 demonstrate transport activity for sulfated steroids. We aimed to evaluate the cellular Universität des Saarlandes Experimentelle und Klinische Pharmakologie und expression of SOAT and the other steroid sulfate carriers and their co-localization with Toxikologie, Gebäude 46, 66421 Homburg, Germany 3 the steroid sulfatase (StS) in human testis. Furthermore we want to perform functional Universität des Saarlandes Institut für Molekulare Zellbiologie, Gebäude 61, 66421 transport studies with the steroid sulfate carriers in stably transfected HEK293 cells. We Homburg, Germany detected SOAT by RT-PCR and Western Blot analysis in the human testis. Single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express TMEM2 proteins show similarities in their primary sequence to motifs that are conserved the SOAT mRNA. SOAT expression in specimens showing maturation arrest at the level amongst various members of the TRP protein family. Based on hydropathy analysis of early round spermatids seems to be severely reduced or absent. StS mRNA was these proteins exhibit 6 to 10 membrane spanning domains. In contrast to TRP channels detected by RT-PCR in testis homogenates. Preliminary immunohistochemical data there is no evidence that these proteins form ion channels in the plasma membrane -/- showed that StS may be expressed in germ cells and interstitial Leydig cells. HEK293 following overexpression of their cDNA in HEK293 cells. TMEM2 mice are viable and cells stably expressing the SOAT carrier protein showed significant transport activity for show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase DHEAS. This was demonstrated by using a radiolabeled [3H]-DHEAS compound as well plasma levels. Microfluorimetric measurements using Fura-2 revealed that the elevation 2+ as by direct analysis of the cell associated uptake fraction by a newly developed LC-MS- of the cytosolic Ca concentration after stimulation with carbachol and the MS method. cholecystokinin analogue caerulein is unchanged in TMEM2-deficient acinar cells. TMEM2 is expressed in several cell types including pancreatic acinar cells, cardiac Supported by DFG Research Group 1369 "Sulfated Steroids in Reproduction” myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. We generated several constructs encoding TMEM2 fusion proteins with fluorescence protein tags by fusing eYFP, mcherry and tagRFP-T to the N-and C-terminus of the protein, respectively. Based on Western blot experiments and expression in HEK293 cells the TMEM2-eYFP construct was most suitable for further colocalisation analysis and 022 generation of viral vectors including Adenovirus and Semliki forrest virus. In contrast to the prediction by the PSORT II algorithm TMEM2-eYFP could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a Functional significance of microRNAs in cancer pain manifestation as revealed by vesicular subcellular localization pattern. We localized TMEM2-eYFP in acidic a comprehensive genome wide screen 1 1 2 1 1 2 compartments and predominantly in lysosomes (Pearson coefficient (PCC) 0,79 ± 0,03, Bali K. K. , Selvaraj D. , Satagopam V. P. , Stösser S. , Gupta P. , Schneider R. , 1 n= 4 using LysoTracker® dye). Analysis of subcellular localization with independent Kuner R. TMEM2 fusion constructs and additional vesicular markers will be presented as a 1Universitaetsklinikum Heidelberg Pharmakologisches Institut, Im Neuenheimer Feld framework to get insights towards the cellular function of TMEM2 and to reveal the 584, 69120 Heidelberg, Germany mechanisms underlying increased amylase release from acinar cells of TMEM2-/- mice. 2EMBL Structural and Computational Biology Unit, Meyerhofstrasse 1, D69117 Heidelberg, Germany
Introduction and back ground: Cancer is a complex disorder and is very closely 020 associated with chronic pain states. 30-40% of cancer patients with ongoing treatment report chronic pain and around 90% of patients in advanced cancer states report cancer pain. miRNAs are small endogenous non-coding RNA molecules which can control The small molecule Bcl-2/Mcl-1 inhibitor TW-37 shows single-agent cytotoxicity in several target proteins either by degrading the mRNA or by preventing the protein neuroblastoma cell lines translation and form a novel set of therapeutic targets owing to the assumption that a set of disease-related proteins leading to the chronic pain pathology may be regulated by a Bachmann H. S.1, Akdeli N.1, Heukamp L.2, Schulte J.3, Siffert W.1 1 single miRNA. While deregulation of miRNAs in different nociceptive states is reported, Institut für Pharmakogenetik, Universitätsklinikum Essen, Hufelandstr. 55, 45147 their expression repertoire and mechanism of action in chronic cancer pain states has Essen, Germany 2 not yet been studied. Present study aims at the genome wide expression profiling of Institut für Pathologie, Universitätsklinikum Köln, Kerpener Str. 62, 50937 Köln, microRNAs in major pain modulating centers i.e., Dorsal Root Ganglia (DRG) via a Germany 3 comprehensive screen and to investigate the functional mechanisms of selected Klinik für Kinderheilkunde III, Universitätsklinikum Essen, Hufelandstr. 55, 45147 microRNAs in cancer pain manifestation. Essen, Germany Methods and Results: miRNA and mRNA screens were performed from the total RNA High-risk neuroblastoma (NB) remains a therapeutic challenge in paediatric oncology. isolated from lumbar DRGs 4 or 8 days after bone cancer induction using pre-spotted Pro-survival Bcl-2 family proteins critically regulate apoptosis, and may represent Illumina chips and a condensed list of miRNA-mRNA pairs, which could have potential important therapeutic targets in NB. Primary NB tumours heterogeneously express Mcl-1 role in this pain model, was identified following very stringent in silico analysis on both or Bcl-2, with high expression correlating to high risk phenotype. Co-expression can be miRNA and mRNA screens. Change in the endogenous expression levels of single detected in approximately 10% and is correlated to reduced survival. Recent studies miRNA in DRGs was achieved by intrathecal administration of custom modified miRNA- with two inhibitors that predominantly target Bcl-2 and other proteins, but not or to a mimic or inhibitor. Inhibiting the over expression of miR-1 resulted in reduced pain lesser extend Mcl-1, elucidated the importance of Mcl-1 inhibition for cytotoxicity in NB. sensitivity while mimicking the miR-370 expression lead to exaggerated pain phenotype. TW-37 is a small molecule inhibitor that showed almost equal affinity to Bcl-2 and Mcl-1. On the other hand, mimicking miR-291b-5p expression did not have any functional To explore the effect of combined Bcl-2/Mcl-1 inhibition on neuroblastoma cells, four cell consequences in pain modulation suggesting its dispensable role in mechanical pain lines (SK-N-AS, IMR-5, SY5Y and Kelly) were treated with TW-37 and changes in modulation in tested pain model. Ongoing studies are focussed on validating potential growth properties were determined. Furthermore, nude mice with Kelly (human mRNA targets for miR-1 and 370 using a qPCR screen and on further functional neuroblastoma cell line) xenografts were treated with TW-37. Using siRNA, we validation of identified mRNA targets. investigated the functional relevance of Mcl-1 and Bcl-2 in Kelly cells. For in vitro cell Conclusion: We for the first time in the field of pain physiology report a comprehensive viability we observed IC50 values of 0.59 ± 0.39 µmol/l. On treatment with 1 µmol/l dose miRNA screen and successful intervention of expression levels of single microRNA in of TW-37, all neuroblastoma cell lines analyzed showed significantly reduced dorsal root ganglion and their impact on pain phenotype. These findings together with proliferation and increased apoptosis rates. Bcl-2 as well as Mcl-1 knockdown induced ongoing intense target and molecular pathway analyses would provide new avenues for apoptosis in Kelly cells. Interestingly, TW-37 was able to reduce, but not to abrogate therapeutic interventions of chronic pain. growth of Kelly neuroblastoma xenografts in nude mice. In conclusion, combined inhibition of Bcl-2 and Mcl-1 using TW-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. Therefore, inhibition of Bcl-2/Mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. 023
Protective effects of thiol compounds on dose-dependent toxic effects of sulfur mustard in HaCaT cells 021 1 1 2 1 1 Balszuweit F. , Schmidt A. , Kehe K. , Thiermann H. , Steinritz D. 1Institut für Pharmakologie und Toxikologie der Bundeswehr, Neuherbergstrasse 11, 80937 München, Germany Localization and functional characterization of membrane transporters for 2 sulfated steroid hormones in the human testis Sanitätsamt der Bundeswehr, Dachauer Straße 128, 80637 München, Germany
Bakhaus K.1, Wapelhorst B.2, Fietz D.2, Döring B.1, Kliesch S.3, Galuska C. G.4, Introduction: Sulfur mustard (SM) is a chemical warfare agent, causing blistering, Hartmann M. F.4, Wudy S. A.4, Bergmann M.2, Geyer J.1 1 inflammation and ulceration of exposed skin. Thiol compounds including n- Institute for Veterinary Pharmacology and Toxicology, JLU Giessen, Frankfurter Straße acetylcysteine (NAC) and glutathione (GSH) have been discussed as potential 107, 35392 Giessen, Germany 2 antidotes. We investigated SM toxicity in HaCaT cells and used NAC and GSH to Institute for Veterinary Anatomy, Histology and Embryology, JLU Giessen, Frankfurter counteract those effects. Straße 94, 35392 Giessen, Germany 3 Methods: Cells were treated with 0, 1, 5 or 10 mM of GSH or NAC for 1 hour, afterwards Center for Reproductive Medicine and Andrology, UK Muenster, Domagkstraße 11, they were exposed to 0, 30, 100 or 300 µM SM for 1 hour. If extracellular interaction with 48129 Münster, Germany 4 SM was to be tested, the selected concentration of GSH or NAC was also present Division of Paediatric Endocrinology & Diabetology, Center of Child and Adolescent during SM exposure. Following 24 hours of post-exposure incubation, cytotoxicity, Medicine, JLU Giessen, Feulgenstraße 12, 35392 Giessen, Germany apoptosis and interleukine production were determined.
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Results: Dose-dependent toxic effects started at 30 µM SM. 100 µM SM led to increased 026 cytotoxicity and apoptosis and very strong inflammation.Those effects were significantly reduced, both by intracellular and extracellular interaction with NAC or GSH. Extracellular reaction of SM with GSH and NAC resulted in more pronounced protective Derived values and Databases for cancer endpoints effects. Protective effects of 1, 5 or 10 mM extracellular GSH or NAC against 100 µM SM were mostly similar. 300 µM SM induced strong cytotoxic and apoptotic effects, Barlow S. whilst inflammation decreased, probably due to limited ability of severely poisoned cells Harrington House, 8 Harrington Road, Brighton, BN1 6RE, Great Britain for interleukine biosynthesis. Intracellular interaction with GSH or NAC could not provide significant protection after exposure to 300 µM SM. Extracellular interaction of either Values for cancer endpoints for the application of the TTC approach have been derived GSH or NAC and SM significantly reduced cytotoxicity and apoptosis. 5 and 10 mM by linear extrapolation of results from animal carcinogenicity studies to calculate GSH or NAC were superior to 1 mM. Rescued cells responded by massive production of “virtually safe doses” (VSDs). A VSD is defined as an exposure that represents an pro-inflammatory cytokines, in particular when treated with 1 mM GSH or NAC only. estimated upper bound increase in risk of 1 in a million of developing cancer during a Conclusions: In summary (1) not severely, but moderately poisoned cells are mostly lifetime. In 1995, from consideration of a range of VSDs for carcinogens, the US Food responsible for inflammation observed after SM exposure; (2) GSH and NAC treatment and Drug Administration (FDA) proposed and adopted a value of 0.5 micrograms/kg of can reduce SM-induced toxic effects; (3) a dose-effects relationship for GSH and NAC diet (0.5 ppb) as a Threshold of Regulation to be used for substances present in food can be observed in cells exposed to 300 µM SL (4) protective effects were more contact materials. The FDA considered that if exposure to a substance in the diet was pronounced when GSH or NAC could already scavenge SM outside the cells; (5) rescue below this value, consumers would be protected “with reasonable certainty of no harm” of severely poisoned cells may result in a strong inflammatory reaction. and no toxicological data on the substance need be submitted. The value of 0.5 ppb is Thus, GSH, NAC or similar compounds may have a role in the future treatment of SM equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g injuries, but additional intervention to prevent adverse effects of interleukine production of fluids diet might be consumed daily. This value was subsequently incorporated into is also needed. the TTC approach to be used for assessment of substances without a structural alert for genotoxicity. In 2004, Kroes and colleagues further explored cancer as an endpoint and recommended a lower TTC value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the TTC approach of certain groups of high potency carcinogens (with VSDs below this value). In this presentation, 024 the data underpinning these TTC values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. Pregnane X Receptor and Yin Yang 1 Contribute to the Differential Tissue Expression and Induction of CYP3A5 and CYP3A4 Baranyai D., Nem D., Qiu H., Gödtel-Armbrust U., Nestler S., Wojnowski L. Universitätsmedizin Mainz Pharmakologisches Institut, Obere Zahlbacher Str. 67, 55101 027 Mainz, Germany
The hepato-intestinal induction of the detoxifying enzymes CYP3A4 and CYP3A5 by the Photorhabdus luminescens toxin TccC5 ADP-ribosylates Rho-GTPases leading to xenosensing pregnane X receptor (PXR) constitutes a key adaptive response to oral actin polymerization and stress fiber formation drugs and dietary xenobiotics. In contrast to CYP3A4, CYP3A5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven Barros Pires V., Lang A. E., Aktories K. disturbances of the steroid homeostasis. Using cell lines and mice transgenic for a Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert- CYP3A5 promoter we demonstrate that the CYP3A5 expression in these organs is non- Ludwigs-Universität Freiburg Abteilung I, Albertstraße 25, 79104 Freiburg, Germany inducible and independent from PXR. Instead, it is enabled by the loss of a suppressing yin yang 1 (YY1)-binding site from the CYP3A5 promoter which occurred in haplorrhine The bacterium Photorhabdus luminescens exists in a symbiotic relationship with primates. This YY1 site is conserved in CYP3A4, but its inhibitory effect can be offset by entomopathogenetic nematodes. After invasion of insect larvae by these nematodes, the PXR acting on response elements such as XREM. Taken together, the loss of YY1 bacteria are released and kill the insects through the action of toxin complexes. Here we binding site from promoters of the CYP3A5 gene lineage during primate evolution may studied the mode of action of the toxin complex PTC5, which comprises TcdA1, TcdB2 have enabled the utilization of CYP3A5 both in the adaptive hepato-intestinal response and TccC5. The toxin component TcdA1 is supposed to be involved in translocation of to xenobiotics and as a constitutively expressed gene in other organs. Our results thus the TccC5 component into the host cytosol. TccC5 ADP-ribosylates Rho-GTPases at constitute a first description of uncoupling induction from constitutive expression for a glutamine-63/61 resulting in constitutive activation of them. major detoxifying enzyme. They also suggest an explanation for the considerable tissue We identified RhoA, RhoB, RhoC, Rac1-3, Cdc42, Tc10 and Rop4 as subtrates for expression differences between CYP3A5 and CYP3A4. TccC5. Similar substrate specificity has been reported for the Rho-activating CNF toxins, which cause deamidation at the identical substrate amino acid (Gln63/61) ADP- ribosylated by TccC5. Modification of Rho-GTPases leads to the activation of SRF/MAL regulated gene transcription. We report the effect of TccC5 on Rho-GTPases and Rho- GTPases mediated signaling in comparison to CNF. 025 References Lang AE, Schmidt G, Schlosser A, Hey TD, Larrinua IM, Sheets JJ, Mannherz HG, Serum albumin adducts as biomarkers for systemic bioavailability of active Aktories K (2010). Photorhabdus luminescens toxins ADP-ribosylate actin and RhoA to metabolites of various glucosinolates in animal models and humans force actin clustering. Science 327:1139-1142 1 2 1 3 3 1 Barknowitz G. , Engst W. , Monien B. H. , Lehmann C. , Lippmann D. , Florian S. , Haack M.3, Brigelius-Flohé R.3, Glatt H.1 1Deutsches Institut für Ernährungsforschung Ernährungstoxikologie, Arthur-Scheunert- Allee 114-116, 14558 Nuthetal, Germany 028 2Deutsches Institut für Ernährungsforschung Analytik, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany 3 Deutsches Institut für Ernährungsforschung Biochemie der Mikronährstoffe, Arthur- Tailored ß-cyclodextrin blocks membrane translocation of binary clostridial toxins Scheunert-Allee 114-116, 14558 Nuthetal, Germany and prevents intoxication of mammalian cells
Barth H.1, Roeder M.1, Nestorovich E.2, Karginov V.3 Glucosinolates (GLS) are natural pesticides of Brassicales, which comprise many 1 important food and feed plants. Upon physical damage to the plant, the enzyme Universität Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein-Allee 11, 89081 Ulm, Germany myrosinase can convert GLS to reactive metabolites (e.g. isothiocyanates). The same 2 reaction can also be catalyzed by enzymes of the intestinal microbiota. Modification of The Catholic University of America Department of Biology, Washington, DC, United States sensor proteins (e.g. Keap-1) by some GLS metabolites leads to adaptive responses, 3 resulting in enhanced detoxification of reactive metabolites and other protective Innovative Biologics, Inc., Herndon, VA, United States reactions. At least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. However, Some pathogenic clostridia including Clostridium botulinum and Clostridium perfringens own research revealed that certain reactive GLS metabolites can covalently bind to DNA produce binary toxins which are associated with severe enteric diseases. The toxins are in vitro and in vivo, involving possible genotoxic and carcinogenic risks. Animal models taken up into the cytosol of mammalian cells where they ADP-ribosylate actin resulting are useful for studying beneficial and adverse effects of GLS. However, it has to be in depolymerization of actin filaments and cell death. Each of these toxins contain a taken into account that the toxicokinetics of GLS may differ between rodent and humans binding/translocation (B) component which forms a complex with a separate enzyme (A) and that the active metabolites are short lived and thus difficult to detect and quantify. component and mediates delivery of the latter into the cytosol by an overall common Likewise, exposure of humans to GLS and their breakdown products may enormously mechanism. Following receptor-mediated endocytosis of the toxin complexes, the B- vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation components mediate the pH-dependent membrane translocation of the A-components of plants consumed and (iii) variations in human xenobiotic metabolizing system and from the lumen of acidified endosomes into the host cell cytosol. Importantly, the B- composition of intestinal microbiota. In order to estimate individual levels of systemic components of C. botulinum C2 toxin and C. perfringens iota toxin form heptameric exposure to reactive GLS metabolites, blood protein adducts may be useful. We have pores in endosomal membranes, which serve as translocation channels for the A- developed LC-MS/MS methods for quantifying serum albumin adducts formed by components. glucoraphanin, glucotropaeolin and neoglucobrassicin. The method involves digestion of Here we provide evidence that tailored pore blockers might represent broad-spectrum the protein to amino acids and the usage of isotope-labelled amino acid adducts as inhibitors of pore-forming exotoxins from pathogenic bacteria. We demonstrate that low internal standards. Serum albumin adducts were detected in mouse models after micromolar concentrations of a 7-fold symmetrical positively charged ß-cyclodextrin feeding broccoli and pak choi respectively, as well as after administration of purified GLS derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cells from and breakdown products. Likewise, GLS adducts were detected in human blood plasma intoxication with clostridial binary toxins. Moreover, this ß-cyclodextrin derivative after consumption of broccoli or cress. This work was financially supported by the inhibited the pH-dependent membrane translocation of the A-components of C2 and iota Bundesministerium für Bildung und Forschung (Grant 0315370D). toxins in intact cells and blocked transmembrane channels formed by the B-components of these toxins in planar lipid bilayers in vitro. The substance had no effect on toxin binding to cells or on the enzyme activity of the toxins. In conclusion, the tailored ß- cyclodextrin derivative specifically blocks translocation pores of the toxins and thus prevent toxin uptake into host cells. Such substances, which were identified by targeted S9
drug design, might represent attractive candidates for novel pharmacological stable conjugates which are recognized by the immune system. In the current approaches against toxin-associated human and animal diseases. In particular, when investigations, the ability of chemical pre-treatment to interfere with antibody-protein the toxin-producing bacteria are (multi)resistant against antibiotics. binding has been investigated using ovalbumin (OVA) as the model protein and naturally occurring anti-OVA IgG from healthy human donors. References Preparation of conjugates: OVA (1 mg/mL) was dissolved in sodium borate buffer (0.1M, Nestorovich EM, Karginov, VA, Popoff MR, Bezrukov SM, Barth H (2011).Tailored ß- pH 9.4). For chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4- cyclodextrin blocks the translocation pores of binary exotoxins from C. botulinum and C. dinitrobenzene (DNFB; sensitizer) or 2,4-dichloro-1-nitrobenzene(DCNB; non-sensitizer) perfringens and protects cells from intoxication. PLoS ONE 6, e23927. was added and stirred for 2 hrs at room temperature. Unbound compound was removed by consecutive dialysis against phosphate buffered saline (PBS) and distilled water. Inhibition ELISA: Plates were coated with 100 µg/mL OVA and blocked with 10% FCS in PBS. OVA samples (native or conjugated; 0.6 – 10 µg/mL) were pre-incubated with 029 polyclonal antibodies (Ab) from pooled normal human serum for 30min and then added to the plates. Human anti-OVA IgG Abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. The concentration of soluble native OVA or Orthosteric and allosteric ligands differentially influence the conformational conjugate required to displace 50% of Ab to plate-bound OVA (IC50) was calculated change of the M muscarinic acetylcholine receptor (minimum of n=3 independent experiments). 2 Treatment of OVA with the chemical sensitizer and protein reactive DNFB resulted in Bätz J.1, Klöckner J.2, Ziegler N.1, Frölich N.1, Zabel U.1, Holzgrabe U.2, Mohr K.3, Lohse 1 1 increased IC50 value, whereas mock treatment resulted in comparable IC50 values to M. J. , Hoffmann C. native OVA. Treatment with DCNB showed that the presence of a chemical per se (and 1 Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Straße 9, 97078 possible denaturation) was not sufficient to alter the IC50 value; conjugation of the Würzburg, Germany compound to the protein was required. The analysis is not test chemical specific 2 Institut für Chemie und Pharmazie Pharmazie, am Hubland, 97074 Würzburg, Germany (specific Ab against compound-protein conjugates are not required) and the colorimetric 3 Pharmazeutisches Institut Pharmakologie und Toxikologie, Gerhard-Domagk Straße 3, analysis is unaffected by absorbance of the compound itself. Thus, this method may 53121 Bonn, Germany have utility for the identification of chemical sensitizers which are directly protein reactive. As has recently been shown different orthosteric ligands can induce distinct changes in the conformation of G protein-coupled receptors (GPCRs). Using the M2 muscarinic acetylcholine receptor (AChR) as a model we investigated whether this observation also holds true for this GPCR-subtype. Since this receptor has well been studied as far as allosteric modulation is concerned, we also investigated the effect allosteric ligands exert 032 on the change of receptor-conformation. Two FRET-sensors were generated that both carried the cyan fluorescent protein fused to their C-terminus. The FlAsH-binding domain was introduced within the third intracellular loop beneath transmembrane 2´-deoxy-cAMP in human cell lines: Another second messenger? 1 1 1 1 2 1 1 domain (TM) 5 or beneath TM6, respectively. After stably expressing the sensors in Beckert C. , Hinz C. , Beste K. , Burhenne H. , Schwede F. , Seifert R. , Kaever V. HEK293 cells, we tested commonly known orthosteric ligands and a set of allosteric 1Medizinische Hochschule Hannover Pharmakologie, Carl-Neuberg Strasse 1, 30625 ligands regarding their effect on the change of receptor conformation of the M2AChR Hannover, Germany using fluorescence resonance energy transfer (FRET)-microscopy. Upon comparison of 2BIOLOG, Bremen, Germany the FRET-signals of the TM5- and TM6-constructs evoked by the orthosteric ligands we found no evidence for a ligand-specific difference in the conformational change of the In a recent study (Naunyn-Schmiedeberg's Arch. Pharmacol.(2011) 383 (Suppl 1):22) M2AChR. However, the results obtained for the allosteric ligands suggest that some of we have shown that 2´-deoxy-cAMP (dcAMP) is synthesized by recombinant human them induce a different receptor conformation than the endogenous ligand acetylcholine soluble adenylyl cyclase (sAC). Here, we report that dcAMP can be detected and alone. quantified by liquid chromatography coupled to mass spectrometry (LC-MS) in various human cell lines. In most cells a ratio of cAMP : dcAMP of ~10 was observed. As a remarkable exception, in HL-60 promyelocytic leukemia cells, the dcAMP concentration exceeded the cAMP concentration more than 3-fold. A differential regulation of cAMP 030 versus dcAMP was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). For example, cAMP was dramatically reduced in HEK293 cells after 24 hours under resting conditions whereas dcAMP was significantly increased. In cellular subfractions of HEK293 cells AC An in vitro test battery for the prediction of skin sensitizers based on key events 2+ 2+ of the toxicity pathway assays (Mn /forskolin- or Mn /bicarbonate-stimulated) with either ATP or dATP as substrate revealed that comparable amounts of cAMP and dcAMP accumulated. In Bauch C.1,2, Eltze T.1, Ramirez-Hernandez T.1, Kolle S.1, Mehling A.3, Kimber I.1, 1 addition to sAC, membranous ACs such as AC V were capable of forming dcAMP with Landsiedel R. v and K values for dATP comparable to those for ATP. We also analyzed the 1 max M BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany substrate-specificity for several human phosphodiesterases. PDE3 and PDE4 2 University of Manchester Faculty of Life Sciences, Manchester, Great Britain hydrolyzed dcAMP more effectively than cAMP. 3 BASF Personal Care and Nutrition GmbH, Düsseldorf, Germany Taken together, these data point to a putative second messenger role of dcAMP in human cells. We are currently investigating the regulatory role(s) of cAMP and dcAMP in Skin sensitization is a common health problem caused by repeated contact with an apoptosis of HEK293 cells. As a novel tool for these studies, we will use the cell- allergen (or hapten or pro-hapten). Currently, skin sensitization relies on animal testing permeant dcAMP-acetoxymethylester which penetrates the plasma membrane and (OECD 406, 429), and so far no validated and regulatory accepted in vitro methods are releases dcAMP intracellularly. available. The purpose of this study was to establish an in vitro test strategy for the prediction of skin sensitizers based on the key events leading from the hapten contact to skin sensitization. For such an integrated approach several in vitro assays are required. We tested a total of 10 assays and – based on their performance – we selected three assays adressing chemical reactivity towards proteins and dendritic cell activation: (1) 033 direct peptide reactivity assay (DPRA) to measure chemical reactivity towards proteins with model peptides with nucleophilic amino acid residues (2) LuSens, an antioxidant response element (ARE) dependent luciferase gene activity in a recombinant cell line (3) Oxidative stress induces an increase of cGMP in the nematode Caenorhabditis myeloid U937 skin sensitization test (MUSST) or human cell line acitvation test (h-CLAT) elegans 1 2 1 1 for measurement of cell surface markers (CD54 and/or CD86) on cells with dendritic cell Beckert U. , Wen Yih A. W. , Burhenne H. , Seifert R. like characteristics. The in-house validation of the in vitro assays was performed with a 1Medizinische Hochschule Hannover Pharmakologie, Carl-Neuberg-Strasse 1, 30625 panel of 59 substances of known sensitizing potential including the 22 performance Hannover, Germany standard substances of the murine local lymph node assay. Out of the 59 substances 54 2University of Kansas Department of Molecular Biosciences, 1200 Sunnyside Avenue, were applicable to the test methods; 5 were insoluble. A combination of DPRA and Lawrence, Kansas 66045, United States LuSens adresses chemical reactivity and offered a sensitivity of 100% (enabling the exclusion of a sensitizing potential). The MUSST adressed dendritic cell activation and Caenorhabditis elegans (C. elegans) is one of the best characterized in-vivo model offered a specificity of 100% (proofing a sensitizing potential). The combination of the organisms whose entire genome is sequenced. C. elegans has already been shown to two elements resulted in an accuracy of 94% for the 54 substances. be a valuable model system for important diseases such as Parkinson’s disease and Alzheimer’s disease and it is also used as a model for ageing. Previous studies revealed the positive influence of low cGMP concentrations on C. elegans lifespan through activation of daf-16 which is the stimulus for sod-3 031 (superoxide-dismutase) transcription, an enzyme which is important for degradation of reactive oxygen species in the nematode [1,2].
Conjugation with protein reactive chemical inhibits anti-protein IgG binding: Based on these data, we tested the hypothesis whether an exposure of wild-type potential for identification of chemical sensitizers C. elegans to oxidative stress such as hydrogen peroxide, UV-light or heat provokes an opposite effect on cGMP in the animal. Therefore, we incubated C. elegans under these Bauch C.1,2, Dearman R. J.2, Eltze T.1, Kimber I.1, Ramirez-Hernandez T.1, van 1 1 three different stress conditions, freeze-cracked the animals in extraction medium and Ravenzwaay B. , Landsiedel R. detected the in-vivo cyclic nucleotide concentration via a highly sensitive mass- 1 BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany spectrometry methodology. 2 University of Manchester Faculty of Life Sciences, Manchester, Great Britain For the first time, we show that shortly before the animals died under stress conditions, Assessment of skin sensitizing potential of chemicals is an important issue for safety the cGMP concentration peaked, while the cAMP concentration was not affected. Under handling/occupational health and there is growing interest in the development of animal- all stress conditions tested, we obtained the same pattern, i.e. a significant increase in free methods for risk assessment. Chemical sensitizers have low molecular weight (< cGMP while the survival rate of living nematodes was decreasing. Under hydrogen 500 g/mol) and therefore are not themselves detected by the immune system. It is peroxide incubation and UV-irradiation, we detected particularly high cGMP levels. known that chemical sensitizers are highly protein reactive towards proteins and form S10
In conclusion, our data suggest that an elevation of cGMP is a death signal in [2] Müller, Haupt, Bildl, et al., Quantitative proteomics of the Cav2 channel C. elegans. Future studies are aiming to elucidate if cGMP and its generators, the nanoenvironments in the mammalian brain. PNAS 107, (2010) 14950-14957. guanylyl cyclases (GC) have a potential regulatory role in C. elegans lifespan when exposed to oxidative stress. By taking advantage of cGMP analogs, PDE-inhibitors, GC- and protein kinase G (PKG) mutants, we will determine in as much as GCs and their downstream target, PKG, are involved in this process. 036
References 1. Hahm, J.-H., Kim, S., and Paik, Y.-K., (2009) Aging Cell 8, 473-483 PMA-mediated downregulation of human cationic amino acid transporters 2. Doonan, R., McElwee, J.J., Matthijssens, F., et al., (2008) Genes and Development (hCATs) requires both, PKC alpha and Cdc42 22, 3236-3241 Bender-Sigel J., Closs E. I. Universitätsmedizin Mainz Institut für Pharmakologie, Obere Zahlbacher Straße 67, 55101 Mainz, Germany
034 Human cationic amino acid transporters (hCAT) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. Our earlier results have demonstrated that activation of either protein kinase Analysis of the signalling pathways of murine H1R and H4R recombinantly C (PKC) by PMA or Cdc42 by EGF leads to an internalization of these transporters. In expressed in HEK 293 cells addition, in a recent collaboration with the group of Alexander Sorkin (University of Beermann S., Seifert R., Neumann D. Colorado Denver) we found that ubiquitination and clathrin-dependent endocytosis are Medizinische Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Str.1, necessary for the down regulation of hCAT-1-mediated arginine transport by PMA (Vina- 30625 Hannover, Germany Vilaseca et al, J Biol Chem 2011 286:8697). This mechanism requires Nedd4 E3 ligases, but hCATs do not contain a PPXY motif to bind the ligases, suggesting that an The biogenic amine histamine is recognized by target cells via four different histamine adaptor protein takes part in this process. However, an involvement of the adaptor receptors subtypes (H1R - H4R), which all belong to the family of G-protein-coupled protein beta-arrestin in this mechanism could be excluded. Using siRNA against PKC seven-transmembrane receptors. Histamine plays a crucial role in allergic reactions alpha we now show that PKC alpha is the major isoform that induces the reduction of such as rhinitis or conjunctivitis and also in allergic asthma. Previously, we showed an arginine transport in human U373 glioblastoma cells overexpressing hCAT-2A-EGFP. In interaction of the effects of antagonists at the H1R and H4R in a mouse model of allergic addition, siRNA-mediated knock down of Cdc42 prevented the decrease of hCAT-2A- asthma. However, not much is known about the signaling pathway activated by murine mediated transport induced by PMA. Taken together PKC seems to negatively influence H1R and H4R. In order to analyze these signaling pathways, we established a cellular the constitutive cycling of CATs by activation the ubiquitination machinery and clathrin- model using transfected HEK 293 cells which stably express recombinant mH1R or mediated endocytosis. Cdc42 is part of this pathway. mH4R. Proper expression of the receptors was verified by western blot analysis and flow cytometry. In functional assays we demonstrated that histamine stimulation results in the 2+ 2+ increase of intracellular Ca concentration ([Ca ]i) in cells expressing either of both, mH1R (pEC50 = 8.2) or mH4R (pEC50 = 6.9). As a second readout, we analyzed the 037 modulation of forskolin-induced cAMP-accumulation. In mH1R-expressing cells the intracellular cAMP concentration was increased by stimulation with histamine, while in mH4R-expressing cells forskolin-induced cAMP accumulation was reduced. The Converging of the classical mitochondria-related pathway in Parkinson and histamine-induced effects in H1R-expressing cells were blocked by the H1R antagonist 2+ nuclear DNA-repair signaling? mepyramine ([Ca ]i: pKb = 8.6) and those in the H4R-expressing cells by the H4R 1,2 2 2 1 2 1,2 antagonist JNJ7777120 ([Ca2+] : pK = 8.8) or by pertussis toxin, which selectively blocks Scherr A. - L. , Benkler T. , Köritzer J. , Althaus F. , Bürkle A. , Beneke S. i b 1 receptor Gi-protein coupling. JNJ7777120, which behaves as a partial mH4R agonist in University of Zurich Institute of Pharmacology and Toxicology / Vetsuisse, the steady-state GTPase assay using membranes of infected SF9 cells, was without Winterthurerstrasse 260, 8057 Zurich, Switzerland 2+ 2 effect on [Ca ]i and forskolin-induced cAMP-accumulation in the mH4R-expressing cells. University of Konstanz Molecular Toxicology, Jacob-Burckhardt-Str. 31, 78457 Currently, we are investigating mitogen-activated protein (MAP)-kinase pathways Konstanz, Germany activated by the H1R and the H4R. Using a phospho-MAP-kinase array, histamine dependent phosphorylation of Erk1/2, p38, JNK, CREB, PKB (Akt), and MKK 3/6 were Parkinson disease is the second most neurological disorder worldwide. Despite the fact detected in cells expressing either of both, mH1R and mH4R. In summary, the HEK 293 that most cases are idiopathic and only few can be traced back to specific genes, cell lines stably expressing selective histamine receptors are very useful tools to general progression between both tracks of the disease is comparable. The variety of investigate HxR signaling pathways in-vitro. clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Several proteins mutated in PD are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). 035 Disturbed calcium- and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to PD progression is not known. On the other hand, experimentally Enhanced fibroblast motility in the absence of the β3 regulatory subunit of elicited Parkinsonism by oxidative stress inducers like paraquat or rotenone and MPTP voltage-activated calcium channels (inhibitors of mitochondrial complex I) lead to damage in nuclear DNA and activation of 1 2 2 1 2 1 Belkacemi A. , Laschke M. W. , Scheuer C. , Beck A. , Menger M. D. , Flockerzi V. the DNA repair protein poly(ADP-ribose) polymerase 1. By consuming substantial 1Universität des Saarlandes Institut für Pharmakologie und Toxikologie, amounts of its substrate NAD+, this enzyme can drastically decrease energy levels and Universitätsklinikum, Geb. 46, 66421 Homburg, Germany disturb the redox balance within a cell, also sensitizing it to stress induced cell death. 2Universität des Saarlandes Institut für Klinisch-Experimentelle Chirurgie, Inhibition of poly(ADP-ribose) polymerase 1 has been proven to be beneficial to some Universitätsklinikum, Geb. 65/66, 66421 Homburg, Germany extent in cell culture models as well as in experimental PD in mice. Our research focuses on a putative crosstalk mechanism we recently discovered 2+ CaVβ subunits of voltage-activated Ca channels are required for trafficking the pore- between the two pathways of experimentally induced cell death in culture models, i.e. forming CaVα1 subunit to the plasma membrane and modulate the kinetics of its current. mitochondrial signaling and PARP1-dependent poly(ADP-ribosyl)ation. Both converge Mouse embryonic fibroblasts (MEFs), acutely isolated cardiac fibroblasts (CFs) and NIH on two mitochondrial chaperones, Mortalin and TRAP1. Whereas mutations in Mortalin 3T3 fibroblasts do express CaVβ2 and CaVβ3 subunits, but we could not detect any have been reported recently to be responsible for some Parkinson disease cases in voltage-activated Ca2+ influx. Whereas in mouse cardiomyocytes or HEK 293 cells co- humans, TRAP1 is a specific target of the kinase Pink1 (PTEN induced putative kinase), 2+ expressing CaVβ3 and CaVα1 subunits a dihydropyridin-sensitive voltage-activated Ca which is often mutated in autosomal-recessive forms of the disorder. Pink1 is a central influx was readily detectable. Apparently, CaVβ subunits serve functions in fibroblasts regulator of the mitophagy process, tagging mitochondria with dissipated membrane unrelated to voltage-activated Ca2+ influx. Among the proteins potentially interacting with potential for destruction. We could show now that both chaperones bind to short-chain CaVβ3 are the inositol 1,4,5-trisphosphate receptors (IP3Rs) [1,2]. We therefore co- poly(ADP-ribose) specifically synthesized by poly(ADP-ribose) polymerase 1. We will expressed mouse CaVβ3 and mouse IP3R type 1, 2 or 3 in COS-7 cells and found co- present our most recent findings about regulation of these two chaperones after immunoprecipitation of IP3Rs using an antibody for CaVβ3 and vice versa. To study the application of Parkinson-inducing toxins. 2+ release of Ca from IP3-sensitive stores we performed FURA-2 measurements on fibroblasts isolated from wild type and CaVβ3-deficient mice either in the presence of thapsigargin or after stimulation of Gq-coupled receptors by PAR-1, LPA or Bradykinin. Receptor-activated Ca2+ release was more pronounced in β3-deficient MEFs and CFs, 038 whereas thapsigargin-induced Ca2+ release was the same in cells from both genotypes. In addition, IP3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. Fibroblasts are migrating cells and involved in Regulation of intracellular cCMP and cUMP concentrations by bicarbonate various physiological and pathophysiological processes. We therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin Beste K., Hinz C., Kaever V., Seifert R. wound healing. Angiogenesis and proliferation were apparently not different in both Medizinische Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Str. 1, genotypes but migration (measured as transwell migration and in scratch assays) and 30625 Hannover, Germany wound healing were affected in different ways. Fluorescent staining of cytoskeleton and quantification of the F-actin/G-actin ratio show similar results in both genotypes, Adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic suggesting that the increased migration rates and wound repair in β3 knockout may monophosphate (cGMP) are well-known second messengers regulating numerous 2+ result, in part, from the increased amount of IP3-releasable Ca . physiological processes like cell differentiation, blood pressure and olfaction. Besides cAMP and cGMP, we have recently shown that cytosine 3′,5′-cyclic monophosphate References (cCMP) and uridine 3′,5′-cyclic monophosphate (cUMP) are present in intact mammalian [1] Berggren, Yang, Murakami, et al., Removal of Ca channel β3 subunit enhances cells, raising the question of their putative second messenger function. Caoscillation frequency and insulin exocytosis. Cell 119, (2004) 273-284. In this study we show that intracellular cAMP, cGMP, cCMP and cUMP concentrations in HEK293 LT cells decrease after treatment with “resting” medium (M7403, Sigma- S11
Aldrich). The effect was reversible within ten minutes when cells were re-incubated in In this context, enzyme activities of oxidizing (CYP; FMO; ADH; ALDH) and conjugating regular cell culture medium. Stimulatory effects were not due to osmolarity or cell stress enzymes (NAT; UGT) were investigated in subcellular fractions of in vitro systems such due to medium exchange. Analysis of different components of both media (Table 1) as keratinocytes and RHEs (epidermis model EpiDermTM (MatTek), full-thickness skin revealed that bicarbonate stimulates accumulation of cCMP and cUMP besides cGMP models EpiDermTMFT (MatTek) and Phenion®FT (Henkel AG)) and compared to those of and cAMP in a time- and concentration-dependent manner. Bicarbonate is known to native human skin. activate soluble adenylyl cyclase (sAC) and particulate guanylyl cyclase G (pGC-G), Activities of CYP 1A, 2B and 3A isoenzymes were measured fluorometrically by regulating sugar metabolism, sperm motility and olfaction by synthesis of cAMP and oxidative desalkylation of alkoxyresorufines. FMO 1/3 activities were evaluated by cGMP, respectively. In order to identify a responsible cyclase for cCMP and cUMP HPLC/FLD detection of N-oxygenated product of Benzydamine [1]. ADH and ALDH generation after bicarbonate treatment, we are currently analyzing transiently and stably activities were investigated by photometrical detection of NADH generation during transfected HEK293 cells overexpressing various known adenylyl and guanylyl cyclases ethanol (ADH) [2] or propanal (ALDH) oxidation [3]. NAT1 activity was followed by (sAC, mAC1, mAC3, mAC9, soluble guanylyl cyclase, pGC-G, pGC-A, and pGC-D) for HPLC/UV detection of acetylated p-aminobenzoic acid. UGT1 activity was quantified their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. fluorimetrically by glucuronidation of methylumbelliferone [1]. In addition, cell fractions will be analyzed for the detection of specific cyclase During the course of this study the following results were observed: compartments. CYP1A CYP2B CYP1A/2B/3A FMO1/3 ALDH ADH NAT1 UGT1 dermal Table 1: Prominent differences of regular medium (left) and “resting” medium (right) system Compound DMEM high glucose Sigma M7403 [nmol/min/mg] Fetal serum albumin [%] 10% - human skin < LOQ < LOQ < LOQ 3.6 6.24 9.06 1.8 0.12 Glucose 4,500 1,081 Phenion®FT < LOQ < LOQ < LOQ 4.02 2.58 7.45 16.99 0.16
CaCl2[µM] 1,800 30 EpiDerm™FT < LOQ < LOQ < LOQ 6.68 2.77 9.18 8.87 0.19 3+ Fe [µM] 247 - EpiDerm™ < LOQ < LOQ < LOQ 5.95 2.01 < LOQ 11.21 1.98 KCl [µM] 5,400 1,500 keratinocytes < LOQ < LOQ < LOQ < LOQ 4.87 < LOQ 0.86 < LOQ Na2HCO3 [µM] 44,000 14,000 (LOQ = limit of quantification) L-Ala [mg/L] 84 8.91 Since the metabolic competence of RHEs is confirmed, these in vitro systems are L-Iso [mg/L] 105 1.968 estimated as suitable for further toxicity tests (e. g. genotoxicity by Comet Assay), where L-Tyr [mg/L] 89.47 3.41 metabolic activation of substances may play a crucial role. However, for the data assessment, the determined metabolic profiles should be taken into account. Nicotinamide [mg/L] 4 0.03663 We acknowledge BMBF funding this project (0315226D). Pyridoxal [mg/L] 4 0.06171 Pantothenic acid [mg/L] 4 0.238 References [1] Jäckh C, Blatz V, Fabian E, Guth K, van Ravenzwaay B, Reisinger K, Landsiedel R (2011) Toxicology in Vitro 25, 1209-1214 [2] Kawashima Y, Someya Y, Sato S, Shirato K, Jinde M, Ishida S, Akimoto S, Kobayashi K, Sakakibara Y, Suzuki Y, Tachiyashiki K and Imaizumi K (2011) J. Toxicol. Sci 36 (1), 101-108 039 [3] Jones CR and Lubet RA (1992) Biochemical Pharmacology 44 (8), 1651-1660
Remodeling of ion-channels and calcium-handling proteins after chronic ventricular pacing 1 2 2 3 4 1 1 041 Blanke K. , Dhein S. , Rastan A. , Hiyasat B. , Janousek J. , Dähnert I. , Salameh A. 1Heart Center, University of Leipzig Department of Pediatric Cardiology, Leipzig, Germany 2+ 2 PARP-1 and PARP-2 regulate cytosolic Ca shifts in H2O2 induced cell death and Heart Center, University of Leipzig Department of Cardiac Surgery, Leipzig, Germany autophagy 3King Hussein Medical Center Department of Cardiac Surgery, Amman Jordan 4University Hospital Motol Kardiocentrum and Cardiovascular Research Center, Prag, Blenn C., Wyrsch P., Bader J., Althaus F. Czech Republic University of Zurich-Vetsuisse Institute of Veterinary Pharmacology and Toxicology, Winterthurerstrasse 260, 8057 Zurich, Switzerland Question: Long term ventricular pacing, especially at the right ventricle (RV), results in 2+ On the cellular level oxidative stress provoke pleiotropic responses ranging from left ventricular (LV) failure. There are several lines of evidence that disturbed Ca - 2+ homeostasis is involved in the pathophysiology of human heart failure. In this study we survival, autophagy to death. All are characterized by perturbations in cytoplasmic Ca examined if ventricular pacing affects the Na+- and Ca2+-channels and the expression of levels resulting from either the insult alone or its interplay with cellular signaling. Here we 2+ demonstrate how the presence or absence of the DNA nick sensors poly(ADP- Ca -handling proteins and investigated if there is a differential effect between right 2+ ventricular free wall (RVFW) pacing and left ventricular apex (LVA) pacing. ribose)polymerase 1 and 2 (PARP-1, -2) interfere with Ca signaling and thereby dictate Methods: After AV-node ablation 14 minipigs underwent ventricular pacing at 120 cell fate after hydrogenperoxide (H2O2) challenge. PARP-1 and PARP-2 show distinct beats/min (DDD mode) for one year. 7 minipigs were paced from the RVFW and 7 roles in transient receptor potential melastatin-like 2 channels (TRPM2) mediated cytosolic Ca2+ shifts originating from extracellular space and lysosomes. The different minipigs from the LVA, respectively. 7 minipigs with normal sinus-rhythm served as 2+ control group. Patch-clamp-experiments were studied to measure Na+-and Ca2+- Ca signals encode for specific subsequent stress kinase responses and thereby 2+ currents. Western-Blots were carried out to investigate the expression of the Ca - modify the ability to undergo cell death. Remarkably we detected H2O2 induced 2+ autophagy in parp-1-/- background, but not in their wildtype counterpart. Gene silencing handling proteins L-type Ca -channel, Serca2 and phospholamban. -/- Results: Both RVFW- and LVA-pacing led to significant decreased Ca2+-current- of the autophagy upstream regulator Beclin-1 protected parp-1 cells partially from cell densities in cardiomyocytes of the LV compared to the control group. The plateau phase death demonstrating an integral role of autophagy in oxidative stress induced cell death of the action potential was significantly shortened after ventricular pacing in relation to in the absence of PARP-1. control minipigs. Furthermore cardiomyocytes of RVFW- and LVA-paced minipigs had significant lower Na+-current-densities than control minipigs. The action potential amplitude was significantly decreased after RVFW- and LVA-pacing whereas the diastolic potential remained unchanged. The expression of the L-type Ca2+-channel was 042 significantly reduced after ventricular pacing, regardless of the pacing site. In contrast RVFW- and LVA-paced minipigs showed significant increased Serca2-expression. The expression of phospholamban remained unchanged after RVFW- and LVA-pacing Adenine receptor: Functionality and possible signalling pathways compared to control minipigs. Bloßfeld M.1, Borrmann T.2, Obst A.1, Bumnaran B.1, Müller C. E.2, Siegert F.1, Nieber Conclusion: In a chronic animal model ventricular pacing leads to remodeling of ion- 1 2+ K. channels and Ca -handling-protein-expression, regardless of the pacing site. 1 University of Leipzig Institute of Pharmacy; Pharmacology for Natural Sciences, Talstraße 33, 04103 Leipzig, Germany 2University of Bonn PharmaCenter Bonn, Pharmaceutical Institute, An der Immernburg 4, 53121 Bonn, Germany 040 Adenine has recently been identified as an endogenous ligand of a novel G-protein coupled receptor in rats. Radioligand binding studies using membrane preparations Investigation on metabolic competence of dermal systems: native human skin, in provide evidence for expression of adenine receptors in rat neuronal tissue which could vitro skin models and keratinocytes be coupled to a Gi-protein. The funcionality and relevance of this new receptor is still Blatz V.1, Jäckh C.1, Reisinger K.2, Fabian E.1, van Ravenzwaay B.1, Landsiedel R.1 unknown. The aim of the present study was to detect the adenine receptor on rat 1BASF SE Experimental Toxicology and Ecology, Carl-Bosch Str. 38, 67056 neuroblastoma cells (B104) and to study the signalling pathways. Additionally the role of Ludwigshafen, Germany adenine receptor activation was investigated under hypoxic conditions using cytotoxicity 2Henkel AG & Co. KGaA KKD- Biological & Clinical research, Henkelstr. 67, 40589 assays and calcium imaging as well as intracellular recordings on cortical pyramidal Düsseldorf, Germany cells in rat brain slices. The adenine receptor mRNA was detected using qualitative RT- PCR. Adenine (10µM to 1mM) did not influence the basal intracellular calcium 2+ The implementation of reconstructed human skin equivalents (RHEs) as an alternative concentration but inhibited the ATP-induced increase of intracellular Ca concentration method for dermal toxicity testing became very prominent in the last decades. Their in B104 cells. Under hypoxic conditions adenine reduced cell death in rat neuroblastoma advantages are e. g. the human cell origin and an organ-like 3D structure. Already cells. In rat brain slices adenine depressed concentration-dependently the electrical regulatory accepted methodologies are widely in use for testing the skin corrosion evoked synaptic potentials but had no effect on membrane potential and input (OECD 431) and irritation (OECD 439) within RHEs. But there are still some questions resistance. The influence of hypoxia on the synaptic transmission was attenuated by open, one of them the metabolic competence of such dermal systems. adenine (1mM). In conclusion our findings indicate a neuroprotective role of adenine receptor activation under hypoxia. The inhibition of different mechanisms of the S12
signalling pathway indicates that in B104 cells the adenine receptor couples to a Gq- 045 protein followed by activation of phospholipase C pathway. These findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. Toxicity of silver nanoparticles in intestinal cells
Boehmert L.1, Hansen U.1,2, Girod M.2, Niemann B.1, Thuenemann A.2, Lampen A.1 1Bundesinstitut für Risikobewertung 5 - Lebensmittelsicherheit, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany 043 2Bundesanstalt für Materialforschung und -prüfung 1.3 - Strukturanalytik, Polymeranalytik, Richard-Willstätter-Straße 11, 12489 Berlin, Germany
Exposure to heavy metals via food consumption Introduction: 1 1 2 2 1 Blume K. , Lindtner O. , Schneider K. , Schwarz M. , Heinemeyer G. The rapid development of nanotechnology has been accompanied by an increased 1Bundesinstitut für Risikobewertung Expositionsschätzung und -standardisierung, concern for the safety of nanomaterials. Especially silver nanoparticles are used in many Diedersdorfer Weg 1, 12277 Berlin, Germany manufacturer identified consumer products including silver coated food contact materials 2Forschungs- und Beratungsinstitut Gefahrstoffe GmbH (FoBiG), Klarastraße 63, 79106 or hydrosol silver supplements. These products lead to an intentional or unintentional Freiburg, Germany oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. The human cell line Caco-2 is a well established model system in studying effects on human In the scope of the project LExUKon ("Foodborne exposure to environmental enterocytes. Although these cells are colon carcinoma cells and exhibit typical features contaminants – Data analysis to support and standardise exposure assessments based of cancer cells when they are kept sub confluent, these cells have the capability to on NVS II") exposure to the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg) via differentiate into polarized cells with morphological and biochemical properties of small food consumption has been assessed for the German adult population based on the enterocyte cells. National Nutrition Study II (NVS II) compiled by the Max Rubner-Institute (MRI) in 2005/2006. The LExUKon project has been conducted from autumn 2008 to the end of Methods: 2010 by the Federal Institute for Risk Assessment (BfR) in co-operation with the We investigated the effects of silver nanoparticles on these colon carcinoma Research and Advisory Institute for Hazardous Substances (FoBiG) and the Department (proliferating) and small intestinal epithelium like (differentiated) Caco-2 cells. The silver of Statistics of the University of Bremen on behalf of the Federal Ministry for the nanoparticles AgPURE were commercially available from rent-a-scientist GmbH. The Environment, Nature Conservation and Nuclear Safety. behaviour of these silver nanoparticles in cell culture medium were characterised using The updated intake assessments show that especially foods regularly consumed such asymmetric flow-fild flow fractionation (A4F), small ankle x-ray scattering (SAXS) and as vegetables and grain contribute mainly to exposure of Cd that is about 1.5 µg/kg body dynamic light scattering (DLS). We investigated the particle toxicity on both cell states weight (bw) per week for average consumers over all food groups. This corresponds to using Cell Titer Blue Assay, xCELLigence impedance measurements, Annexin-V and 58% of the tolerable weekly intake (TWI) of 2.5 µg/kg bw for Cd defined by the European Caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. Food Safety Authority (EFSA) in 2009. Beverages and vegetables are the food groups most relevant for exposure to Pb. About Results: 3.7 µg Pb/kg bw is taken up by average consumers that is below the benchmark dose The AgPURE stock solution is an aqueous suspension of silver particles with a metal for renal toxic effects (4.41 µg/kg bw) defined by EFSA for the weekly Pb intake. core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. For Hg the intake amounts for all population groups examined were significantly below AgPURE silver nanoparticles were toxic for proliferating as well as differentiated Caco-2 the toxicological reference values. For average consumers the weekly intake of Hg is cells in a time and concentration depending manner. The presence of foetal calf serum 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. in the incubation medium has a minor influence on the toxicity. Prior to cell death, However, individual population groups and high consumers reach and/or exceed the morphological abnormal adherence characteristics and morphological changes in the toxicological reference values for the daily intake amounts for Cd and Pb. High cells were observed using microscopy and quantified by xCELLigence impedance consumers almost reach the TWI for the Cd with 94%. For Pb a weekly intake of 5 µg/kg measurement. It is concluded that cell death is caused by an oxidative stress related bw was estimated for high consumers that exceeds the benchmark dose for renal toxic mechanism rather than apoptosis. effects for the weekly Pb intake. The results show that data collection should also focus on highly consumed and not only on highly contaminated foods. Further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. It’s recommended to 046 consider further measures in view of the reduction of contents of environmental contaminants in foods. However, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). The release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin Böhm A.1, Polzin A.2, Lüth A.3, Kleuser B.3, Rassaf T.2, Kelm M.2, Kroemer H. K.1, Schrör K.4, Rauch B. H.1 044 1Universitätsmedizin Greifswald Institut für Pharmakologie, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany 2Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie, Dynamics of G-protein-p63RhoGEF-interactions Universitätsstraße 1, 40225 Düsseldorf, Germany 1 1 2 1 Bodmann E. - L. , Rinne A. , Wieland T. , Bünemann M. 3Universität Potsdam Institut für Ernährungswissenschaft, Arthur-Scheunert-Allee 114- 1Philipps-Universität Marburg Institut für Pharmakologie und Klinische Pharmazie, Karl- 116, 14558 Nuthetal, Germany von-Frisch-Str.1, 35043 Marburg, Germany 4Universitätsklinikum Düsseldorf Institut für Pharmakologie und Klinische 2Ruprecht-Karls-Universität Heidelberg Institut für Experimentelle und Klinische Pharmakologie, Universitätsstraße 1, 40225 Düsseldorf, Germany Pharmakologie und Toxikologie, Maybachstr. 14, 68169 Mannheim, Germany Objective: The sphingosine-derived lipid signaling molecule sphingosine-1-phosphate Major cell biological processes are regulated by Rho-GTPases, actin-mediated (S1P) is an important mediator of vascular homeostasis and stored in large quantities in processes in particular. Amongst others, Rho-GTPases are stimulated by the receptor- platelets. Recently, we have shown that its release from human platelets after activation mediated activation of Gα12/13 and Gαq via specific RhoGEFs. The p63RhoGEF is of the protease-activated receptor-1 (PAR-1) by thrombin is dependent on thromboxane activated by Gαq and plays a major role in the acute response of vascular smooth (TX) formation. In the present study, we observed that the administration of the muscle cells to angiotensin II treatment. The aim of the present study was to establish a cyclooxygenase inhibitor acetylsalicylic acid (ASA, Aspirin) attenuates the release of FRET-assay between Gαq-CFP and Venus-p63RhoGEF and characterize the dynamics S1P in patients with acute coronary syndrome. of p63RhoGEF-Gαq-interaction in single living cells. The fusion of p63RhoGEF with Methods: Blood samples were taken from patients with acute coronary syndrome (ACS) Venus resulted in a functional Gαq-regulated p63RhoGEF-protein as determined by before and after intravenous treatment with 500 mg ASA. S1P was measured in platelet- means of Rho-Luziferase-Assays. Whereas no specific FRET signal was observed richt (PRP), platelet-poor (PPP) and washed platelets (WP) by mass spectrometry; between the two interaction partners in the absence of receptor stimulation, a robust and thrombin-antithrombin (TAT)- complexes by ELISA. Patients (n=25): Age 60±10 years, rapid FRET signal developed in response to stimulation of histaminergic H1- and 60% male, 3±2 cardiac risk factors, 44% were on a permanent treatment with 100 mg cholinergic M3-recptors. The onset of this signal after rapid application of agonist ASA, 8% were treated with additional Clopidogrel (75 mg). Age matched patients (n=10) paralleled Gαq activation kinetics. Similar to the kinetics of Gαq-protein deactivation the with stable coronary syndrome (all on 100 mg ASA) were used as control group. dissociation of p63RhoGEF and Gαq after withdrawal of agonist was slow (tens of Results: Enhanced formation of thrombin in patients with ACS was confirmed by seconds). The specificity of the FRET signal between Gαq-CFP and Venus-p63RhoGEF detection of increased plasma TAT-complexes (15.1±5.6 µg/L) versus control patients was verified by introducing point mutations rendering p63RhoGEF unable to bind to (3.2±0.2 µg/L). The levels of S1P in PRP from ACS patients before and after acute ASA active Gαq. Furtehrmore we observed a robust acceleration of the dissociation of treatment did not differ (866±70 pmol/mL vs 927±82 pmol/mL). In contrast, ASA p63RhoGEF and Gαq upon cotransfection of RGS2, suggesting a very short lifetime of treatment significantly decreased S1P levels was in PPP (621±51 pmol/mL vs 548±46 the p63RhoGEF-Gαq-complex or the ability of RGS2 to bind to p63RhoGEF-associated pmol/mL) and increased S1P in WP (199±25 pmol/mL vs 382±45 pmol/mL). In Gαq. Taken together, FRET-based imaging of the interactions between p63RhoGEF and comparison, considerably higher S1P concentrations were observed in control patients: Gαq revealed fast interaction kinetics closely resembling G-protein activation kinetics, 1768±47 pmol/mL in PRP, 1245±34 pmol/mL in PPP, and 634±39 pmol/mL in WP. In both of which can be regulated by RGS2. addition, in vitro data suggest that the release of S1P from platelets involves signaling via classical protein kinase C isoforms. Conclusions: Acute coronary syndrome leads to S1P release from human platelets. This can be attenuated by intravenous treatment with 500 mg ASA. The clinical implications require further clarification.
S13
047 paraneoplastic hypercoagulability. Taken together, this record does not fulfill SUSAR criteria as defined by GCP guidelines.
Drug-induced Cancer as SUSAR Signal in Clinical Trials (Part I): Lung Cancer in a References Patient with Rheumatoid Arthritis and Long-term MTX Use – A Case Report from 1. Eckhardt et al., Bundesgesunheitblatt Gesundheitsforschung Gesunheitschutz, 48(2), Pharmacovigilance Practice of an Academic Ethics Committee 2005. 2. NAVIGATOR Study Group, McMurray et al., N. Engl. J. Med., 362(16), 2010. Böhme A., Siegert J., Kirch W. 3. NAVIGATOR Study Group, Holman et al., N. Engl. J.Med., 362(16), 2010. Technische Universität Dresden Ethikkommission, Fetscherstrasse 74, 01307 Dresden, 4. Prescribing information for Starlix® (nateglinide tablets, Novartis), Diovan® (valsartan Germany tablets, Novartis) and Norvasc® (amlodipine tablets, Pfizer) 5. Gonzalez et al., Am. Surg., 76(12), 2010. In compliance with Good Clinical Practice (GCP), the sponsors of clinical trials are 6. Hanahan, Weinberg, Cell, 100(1), 2000. obliged to forward all suspected, unexpected, serious adverse reactions (SUSAR) to 7. Hanahan, Weinberg, Cell, 144(5), 2011. competent authorities (including all involved ethics committees) within statutory periods of 7 (death) and 15 days (non-fatal events), respectively. Severe adverse events (SAE) and reactions (SAR) have to be communicated periodically to investigators and involved public instances by manufacturers. Empirically, maximal 15 percent of incoming report forms are assessed as relevant SUSARs by content. 049 We discuss causality of an authentic case from floods of SUSAR notification forms received by our bureau on every day. A 53 years old male was primarily diagnosed with small cell lung cancer in August 2011. Cytoprotective effects of aqueous extracts from Marshmallow roots (Althaea It was accompanied by membranous nephropathy with nephrotic syndrome, leg edema officinalis L.) 1 1 2 3 3 1 and rapid weight gain. Because of rheumatoid arthritis (known since 2008) the patient Böker I. , Sendker J. , Stark T. , Kelber O. , Fink C. , Hensel A. received both methotrexate (MTX) and prednisolone for approximately 3 years 7 1University of Münster Institute of Pharmaceutical Biology and Phytochemistry, months. Moreover, Tofacitinib, an investigational inhibitor of JAK kinase (formerly known Hittorfstraße 56, 48149 Münster, Germany as Tasocitinib) completed long-term immunosuppressive strategy since 1099 days. The 2Technical University of München Chair of Food Chemistry and Molecular Sensory subject smoked 40 to 60 cigarettes a day for more than 33 years, suffered from Science, Lise-Meitner-Straße 34, 85354 Freising, Germany interstitial pneumonia since March 2007, fully developed lung emphysema since 3Steigerwald Arneimittel GmbH, Havelstraße 5, 64295 Darmstadt, Germany February 2008 and was occupationally exposed to noxious agents within a printing house (corn starch dust, solvents) during several decades. Extracts from the roots of Althaea officinalis L. (Marshmallow) are traditionally used for Permanent treatment with immune modulators (especially MTX, also Tofacitinib, and treatment of dry cough and irritation of oral, pharyngeal or gastric mucosal membranes. glucocorticoids) heightens the individual cancer risk substantially. In this context, This effect is due to the high content of mucilage polysaccharides. These aforementioned membranous nephropathy should be regarded most likely as polysaccharides are regarded as active compounds by forming bioadhesive layers on paraneoplastic syndrome. Generally accepted “Hallmarks of Cancer” Conception by irritated mucus membranes [1] and by stimulating keratinocytes’ and fibroblasts’ cellular Hanahan and Weinberg (2000, 2011) holds true for small cell lung cancer as well. activity and proliferation rate [2]. Pathogenesis of solid tumors has to be perceived as a multistep process whereas it Within our present study, marshmallow roots were investigated for the occurrence of usually takes decades of continuous exposure to various risk factors (e. g. smoking, further secondary compounds which might be responsible for the tissue protective carcinogenic pollutants, radiation) to acquire the characteristic traits of fully transformed activity of aqueous extracts. Different homologues of N-Phenylpropenoyl-L-amino acids malignant cells. Only moderate duration of drug exposure (less than 4 years) widely (NPA) [3] with keratinocyte stimulating activity were found for the first time in the plant excludes putative adverse drug reactions as a sufficient cause of disease. Coincident material and quantified by LC-MS/MS. Additionally, a high content of glycine betaine study participation at the point of diagnosis led to aberrant announcement of the entire was found by LC-MS, beside higher amounts of the oligosaccharide raffinose. About tumor-related symptomatology as a presumed SUSAR signal. 10% of the water extract was found to be constituted by free amino acids with proline, arginine and aspartatic acid being the main components. These polar, low-molecular, References water soluble compounds belong to the group of compatible solutes, for which protective 1. Eckhardt et al., Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz, effects against proteine membrane systems during stress conditions are known. 48(2), 2005. Therefore extracts and pure compounds were investigated for protective effects against 2. Böhme et al., Br. J. Clin. Pharmacol., 70(Suppl.1), 2010. the model enzymes lactate dehydrogenase (LDH) and glucose oxidase (GOD) under 3. Investigator's Brochure Tofacitinib (CP-690,550) heat and dry stress conditions [4]. Especially the aqueous extract and proline caused a 4. Prescribing Information for Trexall® (methotrexate tablets) and generic prednisolone higher residual activity related to an untreated control. Therefore protective properties of tablets (both available on Micromedex® database) these agents against proteins under the stress conditions can be claimed. 5. Buchbinder et al., Arthritis Rheum., 59(6), 2008. In vitro investigations on a human nasopharyngeal cell line (KB-cells) by MTT-assay 6. Hanahan, Weinberg, Cell, 100(1), 2000. showed that neither the extract of marshmallow roots nor the compounds had any toxic 7. Hanahan, Weinberg, Cell, 144(5), 2011. effect on the KB-cells. From these data cytoprotective effects of the marshmallow extract may be explainable.
References 048 1. Schmidgall et al., Planta Medica, 2000, 66, 48 – 53. 2. Deters et al., Journal of Ethnopharmacology, 2010, 127, 62-69. 3. Hofmann et al., Planta Medica, 2007, 73, 142-150. Drug-induced Cancer as SUSAR Signal in Clinical Trials (Part II): Renal Cancer in 4. Borges et al., Extremophiles, 2002, 6, 209-216 a Patient with Short-term Use of common Antihypertensives and Nateglinide – A Case Report from Pharmacovigilance Practice of an Academic Ethics Committee Böhme A., Siegert J., Kirch W. Technische Universität Dresden Ethikkommission, Fetscherstrasse 74, 01307 Dresden, 050 Germany
In the period from October 2007 to November 2011 among 3.524 SUSAR forms Is a Chronic Inflammation the Main Trigger for Cardiovascular Diseases? 1 2 3 2 3 2 2 2 (referred to EU directive 2001/20/EG and GCP) a small proportion of 106 reports Bollmann F. , Wu Z. , Oelze M. , Siuda D. , Daiber A. , Kleinert H. , Li H. , Pautz A. (approximately 3 per cent) included suspicion of drug-induced cancer (unpublished 1University Medical Center Mainz Center of Thrombosis and Hemostasis (CTH), data). The majority of pharmacovigilance signals – i. e. serious adverse events (SAE) or Langenbeckstraße 1, 55131 Mainz, Germany reactions (SAR) – should be analyzed statistically by the sponsor’s post-marketing 2Medical Center of the Johannes Gutenberg University Mainz Department of surveillance. Pharmacology, Obere Zahlbacher Straße 67, 55131 Mainz, Germany We demonstrate a typical example of misinterpreting the initial manifestation of 3Medical Center of the Johannes Gutenberg University Mainz 2nd Medical Clinic, malignant disease as a SUSAR in a female participating in a study coincidentally. Department of Cardiology, Langenbeckstraße 1, 55131 Mainz, Germany A female of unknown age took part in a multinational study targeted on secondary prevention of type 2 diabetes mellitus or cardiovascular incidents in patients with Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease associated impaired glucose tolerance by Nateglinide and Valsartan (NAVIGATOR study). with an increased mortality from cardiovascular disorders, resulting from enhanced Concomitantly, she received amlodipine. At the end of participation period that lasted no atherosclerotic and thrombotic events. Until now it is not completely understood in which more than 335 days the subject was initially diagnosed with renal cell carcinoma that way the abnormal expression of pro-inflammatory mediators in RA patients contribute to was flanked by poor general condition, increased susceptibility to infection (e. g. this cardiovascular risk. Tristetraprolin (TTP) is a RNA-binding protein responsible for catheter-related vesico-urethritis) and cachexia (only 44 kg!). Predisposed by previously the destabilisation of many important pro-inflammatory genes, such as TNF-a or Mip1-a. detected essential hypertension, hyperlipidemia, cerebrovascular disease and It has been shown that a TTP-deficiency in mice leads to an overexpression of multitude hypertrophic cardiomyopathy, study attendant developed ischemic cerebellar stroke with inflammatory genes and therefore to a severe chronic inflammation and arthritis. In this consecutive dysarthria, impaired chewing, ataxia, facial nerve paresis, brain edema, study we investigated the relationship between chronic inflammation and the nausea and vomiting. Further follow up information was not submitted. development of cardiovascular diseases, using the TTP knock out model. Hanahan and Weinberg postulated stepwise accumulation of malignant traits by tumor In this mouse model we observed the upregulation of known TTP-targets as well as cells, whereas cancer-causing agents and conditions usually impact for decennia with increased mRNA levels of atherosclerotic and thrombotic marker genes, e.g. Cathepsin perpetual intensity. Both pharmacoepidemiological and animal-testing data revealed no S, Osteopontin, tissue factor, and von Willebrand factor. Those results implicate the relevant carcinogenic potential for administered drug regimen so far. In consideration of relation between a chronic inflammation, due to a TTP-deficiency, and cardiovascular the short exposure period a causal relationship with study regimen can be reasonably diseases like atherosclerosis. excluded. Even if the outstanding cerebrovascular high-risk constellation would be We further observed that a TTP knock out leads to a massive but cholesterol- managed rigidly, one can merely decelerate the progression of atherosclerosis. Thus, independent endothelial dysfunction. We could show, that the formation of reactive despite antihypertensive and antidiabetic prophylaxis the incidence of brain insult grows oxigen and nitrogen species is strongly increased in the heart and blood of TTP deficient moderately with age. Besides, renal cell carcinoma might also contribute to cerebellar mice. This might contribute to the dysfunction of the endothelium. Although asymmetric infarction by release of bulked tumor cell leading to paradoxical embolism and dimethylarginine (ADMA) is considered to be a cardiovascular risk marker gene, ADMA serum levels are not increased in mice lacking TTP. S14
In addition to atherosclerosis TTP knock out mice develop more cardiovascular formation. Interestingly, co-administration of the ROS scavenger N-acetylcysteine, the dysfunctions. In tail vein bleeding assays we monitored a significant difference in the glutathione peroxidase mimetic ebselen and the NADPH oxidase inhibitor diphenylen bleeding times of TTP deficient mice in comparison to wildtype mice, triggered by a iodonium chloride (DPI) drastically reduced PDGF-induced CSE expression, indicating a stronger granulopoeisis. role for endogenously produced ROS in mediating regulation of CSE. As demonstrated Our results leed us to the assumption that the chonic inflammation seems to be more by electrophoretic mobility shift (EMSA) experiments PDGF-BB induces binding of the improtant for the development of cardiovascular diseases in RA patients than the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) to a consensus traditional risk factors. antioxidant response element and this effect was also diminished by co-administration of antioxidants (DPI, NAC, ebselen). Furthermore, LPS/IFNγ- as well as PDGF-BB-induced CSE upregulation was nearly completely abolished in Nrf2-/- spleen macrophages and mesangial cells, respectively. As a consequence of the elevated CSE levels we could 051 demonstrate increased H2S levels and a higher CSE enzyme activity in mesangial cells after stimulation with PDGF-BB by using the colorimetric methylenblue method and a CSE activity assay. Importantly, in a rat model of anti-Thy-1-induced proliferative Differentially expressed cardiac genes in a mouse model with heart-specific glomerulonephritis we observed a marked upregulation of CSE protein during the course overexpression of PP2A of the disease paralleled by a stabilization of Nrf2 protein. From our data, we hypothesize that PDGF-BB-mediated regulation of CSE via a redox-mediated activation Bollmann P., Makarova E. A., Gergs U., Neumann J. of Nrf2 may constitute a protective mechanism during glomerular inflammatory disease. Institute for Pharmacology and Toxicology Medical Faculty, Martin-Luther-University Halle-Wittenberg, Magdeburger Str. 4, 06112 Halle (Saale), Germany
In transgenic (TG) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2A (PP2A) reduced cardiac protein phosphorylation, 054 cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (WT) littermates. The hearts of TG mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. Analyses of mRNAs expressed in TG Rac1 knockout protects from acute hepatic damage following doxorubicin and WT hearts (n=3) using Affimetrix mouse genome microarray chips resulted in treatment several candidate genes possibly differentially regulated. In this study, we focussed on Bopp A., Wartlick F., Fritz G. verifying the mRNA data of selected genes important for stress response and signal Heinrich-Heine-Universität Institut für Toxikologie, Universitätsstr. 1, 40225 Düsseldorf, transduction on protein level in cardiac homogenates by Western blotting. Hearts from Germany WT littermates were used as control. Compared to WT heat shock protein 25 (HSP25) and calcium calmodulin dependent protein kinase type II (CaMKII) mRNAs were Rac1 belongs to the best characterized members of the Ras-homologous (Rho) family of upregulated in TG but only HSP25 protein was increased (p<0.05, n=7-8) but not small GTPases, which are key regulators of the actin cytoskeleton. Furthermore, Rac1 is CaMKII (p>0.05). Protein phosphatase type 5 (PP5) and superoxide dismutase (SOD) part of the activation of the NADPH oxidase, which produces reactive oxygen species were downregulated on mRNA level in TG but on protein level this could be found only and regulates the activity of stress kinases (e.g. SAPK/JNK) and transcription factors for SOD (p<0.05, n=6-7). In contrast, PP5 protein was upregulated (p<0.05, n=7-8) in such as NF-κB and AP1. Anticancer drugs cause DNA damage, which in turn stimulates TG compared to WT. For comparison the regulatory A-subunit of PP2A and HSP90 the DNA damage response (DDR) regulating DNA repair, cell cycle progression and, in were studied. Both genes were unchanged on mRNA level in TG: Western blotting case of non-repairable DNA damage, triggers apoptosis. So far, a role of Rac1 in the revealed the same results for the corresponding proteins. In summary, mRNA DDR has not been reported. Based on its exceptional function as a regulator of expression data could only partially be confirmed on protein level elucidating the transcription and because of its recently found ability to translocate to the nucleus, we importance of Western blotting studies. These data indicate that increased PP2A activity hypothesize that Rac1 may be involved in the DDR. is associated with modified gene expression in TG hearts possibly affecting stress To study the in vivo function of Rac1 we used an inducible Cre-based knockout mouse response and regulation of cell signalling. (Supported by the Deutsche model (Rac1flox/flox/MxCre). Mice were treated with different doses of doxorubicin for Forschungsgemeinschaft) different periods of time. We monitored gH2AX foci formation as a marker of DNA strand breaks, used the Masson-Goldner staining for the detection of collagen accumulation, analyzed phosphorylated Histone 3 as a marker of mitotic events and performed a Tunel assay to detect apoptotic cells. 052 In the absence of Rac1 the basal mRNA expression of pro-fibrotic CTGF was decreased. Collagen levels were increased and MMP1 mRNA expression was reduced in the liver of Rac-/- animals as compared with Rac1 proficient animals. In addition we -/- Current Status of cellular research in diabetology found more apoptotic cells in Rac1 mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gH2AX foci in Rac1-/- animals was Bonifacio E. reduced in comparison to Rac1+/+ animals. We also found lower level of CTGF mRNA Center for Regenerative Therapies, Technische Universtität, Dresden, Germany expression and reduced amount of collagen in Rac1-/- mice. None of these protective effects resulting from Rac1 deficiency could be detected after administration of three Cell therapy in the form of beta cell replacement to cure diabetes has been practiced for consecutive doxorubicin injections over a time period of 21 days. There were no decades without become a routine clinical therapy. More widespread clinical application significant differences in the number of gH2AX foci or collagen accumulation. The is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells mRNA expression of CTGF was even higher in Rac1-/- animals. Furthermore the number within the first days post-transplant, the requirement of long term immunosuppression to of mitotic events was almost two times higher in the Rac1-/- mice compared to the maintain graft survival, and despite this, a loss of graft function from a recurrence of Rac1+/+ mice. autoimmunity in some patients. Research is currently dedicated to overcome each of Summarizing, our findings show that impaired hepatic expression of Rac1 protein is these limitations. Additional beta cell sources investigated include embryonic stem cell hepatoprotective against acute damage following doxorubicin exposure, but does not derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta protect against doxorubicin-induced subacute toxicity. cells. Parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. Additional therapy to reduce immune suppression includes the infusion of T regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor 055 directed compounds aimed at blocking early inflammation or autoimmunity. These efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable In vitro cytotoxicity of tBHQ (tert-butyl-hydroquinone) in patients with diabetes. Braeuning A., Vetter S., Schwarz M. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Toxikologie, Wilhelmstrasse 56, 72074 Tübingen, Germany
053 At high concentrations, tert.-butyl-hydroquinone (tBHQ), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. Here we describe that treatment of murine 3T3 cells with tBHQ in 96-well PDGF-BB induces the H2S producing enzyme Cystathionine-γ-lyase via a ROS- dependent mechanism in rat renal mesangial cells culture plates induces the death of untreated cells in neighboring wells on the same plate. The mechanisms underlying that effect were investigated. Death of the seemingly Boosen M.1, Hassan M.1, Schaefer L.1, Eisel F.1, Beck M.1, von Knethen A.2, Beck K. - 1 1 untreated neighboring cells was caused by a more toxic and volatile tBHQ oxidation F. , Pfeilschifter J. product which was formed in a non-enzymatic process involving metal ions and oxygen. 1 Klinikum der Johann Wolfgang-Goethe Universität Institut für Allgemeine The unexpected perturbation of cytotoxicity testing by the volatile tBHQ metabolite Pharmakologie und Toxikologie, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, shows that not only metabolic processes, but also non-enzymatic mechanisms have to Germany be considered as important parameters for in vitro assays. Furthermore, our data show 2 Klinikum der Johann Wolfgang-Goethe Universität Institut für Biochemie I, that even cells several wells distant from the site of treatment do not necessarily Pathobiochemie, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany constitute proper “untreated” controls when cells are treated with tBHQ, e.g. in assays aimed to analyze the activity of the tBHQ-inducible Nrf2 pathway. There is increasing evidence that hydrogen sulfide (H2S) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. In the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (CSE) in cultured rat renal mesangial cells. As demonstrated by qPCR and Western blot experiments, mesangial cells showed a marked time- and dose-dependent upregulation of CSE mRNA and protein levels after treatment with platelet-derived growth factor (PDGF-BB). The CSE upregulation by PDGF-BB is accompanied by a marked increase in reactive oxygen species (ROS) S15
056 predominantly expressed isoforms in the heart. In the heart, ACs integrate β-adrenergic (β-AR) signaling as the main physiological mechanism to improve cardiac performance. Although AC5 and AC6 share high sequence homology, opposing effects on Angiotensin II causes oxidative stress and DNA damage in mouse kidneys via the cardioprotection have been reported, where disruption of AC5, as well as angiotensin II type 1 receptor overexpression of AC6 both exerting beneficial effects in heart failure. Prospective pharmacological treatment of heart failure on the level of AC is under investigation. Brand S.1, Amann K.2, Schupp N.1 1 Our study explored the impact of AC5 KO on AC-activities in the heart at a functional Universität Würzburg Institut für Pharmakologie und Toxikologie, Versbacherstr. 9, level. 97078 Würzburg, Germany 2 Complementary, mRNA expression studies of the β-AR-G-protein-AC signaling cascade Universität Erlangen-Nürnberg Institut für Pathologie, Krankenhausstraße 8, 91054 were performed to detect possible compensatory alterations. Erlangen, Germany Hearts from 16-20 week old homozygote AC5 knockout and wild-type male littermates were examined in this study. AC activities where measured in cardiac membrane Angiotensin II (Ang II), the reactive peptide of the renin-angiotensin-system, causes preparations from left ventricles. AC activities were assessed under β-AR and G-protein vasoconstriction and, in higher levels hypertension, which is connected with an (Gs) stimulation by isoproterenol, guanosine 5’-triphosphate (GTP) and 5’-O-(3- increased cancer risk in the kidney. Treatment of male C57BL/6 mice with Ang II results thiotriphosphate) (GTPγS) as well as for direct activation by forskolin. Relative mRNA in the formation of superoxide radicals and DNA damage in the kidney as well as in the expressions for AC1-9, Gs-, Gi-a and β1-, β2-AR where measured by quantitative real- heart. To answer the question if the DNA damage is caused by hypertension or by time PCR. elevated Ang II concentrations, mice were treated with different compounds: the Surprisingly, assessment of basal, β-AR and G-protein-mediated AC-stimulation as well angiotensin-converting-enzyme blocker ramipril, the Ang II receptor blocker ramipril, the as direct activation by forskolin revealed no changes in AC activities. Besides from Ang II receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. The detection of the AC5 knockout, mRNA expressions analysis of AC1-9, Gs-, Gi-a and β1-, effect on blood pressure and renal function of Ang II-treated C57Bl/6 mice was β2-AR did not detect any compensatory alteration. examined. These findings suggest that proximal adrenergic signaling in the heart does not Treatment with Ang II led to a significant increase in blood pressure. Candesartan and necessarily require AC5. Whether physiological integration of beta adrenergic signaling hydralazine led to a decrease, whereas intervention with ramipril and tempol had no in the heart is mediated by both isoforms AC5 and AC6, or can be attributed to one main effect. isoform remains to be elucidated. Equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. In addition, histopathological changes were investigated. There was significant glomerular damage and tubulointerstitial damage in Ang II-treated animals compared to control animals, which was significantly improved by candesartan and tempol. 059 Hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. Furthermore, the Ang II-induced formation of superoxide radicals in the kidney and the Melanocortin-promoted PKA activation decreases AMPK activity via ERK-1/2 and heart was slightly affected by all interventions. Genomic damage, in the form of double LKB-1 in hypothalamic GT1-7 cells strand breaks was prevented by the Ang II receptor antagonist candesartan and the Breit A., Ellen D., Gudermann T. antioxidant tempol. Ludwig-Maximilians-Universität Walther-Straub-Institut, Goethestrasse 33, 80336 To sum up, the results from this study show that Ang II induces the elevation of markers München, Germany of kidney failure and DNA damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of DNA α-melanocyte stimulating hormone (α-MSH)-induced activation of the melanocortin-4 damage. Actually by substances not lowering blood pressure like tempol, the oxidative receptor (MC4R) in hypothalamic neurons increases energy expenditure and inhibits stress and DNA damage was ameliorated, showing the involvement of reactive oxygen food intake. Intrahypothalamic injection of melanocortins decreased food intake due to species. the inhibition of AMP-activated protein kinase (AMPK) that has recently been reported to enhance food intake in rodents. Until now, it is not clear if α-MSH affects AMPK via direct intracellular signaling cascades or if the release of paracrine factors is involved. Herein, we used a murine, hypothalamic cell line (GT1-7 cells) and monitored AMPK 057 phosphorylation at Thr172 which has been suggested to increase AMPK activity. We found that α-MSH dephosphorylated AMPK at Thr172 and consequently decreased phosphorylation of the established AMPK substrate acetyl-CoA-carboxylase at Ser79. Optimization of the BALB/c-3T3 cell transformation assay by coupling a drug Inhibitory effects of α-MSH on AMPK were blocked by specific inhibitors of protein metabolizing system kinase A (PKA) or extracellular-regulated kinases-1/2 (ERK-1/2), pointing to an important role of both kinases in this process. Since α-MSH-induced activation of ERK- Brauneis M. D., Steinberg P. 1/2 was blunted by PKA inhibitors, we propose that ERK-1/2 serves as a link between Stiftung Tierärztliche Hochschule Hannover Institut für Lebensmitteltoxikologie und PKA and AMPK in GT1-7 cells. Furthermore, down-regulation of liver kinase B-1 (LKB- Chemische Analytik, Bischofsholer Damm 15, 30173 Hannover, Germany 1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of AMPK and its The analysis of the carcinogenic potential of chemicals plays an important role in dephosphorylation induced by α-MSH. Thus, we propose that α-MSH inhibits AMPK toxicology. Up to now the acquisition of such data requires a large amount of animal activity via a linear pathway including PKA, ERK-1/2 and LKB-1 in GT1-7 cells. Given experiments. The aim of this study is to reduce the number of experimental animals the importance of the melanocortin system in the formation of adipositas detailed being used by further optimizing the BALB/c-3T3 cell transformation assay, an already knowledge about this pathway might help to develop drugs targeting obesity. well-established in vitro method. This method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. The incubation of BALB/c-3T3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. These foci can be 060 distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. A major disadvantage of the “classic” BALB/c-3T3 Autistic patients exhibit rare missense mutations in the calcium channel β2 cell transformation assay is that a number of compounds initially require a metabolic subunit gene which result in a reduced time-dependent inactivation of CaV1.2. 1 2 3 1 transformation to gain their full genotoxic potential. Hence, without prior metabolic Breitenkamp A. F. S. , Sinzig J. , Nürnberg P. , Herzig S. transformation many chemicals are not detected as carcinogenic in the above- 1Department of Pharmacology, University of Cologne, Gleuelerstr 24, 50931 Cologne, mentioned test system. To overcome this drawback the BALB/c-3T3 cell transformation Germany assay has been coupled to a drug metabolizing system, in this case the so-called liver 2Department of Child & Adolescent Psychiatry and Psychotherapy, University of S9. In a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin B1 and N- Cologne, Robert-Koch-Straße 10, 50931 Cologne, Germany nitrosodimethylamine were tested in this assay. All three compounds led to a 3Cologne Center for Genomics, University of Cologne, Weyertal 115b, 50931 Cologne, concentration-dependent increase in the number of foci formed, whereby this Germany concentration-dependent increase was observed in a non-cytotoxic concentration range. In a next step the BALB/c-3T3 cell transformation assay will be coupled to further drug Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with metabolizing systems as well as to the soft agar assay. dysfunction of social interaction and communication. A hitherto unknown complex- genetic principle of origin probably underlies ASD. So far, more than 100 candidate This study is being financially supported by the Stiftung SET and the Doerenkamp- genes were identified in literature. The patients affected with the monogenic Timothy Zbinden Foundation. syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. This single gene disorder serves as a model disease for ASD, giving insights in a possible pathophysiology. Here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium 058 channel (CaV) CaV1.2 gene (CACNA1C) results in incomplete inactivation of the L-type calcium currents (Splawski et al., Cell 2004;119:19-31). Functionally similar biophysical effects can be induced by structural variation β1- and β2-subunits of the voltage- Characterization of cardiac adenylyl cyclase in AC5 knockout mice dependent calcium channels (Herzig et al., FASEB J. 2007;21:1527-38; Jangsangthong et al., Pflugers Arch. 2010;459:399-411). Supported by findings in a meta-analysis of Bräunig J. H.1, Schweda F.2, Han P. - L.3, Seifert R.1 1 linkage data of ASD patients (Trikalinos et al., Mol Psychiatry. 2006;11:29-36), we are Medizinische Hochschule Hannover Pharmakologie, Carl-Neuberg-Str. 1, 30623 investigating a function-based candidate gene hypothesis linking the β2 subunit gene Hannover, Germany (CACNB2) with ASD. We performed a case control study sequencing all exons and 2Universität Regensburg Physiologie, Universitätsstr. 31, 93040 Regensburg, Germany 3 flanking intronic regions of CACNB2 in 155 patients with ASD. We found three rare Ewha Womans University Brain and Cognitive Science, Daehyun-Dong 11-1, Seoul missense mutations in ASD patients, but not in 259 unaffected controls. All three 120-750, Korea Republic (South) mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational Adenylyl cyclases (ACs) synthesize the second messenger cAMP. The family of ACs modification by phosphorylation. So far, we characterized two of these mutations and consists of nine membranous and one soluble isoforms with AC5 and AC6 being the S16
also a phosphorylation-mimicking mutant in electrophysiological studies. All variants 063 show a decelerated and incomplete time-dependent inactivation of the co-transfected CaV1.2 subunit. Furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. Chronic myeloid leukemia: Prediction of response to imatinib therapy using We here present mutations in the β2 subunit gene of ASD patients that result in a microRNA expression profiles retardation of inactivation behavior, thus phenocopying the monogenic Timothy Bruhn O.1, Diewock T.1, Pott C.2, Kneba M.2, Cascorbi I.1, Haenisch S.1 syndrome mutations of CaV1.2. β2 subunit mutations may influence neuronal function or 1 development in some ASD patients. Institut für Experimentelle und Klinische Pharmakologie, Hospitalstr. 4, 24105 Kiel, Germany 2 References Universitätsklinikum S-H II. Medizinische Klinik und Poliklinik im Städtischen Jangsangthong, W., Kuzmenkina, E., Khan, I.F., Matthes, J., Hullin, R., and Herzig, S. Krankenhaus, Chemnitzstraße 33, 24116 Kiel, Germany (2010). Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits. Pflugers Arch 459, 399-411. Introduction: Despite the remarkable success of imatinib treatment of chronic myeloid Herzig, S., Khan, I.F., Grundemann, D., Matthes, J., Ludwig, A., Michels, G., Hoppe, leukemia (CML), therapy resistance emerged as a major clinical problem. The aim of U.C., Chaudhuri, D., Schwartz, A., Yue, D.T., et al. (2007). Mechanism of Ca(v)1.2 this study was to identify microRNAs, which may serve as biomarkers for therapy channel modulation by the amino terminus of cardiac beta2-subunits. FASEB J 21, response or predict pathways involved in pharmacoresistance of imatinib treatment. 1527-1538. Methods: Blood was collected from 21 CML-patients, ten of whom responded to Splawski, I., Timothy, K.W., Sharpe, L.M., Decher, N., Kumar, P., Bloise, R., Napolitano, C., imatinib therapy. After RNA extraction from leukocytes, we performed a TaqMan Low- Density Array screen to determine the expression of 667 microRNAs. Statistical analysis Schwartz, P.J., Joseph, R.M., Condouris, K., et al. (2004). Ca(V)1.2 calcium channel -∆Ct dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119, 19- using the 2 method was performed. MicroRNAs showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. In addition, by using 31. 1 2 3 4 Trikalinos, T.A., Karvouni, A., Zintzaras, E., Ylisaukko-oja, T., Peltonen, L., Jarvela, I., microRNA target prediction databases (TargetScan , mirDB , PicTar , MicroCosm , Diana microT5), selected putative target genes were further functionally investigated by and Ioannidis, J.P. (2006). A heterogeneity-based genome search meta-analysis for 6 autism-spectrum disorders. Mol Psychiatry 11, 29-36. the DAVID bioinformatics database . Results: Comparing treatment-naïve responders and non-responders four microRNAs were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). Pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in CML (SMAD4, NRAS, RB1, 061 RAF1). When comparing patients' expression profiles before and under treatment, seven microRNAs were identified to be deregulated in responders and five microRNAs in non- responders. Ninety-nine targets of the latter include transcription regulators (19%), but 5-HT1A autoreceptors mediate the hyperphagic effect of 8-OH-DPAT in mice also cellular transporters (18%, especially uptake transporters of the SLC-family). Most Brosda J., Müller N., Bert B., Fink H. target genes are involved in MAPK signalling or endocytosis pathways. Freie Universität Berlin Fachbereich Veterinärmedizin - Institut für Pharmakologie und Conclusion: Analysis of microRNA expression profiles revealed four microRNAs Toxikologie, Koserstr. 20, 14195 Berlin, Germany involved in imatinib-response and 12 microRNAs deregulated during imatinib treatment. Predicted target genes code mainly for transcription factors as well as oncogenes Background: Brain serotonin (5-HT) has been implicated in the regulation of food-intake. relevant for CML and are involved in transporter expression and endocytotic processes. The ingestive effects of 5-HT are mediated by a number of different receptor subtypes under which the 5-HT1A-receptor plays a central role. Former in vivo studies have shown References an increased intake of food, elicted by 5-HT-receptor agonists. The aim of this 1http://www.targetscan.org/ behavioural pharmacologic project was to determine if the hyperphagic effect is 2http://mirdb.org/ 3 mediated by presynaptic 5-HT1A autoreceptors in the raphe nuclei or by postsynaptic 5- http://pictar.mdc-berlin.de/ 4 HT1A heteroreceptors in serotonergic terminal structures. http://www.ebi.ac.uk/enright-srv/microcosm/ 5 Methods: The effect of the 5-HT1A receptor agonist 8-OH-DPAT (0.1, 0.5 or 1.0mg/kg) was http://diana.cslab.ece.ntua.gr/microT/ investigated on feeding behaviour in non-food-deprived young-adult and adult NMRI and 6http://david.abcc.ncifcrf.gov/ transgenic L35 mice. L35 mice are characterized by an overexpression of postsynaptic 5- HT1A receptors. Results: The administration of the 5-HT1A receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. 064 Conclusion: The results confirm a key role of the 5-HT1A receptor in food intake. Further, we make the assumption that the hyperphagic effect of 8-OH-DPAT is mediated by presynaptic 5-HT1A autoreceptors in the raphe nuclei which decreases 5-HT function in the central Dissociations in the effects of beta2-adrenergic receptor agonists on cAMP nervous system. It can be speculated that the aberrant feeding behaviour of the adult formation and superoxide production in human neutrophils transgenic mice refers to a possible opposite role of the postsynaptic 5-HT receptors. 1A Brunskole I.1, Buschauer A.1, Kälble S.2, Burhenne H.2, Seifert R.2 These receptors might affect the release of neuropeptides in the hypothalamus. 1 Universität Regensburg Lehrstuhl für Pharmazeutische/Medizinische Chemie II, Universitätsstraße 31, 93053 Regensburg, Germany 2Medizinishe Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Straße 1, 30625 Hannover, Germany 062 Activation of the β2-adrenergic receptor (β2AR), a classically Gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1], which Induction of ABCC2 gene expression in a time and concentration dependent could be further therapeutically exploited. The aim of the present study was to evaluate manner by IL 1β via p38 MAPK and AKT pathway in various cell lines the effects of various β2AR ligands on cyclic adenosine 3',5'-monophosphate Bruckmüller H., Werk A., Haenisch S., Cascorbi I. accumulation (cAMP assay) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)- •- Institute for Experimental and Clinical Pharmacology, Arnold Heller Straße 3 Haus 30, induced superoxide anion production (O2 assay) in isolated human neutrophils, which 24105 Kiel, Germany are a physiologically relevant native test system. cAMP concentration in neutrophils was •- determined by HPLC/tandem mass spectrometry, and O2 formation was assessed by The efflux transporter ABCC2 (MRP2) expressed at different compartment barriers is monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. important for the elimination of various endogenous and exogenous compounds. With (-)-Isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in •- some evidence inflammatory processes regulate ABCC2 expression and cause changes inhibiting fMLP-induced O2 production than in stimulating cAMP accumulation. (-)- of absorption, distribution and clearance of a number of xenobiotics. Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP •- The investigation of the influence of interleukin (IL) 1β on ABCC2 mRNA and protein assay, but could partially inhibit fMLP-induced O2 production at higher concentrations. expression in various cell lines representing specific tissues is the aim of our study. A Moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas •- further aim is to characterize the signaling pathways regulating ABCC2 expression while the efficacy of salbutamol was more than two fold higher in the O2 assay. This suggests inflammation. that salbutamol is able to stabilize a different receptor conformation than the other two Three different cell lines a) HepG2 cells (liver tissue) and b) CaCo2 (colon tissue) both ligands. Thus, ligand-directed signaling via β2AR can also occur in human neutrophils. In without naïve IL1β expression and c) SkHep1 cells representing physiological liver addition, differences between the data from neutrophils and recombinant test systems tissue with naive IL1β expression, were stimulated with different concentrations of IL1β [2,3] were noticed, pointing to the problem of insufficient comparability of effects in (range 10 pg/ml to 10 ng/ml). Over a period of 48h samples were taken at defined time recombinant and native test systems. points. ABCC2 mRNA and protein expression were quantified by qRT-PCR and The investigation of β2AR antagonists on neutrophil granulocytes is subject of ongoing Western blot analysis, respectively. By using small molecule kinase inhibitors for signal work, in order to find out whether pKB values of β2AR antagonists in the cAMP assay •- transduction proteins (p38 MAPK, AKT, ERK1 and JNK) we analysed the signal and the O2 assay are different. Such differences were previously reported for β2AR transduction pathways associated with IL1β-mediated transcriptional ABCC2 regulation. antagonists in other test systems [4]. Moreover, studies with protein kinase A inhibitors On ABCC2 mRNA level an up-regulation in CaCo2 cells (1,27- 1,35 fold) and HepG2 should give deeper insight into the signaling events in neutrophils that result in inhibition •- cells (3,6- 4,6 fold) within the first hour after stimulation with 1 ng/ml IL1β was shown. In of fMLP-stimulated O2 production and clarify how cAMP increase interferes with this contrast SkHep1 cells demonstrated a decreased ABCC2 mRNA expression (0,59-0,62 events. fold) in comparison to unstimulated controls. The ABCC2 protein expression exhibited a time and IL1β dependent regulation as well. The analysis for the signal transduction References showed for p38MAPK a moderat time dependet down regulated phosphorylation (15%) [1] Johnson M, J Allergy Clin Immunol, 2002; 110:S282-290. in HepG2 cells whereas it showed no effect in CaCo2 cells. [2] Seifert R et al., J Biol Chem, 1998; 273:5109-5116. Concluding, the expression of ABCC2 is regulated moderately by IL1b in a concentration [3] Weitl N and Seifert R, J Pharmacol Exp Ther, 2008; 327:760-769. and time-dependent manner. Interestingly, the effects are strongly tissue-dependent [4] Baker JG et al., Mol Pharmacol, 2003; 64:679-688. concerning ABCC2 expression and signal transduction pathways and show partly contradictory results. The regulation of the different signaling pathways is currently subject of ongoing investigations.
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065 However, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in C. elegans. The methylated myricetin derivatives (100 µM) showed a decrease in lipofuscin accumulation and ROS induction and they further improved the Agonist-selective internalization of the human 5-HT receptor heat stress resistance. 2A In order to elucidate the basis of the life prolonging action of myricetin, we investigated Buchborn T., Kahl E., Höllt V., Koch T. its influence on factors known to have important functions in stress response and the Otto-von-Guericke-Universität Magdeburg Institut für Pharmakologie und Toxikologie, regulation of aging, namely the FoxO homologue DAF-16, the NAD+-dependent protein Leipziger Straße 44, 39120 Magdeburg, Germany deacetylase SIR-2.1 and the heat-shock transcription factor HSF-1, respectively. Lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant The serotonin 2A (5-HT2A) receptor is a G protein coupled receptor and the molecular strains, showing the effect is at least partially dependent on these signaling molecules. target of LSD-like hallucinogens. Downregulation of 5-HT2A receptors is an adaptive By using a hsf-1 loss of function mutant strain of C. elegans, it was further shown that process considered relevant for the therapeutic action of diverse serotonergic the life prolonging effect of myricetin is independent of hsf-1. antidepressants, such as SSRIs. Since the antidepressant targeting of 5-HT2A receptors, In conclusion, our results indicate that the life prolonging effect of myricetin is at least in however, is largely restricted to indirect agonists and/or antagonists, little is known about part dependent on daf-16 and sir-2.1, probably due to a modified expression of target the mechanisms and implications of their regulation by direct agonists. In the present genes. study we, therefore, investigated the capacity of various agonists to regulate the human HA-tagged 5-HT2A receptor by internalization. Using immunocytochemical techniques in stably transfected HEK293 cells, we show that agonists differ in their capacity to internalize the receptor. Serotonin, quipazine and DOI are the agonists most efficaciously internalizing the receptor, DMT and methysergide, on the other hand, 068 hardly internalize; other agonists like psilocin, ergotamine and LSD induce low to intermediate internalization. The specificity of the agonistic effect was demonstrated by Stimulatory and inhibitory control of phospholipase C-gamma 2 the 5-HT2A selective antagonist ketanserin, which blocked the agonist-induced internalization. In additional experiments, we show that the internalized 5-HT2A receptors Bühler A., Walliser C., Becker L., Gierschik P. colocalize with A488-labelled transferrin receptors, and that the internalization can be Universität Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein-Allee 11, blocked by high molar sucrose; these results are indicative of a clathrin associated 89081 Ulm, Germany sequestration of 5-HT2A receptors in recycling endosomes. Also, we demonstrate that the proteinkinase C activator PMA efficaciously induces 5-HT2A internalization in the Activation of phospholipase C-γ2 (PLCγ2) upon B cell antigen receptor (BCR) stimulation absence of an agonist, and that the DOI-induced internalization can be blocked by the has been implicated to be a critical step in the BCR-mediated calcium signaling. proteinkinase inhibitor staurosporine. We, thus, confirm previous findings that the Therefore it is important to understand the mechanisms of how the activity of PLCγ2 is activation of proteinkinases seems to be necessitated for the 5-HT2A internalization to stimulated and inhibited. occur. Overall, we conclude that the internalization of the human 5-HT2A receptor is The mammalian PLCs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. agonist-selective, and employs a proteinkinase (possibly PKC) dependent, clathrin- Within the PLCγ subfamily, the two PLCγ isoforms share a number of features that are coated endosome associated pathway. As there is recent evidence that the regulation of distinct from those of the other PLC subfamilies. The most striking difference is the 5-HT2(A) receptors by agonists might have antidepressant(-like) properties, knowledge insertion of additional domains between the catalytic subdomains X and Y. This specific about the agonist-selective processing of 5-HT2A receptors could help to identify array (SA) contains a second, split pleckstrin homology (spPH) domain, consisting of agonists most promising for future (pre-)clinical research. two halves separated by two Src homology 2 (SH2) domains and one Src homology 3 (SH3) domain. There is abundant evidence in the literature that PLCs are autoinhibited in their basal state by structural elements within their X/Y linker, pointing to a conserved role of the 066 X/Y linker in autoinhibitory regulation of PLC isozymes. Data from our group show that PLCγ2 is also regulated by autoinhibitory elements within its specific array (Walliser et al., 2008; Everett et al., 2011). Our recent data demonstrated that PLCγ2 is negatively Non-clinical safety assessment of homeopathic medicinal products: criteria for regulated by its SA. Specifically, within the SH domain tandem, the C-terminal SH2 establishing a first safe dilution (SH2C) and the SH3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of PLCγ . Buchholzer M. - L., Werner C., Knoess W. 2 PLCγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon BfArM Bundesinstitut für Arzneimittel und Medizinprodukte Zulassung 4, Kurt-Georg- BCR stimulation (Kim et al., 2004). Both tyrosines are located in the linker between the Kiesinger-Allee 3, 53175 Bonn, Germany SH2C and the SH3 domain, which we have shown to be the major elements involved in
autoinhibitory regulation of PLCγ2. Interestingly, a novel phosphorylation site in PLCγ2 Like all human medicinal products the homeopathic medicinal products for human use was found in non-small cell lung cancer (NSCLC) tissue which is located at tyrosine must demonstrate adequate safety. In general, they are regulated according to the residue 733 (Rikova et al., 2007). In this work, we demonstrate, for the first time, the analogue non-clinical safety principles (Points to Consider on Non-Clinical Safety of activation of PLCγ2 by phosphomimetic mutations in these three positions and the Homeopathic Medicinal Products of Botanical, Mineral and Chemical Origin, adoption by functional interplay of the three tyrosine phosphorylation targets. Most interestingly, HMA 2007). One particular approach is the recently introduced concept of a first safe mimicking phosphorylation of Tyr733 is critical to fully activate the enzyme. The results dilution (FSD; INTRODUCTION TO THE LIST OF FIRST SAFE DILUTIONS, adoption not only point to a crucial role of PLCγ2 in pulmonary tumorigenesis, but also prompt and by HMA 2010). stimulate the search for the protein kinase involved in phosphorylating PLCγ2 at Tyr733. This contribution summarizes the first experiences in establishing FSDs of a selection of given homeopathic preparations by BfArM. For a given preparation the major toxicological concern and available data set is identified. This determines the safety assessment route: food regulation, permitted daily exposure (PDE), threshold of toxicological concern (TTC) or lowest human 069 recommended dose (LHRD/100). Finally the acceptable amount/tolerable daily intake is derived and the respective FSD is calculated. For example the draft evaluation for Reserpinum (Ph. Eur. method 4.1.1) and for Molecular characterization of hepatotoxic effects of perfluorooctanoic acid Atropine (Ph. Eur. method 3.1.1 or 4.1.1) based on LHRD leads in each case to a (PFOA) suggested FSD of D7 related to 10 g of preparation. Furthermore, the draft evaluation Buhrke T., Scharmach E., Lampen A. for Potassium iodide (Ph. Eur. method 3.1.1 or 4.1.1) based on food legislation emerged Bundesinstitut für Risikobewertung (BfR) Lebensmittelsicherheit, Max-Dohrn-Str. 8-10, a proposed FSD of D6 related to 10 g of preparation. 10589 Berlin, Germany The concept of FSD combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. Perfluorooctanoic acid (PFOA) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt- and water-repellent properties. PFOA is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. The toxicological data of PFOA give cause for 067 concern as the substance was shown to damage the liver of rodents and to impair embryo development. Currently, the hazard potential of PFOA for humans is controversially discussed. Impact of myricetin and its methylated derivatives laricitrin, syringetin and In this study the human liver cell line HepG2 was employed to analyse the hepatotoxic myricetin-3`,4`,5`-trimethylether in C. elegans effects of PFOA on the cellular and on the molecular level. PFOA was shown to stimulate cellular proliferation at concentrations in a range between 5 µM and 25 µM. At Büchter C.1, Ackermann D.1,2, Chovolou Y.1, Fritz G.1, Wätjen W.1 1 concentrations higher than 25 µM the substance was cytotoxic to the cells (IC50 47µM). Heinrich-Heine-Universität Institut für Toxikologie, Universitätsstr.1, 40225 Düsseldorf, Cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity Germany 2 was detected up to a level of 100 µM PFOA. On the molecular level PFOA is known to Heinrich-Heine-Universität Institut für Pharmazeutische Biologie und Biotechnologie, act as an agonist of the peroxisome proliferator-activated receptor alpha (PPARα), and Universitätsstr.1, 40225 Düsseldorf, Germany the observed hepatotoxic effects in rodents are associated with PFOA-mediated PPARα activation. Here we show that PFOA has the capacity also to activate the human isoform Polyphenolic compounds ubiquitously present in herbal food are discussed to contribute of PPARα. Additional human nuclear receptors were tested for activation by PFOA, and to the health beneficial effects of a diet rich in vegetables and fruits. Additional to a PPARγ as well as the pregnane X receptor (PXR) were shown to be activated at high strong antioxidative activity of various flavonoids, most of these substances display a concentrations of PFOA whereas PPARδ and the liver X receptor alpha (LXRα) were variety of other pharmacological properties. We investigated the flavonoid myricetin insensitive to activation by PFOA. Notably, we observed a significant inhibition of the found in several species of berries, as well as the methylated derivatives laricitrin, activity of the hepatocyte nuclear factor 4α (HNF4α) by incubating the cells already with syringetin and myricetin-3`,4`,5`-trimethylether. moderate concentrations of PFOA at a level of about 1 µM. These findings indicate that In this study Caenorhabditis elegans was used as a model to explore the impact of additional, PPARα-independent mechanisms may contribute to the observed myricetin and its methylated derivatives in vivo and to investigate molecular modes of hepatotoxicity of PFOA. The elucidation of novel modes of action of PFOA is relevant for action. the ongoing risk assessment of the substance. Myricetin (100 µM) caused an increase in mean and median adult lifespan of C. elegans. This longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ROS induction, measured by using the H2DCF-DA assay. S18
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Human breast stem cells as a toxicological model for endocrine disruptors, such G protein-coupled receptor kinase 5 functions as a candidate gene in congenital as soy isoflavones heart disease Stempin S.1, Bumke Scheer M.1, Chang C. C.2, Trosko J.2, Lampen A.1 Burkhalter M.1, Fralish G.2, Caron M.2, Philipp M.3 1Bundesinstitut für Risikobewertung - BfR Lebensmittelsicherheit, Max-Dohrn-Str. 8-10, 1University of Ulm Molecular Medicine and Max-Planck-Research-Group on Stem Cell 10589 Berlin, Germany Aging, Albert-Einstein-Allee 11, 89081 Ulm, Germany 2Michigan State University, East Lansing, MI United States 2Duke University Medical Center Cell Biology and Medicine and Neurobiology, Reserach Dr, Durham, NC 27710, United States The adult mammary gland requires stem cells, or a stem cell-like activity. Recently a 3University of Ulm Biochemistry and Molecular Biology, Albert-Einstein-Allee 11, 89081 human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics has been Ulm, Germany isolated. The SV40 (simian virus) immortalized Type 1 HBEC cell line (M13SV1) was non-tumorigenic (Kao et al., 1995). Two daughter cell lines were developed from The functionality of the heart greatly depends on strict homeostasis and interplay of a M13SV1 after X-ray irradiation (M13SV1 R2) and an additional transfection with a range of signalling cascades. Deregulation of either one is always harmful and mutated ERBB2 oncogene (M13SV1 R2-N1), resulting in high and low tumorigenicity eventually detrimental for life. Some of the most relevant signals in the adult heart are respectively and showing a change in estrogen response after growth in minimal media triggered by the stimulation of G protein-coupled receptors such as adrenergic or (Wang et al., 2010). Angiotensin receptors. Those in turn are modulated by a small subset of kinases, the G Isolated isoflavones are currently widely used in the treatment of postmenopausal protein-coupled receptor kinases (GRKs). Interestingly, GRKs, which for the longest time symptoms of women. According to the stem cell theory of carcinogenesis, breast stem were believed to regulate only G protein-coupled receptors were shown to modulate also cells are the ideal target for the proposed research. However, the epidemiological data non-receptor-mediated signalling pathways. By now it is well documented that GRK5 to the effect of isoflavone intake on breast cancer is contradictory. Therefore, we want to plays important roles in both the physiological as well pathological setting of the adult develop a toxicological model using these human breast stem cell lines to test the effect heart. of endocrine disruptors, such as soy isoflavones in a human relevant model. In spite of the important functions of GRKs in the adult heart, it must be assumed that In the present study we analyzed the effect of the phytoestrogen genistein, the most GRK5, one of the two main cardiac GRKs, may also be involved in signal modulation in intensively studied soy protein, on the differentiation of the 3 HBEC lines. The the course of heart development. Deregulation of GRK5-dependent pathways may very expression of different luminal epithelial cell markers, estrogen receptors and stem cell well be causal for impaired cardiac development up to congenital heart disease. In fact, markers was measured on the mRNA level by quantitative real time PCR. The analysis GRK5 is already expressed during embryogenesis and we can detect it in the of several of these markers was also performed on the protein level using western blot. developing heart. However, GRK5 has not been studied yet for a potential function Additionally, a broad number of genes related to breast cancer and estrogen receptor- during embryonic development in general or heart formation in particular. dependent signal transduction were studied using a commercial PCR-Array. In parallel We have established zebrafish as a very time and cost efficient vertebrate model to we are also analyzing the changes on the protein level using 2D gel electrophoresis. investigate the role of GRK5 on cardiac signalling and development. Tools for GRK5 We want to use this panel of different markers to establish a toxicological model that can specific loss- as well as gain-of-function analyses have been developed in our lab. We be used in the future to analyze a wide range of different endocrine disruptors. revealed an unexpected role of GRK5 in the development of left-right asymmetry in zebrafish. Clinically, this has been associated with disorders such as heterotaxy and References other syndromes linked to ciliary dysfunction. Many of those disorders are known to Kao CY, Nomata K, Oakley CS, Welsch CW, Chang CC. (1995) Two types of normal affect proper heart development resulting for example in septum defects or in human breast epithelial cells derived from reduction mammoplasty: phenotypic detrimental translocation of the outflow tract. Precisely, depletion of the close homolog of characterization and response to SV40 transfection. Carcinogenesis (3): 531-538. human GRK5 in zebrafish mirrors the human syndrome called heterotaxy by displaying Wang KH, Kao AP, Chang CC, Lee JN, Chai CY, Hou MF, Liu CM, Tsai EM (2010) randomized placement of inner organs, aberrant heart looping and disrupted valve Modulation of tumorigenesis and oestrogen receptor-α expression by cell culture formation in the heart. In addition, loss of zebrafish GRK5 results in a lower heart rate as conditions in a stem cell derived breast epithelial cell line. Biol Cell. 102(3):159-72. well as dilatation of the embryonic heart at later stages of development. Therefore, we believe that GRK5 may potentially serve as a candidate gene for congenital heart disease.
071 073 Versatile HPLC-MS/MS method for pharmacokinetic studies of three histamine H3- and H4-receptor ligands Burhenne H., Beermann S., Hartwig C., Neumann D., Seifert R. Identification of a HCN3 interacting protein in mouse brain MHH Pharmakologie, Carl-Neuberg Straße 1, 30625 Hannover, Germany Cao-Ehlker X., Hammelmann V., Zong X., Fenske S., Biel M. Department of Pharmacy, Center for Drug Research Ludwig-Maximilians-Universität, Bronchial asthma is a common inflammatory disease of the airways whose occurrence Munich Center for Integrated Protein Science CIPSM, Butenandtstr. 5-13, 81377 has increased dramatically over the past decades. Histamine plays an important role in München, Germany mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. Since antagonizing the histamine Hyperpolarization activated cyclic nucleotide-gated cation channels (HCN) pass a H1-receptor (H1R) shows no ameliorating effects on asthmatic symptoms, H4R depolarizing current (Ih) that is involved in cardiac pacemaking and the control of antagonists may be new drugs for asthma therapy. In addition to the H3R- and H4R- numerous basic functions in neuronal circuits. The four HCN channel types (HCN1-4) selective antagonist thioperamide, the selective H4R antagonist 1-[(5-chloro-1H-indol-2- display specific expression pattern in brain suggesting that each channel fulfills a distinct yl)carbonyl]-4-methylperazine (JNJ7777120) is used in pharmacodynamic studies. physiological function. While HCN1, HCN2 and HCN4 channels have been studied in A correct interpretation of the collected data requires the detailed knowledge of the quite some detail there is only little information on the particular role of the HCN3 pharmacokinetics of the applied substances. For this reason, we developed a fast and channel. As an important step towards achieving a better understanding of HCN3 robust method based on high performance liquid chromatography coupled to tandem function we set out in this study to identify proteins that are assembled with HCN3 in mass spectrometry (HPLC-MS/MS) which allows the simultaneous quantitation of brain tissue. To this end we performed a yeast two hybrid screen with a mouse cDNA thioperamide and JNJ7777120 as well as the selective H3R antagonist 1-({4-[3- library using the HCN3 C-terminal domain as bait. Several proteins were obtained and (piperidin-1-yl)propoxy]phenyl}methyl)piperidine (JNJ5207852) in murine plasma and confirmed for interaction with HCN3 using heterologous coexpression in HEK293 cells. lung tissue. The treatment of plasma samples based on protein precipitation performed Here, we provide an in-depth analysis of the functional interaction between HCN3 and with a mixture of methanol and 0.2 M ZnSO4 using 30 µL of plasma. Analyte extraction one of the identified interacting proteins (HIP3.1). We show that HIP3.1 physically binds from lung tissue was achieved by treating 150 - 200 mg of tissue with a mixture of to HCN3 channels in vitro and in vivo. While HIP3.1 does not change the principal ethanol and water followed by rigorous mixing using a FastPrep-System. activation properties of HCN3, it profoundly upregulates HCN3 current densities (Imax). Ten µL of the extracted samples were transferred to a Synergy Polar-RP 80A Mercury Cell surface labeling suggests that the increase in Imax is caused by the enhancement column (10 x 2mm; 4µm) connected to a Polar-RP Security Guard. Chromatographic of HCN3 cell surface expression when HIP3.1 is present. Immunohistochemistry and in separation was performed via a gradient using an acetate buffer (pH 5) and methanol at situ hybridization experiments indicate that HIP3.1 is expressed in several brain regions, a flow rate of 0.4 mL/min. The analytical run-time was 5 minutes. For plasma samples including olfactory bulb, thalamus and hypothalamus. Our data suggest that HIP3.1 is a the assay was linear over a concentration range of 0.078 - 40 µg/mL for JNJ7777120 regulator of neuronal HCN3. and JNJ5207852, and 0.313 - 40 µg/mL for thioperamide, respectively. In tissues, thioperamide could be quantified in a concentration range of 0.008 - 2 µg/sample, JNJ7777120 and JNJ5207852 in a range of 0.004 - 2 µg/sample. Our results show that the developed HPLC-MS/MS method is suitable for the 074 quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue.
Towards the molecular and vesicular environment of TPC1 channels Castonguay J., Wilmes T., Sleman F., Mallmann R., Klugbauer N. Albert-Ludwigs-Universität Freiburg Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstr. 25, 79104 Freiburg, Germany
Two Pore Domain Channels (TPCs) are calcium channels which consist of two transmembrane domains each with six transmembrane segments. The functional channel is formed upon homodimerisation of TPC monomers to result in a typical tetrameric calcium channel domain structure. TPC channels are not detectable on the cytoplasma membrane, but demonstrate an endolysosomal expression pattern. It has been shown that nicotinic acid adenine dinucleotide phosphate (NAADP) a very potent second messenger can release calcium from these endolysosomal compartments by activating TPCs. Overall, the family of mammalian TPC channels comprises two S19
members, TPC1 and TPC2. Whereas the localization of TPC2 on lysosomes has been ELISA assays. In addition, MCF-7 cells can be sentisized to TNF-α mediated cytotoxicity demonstrated, the precise localization of TPC1 is unclear. which is assocciated with a dimineshed activation of NF-κB. Altogether, we identified Still several open issues remain to be clarified i.e. the precise function of TPC1 and its A20, an ubiquitin modifying enzyme, as a novel FoxO4 target gene. Our data implicate tissue-specific and subcellular distribution. Therefore we established a mouse model that sustained FoxO4 expression may be involved in regulation of TNF receptor/NF-κB with a general deletion of TPCN1 and generated a series of TPC1 antibodies. Using pathway and leading to reduced cell survival. these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (FAOS), total internal reflection fluorescence 077 (TIRF) and confocal microscopy. So far, we confirmed the TPC1 knock-out model by our self-generated TPC1 antibodies. TPC1 knockout mice are viable and do not show any obvious deficits. To isolate TPC1 Role of forkhead transcription factor 1 on doxorubicin-mediated cytotoxicity in containing vesicles or protein complexes, tissue or cell culture derived material was pre- human ovarian cancer cells purified by sucrose density gradient centrifugation. For a subsequent mass spectrometric analysis this preparation was taken as a source material for co- Wurth F., Chovolou Y., Wätjen W., Fritz G. immunoprecipitation or FAOS respectively. Heinrich-Heine-Universität Institut für Toxikologie, P.O.Box 101007, 40225 Düsseldorf, In another approach the migration pattern of TPC1 containing endosomes on linear Germany density gradients was compared with a series of endolysosomal markers i.e. different Rab- , ER- , Golgi- and lysosomal antibodies. Potentially interesting markers were then The subclass O of forkhead box transcription factors (FOXO) contains four family in turn analyzed for their co-localization with TPC1 by confocal microscopy/TIRF. By members in vertebrates: FOXO1, FOXO3a, FOXO4 and FOXO6. Especially FOXO1, combining these results with that from mass spectrometric analysis of FAOS samples FOXO3a and FOXO4 seem to play an important role in mechanisms like DNA-repair, we collect data to get detailed information on the precise endolysosomal distribution cell cycle arrest and apoptosis which are frequently deregulated in cancer cells. A major pattern of TPC1. problem in cancer therapy is the appearance of drug resistance.Therefore, we investigated a potential association between drug resistance and FOXO. As model we References used the adriamycin resistant cell line A2780 Adr which is developed from human 1. Zong et al., Pflügers Archiv - European Journal of Physiology 458, 891–899 (2009) ovarian cancer cell line A2780 Sense by treatment with adriamycin. Adriamycin 2. Brailoiu et al., Journal of Biological Chemistry 186(2), 201-209 (2009). mediated cytotoxicity was assessed by MTT assay. We found that A2780 Sense cells 3. Calcraft et al., Nature 459, 596-601(2009). were more susceptible to doxorubicin-mediated cytotoxicity, compared to A2780 Adr cells with EC50 values (µM) of 0.55 and 9.95 respectively after treatment for 72h. The anthracycline adriamycin mediates its cytotoxic effects by various mechanisms, e.g. through DNA intercalation and topoisomerase II inhibition. It is known that adriamycin may cause cell cycle arrest, so we performed a cell cycle analysis which indicates that 075 incubation with adriamycin in A2780 Sense cells leads to G2/M arrest. However, A2780 Adr cells do not enter into arrest after treatment with adriamycin. Next we examined the expression of FOXO transcription factors on mRNA level in both cell lines by realtime Identification and characterization of FOXO4 target genes in human colon PCR. A ~16-fold increased FOXO1 expression could be detected in adriamycin resistant carcinoma cells cells compared to the parental sensitive cell line. Immunoblot experiments confirmed at Hanhsen B., Chovolou Y., Wätjen W., Fritz G. protein level the increased expression of FOXO1 in A2780 Adr cells. To check whether Heinrich Heine Universität Institut Toxikologie, P.O. Box 101007, 40001 Düsseldorf, the increased FOXO1 expression is associated with an increased FOXO activity a Germany reporter gene assay was performed. First experiments show that FOXO transcriptional activity was elevated in adriamycin resistant A2780 cells compared to A2780 Sense The forkhead superfamily of transcription factors are involved in the regulation of crucial cells. In addition, we assessed the expression of FOXO target genes. Preliminary results cellular processes like controlling of cell cycle, apoptosis, oxidative stress, cell indicate that various FOXO target genes are differentially expressed in A2780 Adr cells. differentiation and DNA repair (Ho et al., 2008). Altogether our results suggest an association between overexpression of FOXO1 and The activity of FOXO transcription factors are negatively regulated by the PI3K/AKT adriamycin resistance in human ovarian cancer cells. pathway. The PI3K/AKT pathway is an oncogenic signaling pathway linked to cellular survival and proliferation by phosphorylating multiple downstream targets on serine and threonine residues. In the absence of AKT activation, FOXOs are located in the nucleus, where they function as transcription factors. Upon AKT activation, FOXOs become 078 phosphorylated and inactivated. In our previous work we found that treatment of human cancer cells with the anthracyclic drug doxorubicin results in phosphorylation of AKT which in turn phosphorylates FOXO4. Phosphorylated FOXO4 is inactivated and Pharmacological targeting of the endogenous TRPM7 currents by NS8593 excluded from the nucleus. Furthermore, we showed that transient overexpression of Chubanov V.1, Mederos Y Schnitzler M.1, Meißner M.1, Schäfer S.1, Abstiens K.1, FOXO4 enhances sensitizing of the cells towards doxorubicin by inducing cell cycle 2 1 arrest and finally apoptosis (Lüpertz et al., 2008). The aim of this study was therefore the Hofmann T. , Gudermann T. 1 identification and characterization of FOXO specific transcriptional targets by real time Ludwig-Maximilians-Universität München Walther-Straub-Institut für Pharmakologie und PCR. For this purpose human colon carcinoma cells were transiently transfected with Toxikologie, Goethestrasse 33, 80336 München, Germany 2 FOXO4 and the expression of several FOXO target genes was assessed by real time Philipps-Universität Marburg Biochemisch-Pharmakologisches Centrum Marburg, Karl- PCR. Our results but also results from other groups indicate that transient von-Frisch-Straße 1, 35032 Marburg, Germany overexpression of FOXO transcription factors may initially trigger a program of cell cycle arrest and cell death in response to doxorubicin, while sustained FOXO activation TRPM7 is a bi-functional protein consisting of a transient receptor potential ion channel promotes drug resistance and survival of cells. To test this hypothesis a stable cell line segment linked to an α-type protein kinase domain. TRPM7 is essential for motility, model overexpressing FOXO4 was generated. The impact of sustained and constitutive proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic overexpression of FOXO4 in modulating cellular response to doxorubicin in colon neuronal death, cardiac fibrosis and tumor cell proliferation. Recently, we have carcinoma cells are still under investigation. The aim is to understand better the demonstrated that the recombinant TRPM7 channel is inhibited by the known molecular signaling pathways of cancer cells. modulators of SK1-3 channels such as antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31, UCL 1684. The most potent of these compounds, 2+ NS8593 (IC50 1.6 µM), interferes with the regulation of TRPM7 by cytosolic Mg . Here we show that NS8593 (10 µM) fully and reversibly inhibits native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular 076 myocytes. Furthermore, we examined whether targeting of the native TRPM7 currents by NS8593 would impact cellular processes known to be affected by a genetic inactivation of TRPM7. We found that NS8593 (10-30 µM) suppressed motility of HEK Regulation of NF-κappa B pathway by forkhead transkription factors 293 cells without a detectable effect on cell viability. Taken together, our findings Chovolou Y., Hanhsen B., Wurth F., Wätjen W., Fritz G. indicate that NS8593 is a potent and reversible inhibitor of endogenous TRPM7 currents Heinrich-Heine Universität Düsseldorf Institut für Toxikologie, Universitätsstr. 1, 40225 and may be a good candidate drug for pharmacological targeting of TRPM7. Düsseldorf, Germany
The PI3K/Akt signalling pathway is a well known cellular survival pathway often deregulated in cancer cells. One feasible Akt downstream target are forkhead box class 079 O (FOXO) transcription factors. Activation of PI3K/Akt signalling results in phosphorylation, nuclear exclusion and inactivation of FOXO transcription factors. FOXOs are involved in a wide spectrum of cellular functions, including cell proliferation, Small GTPases of the Rab family play a role in lipid absorption in the intestine apoptosis and regulation of oxidative stress. In order to identify novel target genes of FOXO transcription factors and to achieve Chung B., Jaschke A., Schürmann A. further insight into their role in cancer cells, DNA microarray analysis was performed Deutsches Institut für Ernährungsforschung Experimentelle Diabetologie, Arthur- using wild type MCF-7 breast cancer cells and MCF-7 cells overexpressing FOXO. We Scheunert-Allee 114-116, 14558 Nuthetal, Germany found that several genes involved in the TNF receptor/NF-κB pathway were differentially regulated. One of the genes that was identified to be up-regulated in FoxO4 Background: Intestinal lipid absorption is comprised of multi-steps including vesicle overexpressing cells was A20 a negative regulator of NF-κB signaling pathway. At both trafficking. We have recently shown that the GTPase ARFRP1 controls the lipidation of mRNA and protein level FOXO4-dependent up-regulation of this ubiquitin modifying chylomicrons in the Golgi. Since ARFRP1 recruits Golgin proteins -which itself bind enzyme was confirmed. To determine whether A20 is a direct target of FOXO4, a several Rab proteins (e.g. Rab2 and Rab6)- to the Golgi apparatus, the aim of the study luciferase reporter containing a 1.2 kb of the A20 promoter was co-transfected with was to investigate the role of Rab2 in the secretion of chylomicrons in the intestine. different amounts of FOXO4 wild-type expression construct. FOXO4 induced a dose- Methods: Mouse intestinal sections were stained for Rab2 or Rab6 antibody to show dependent increase in A20 promoter activity, supporting the assumption that A20 is a their localization. Rab2 expression was suppressed by transfection of specific siRNA in direct transcriptional target of FOXO4. Overexpression of FOXO4 led to decreased Caco-2 cells, an intestinal in vitro model, and lipid absorption and lipoprotein production activity of NF-kB signaling pathway as confirmed by reporter gene and NF-kB specific were examined. S20
Results: Immunohistochemistry of mouse intestinal sections showed that Rab2 and Rab6 082 are co-localized in the Golgi complex of epithelial cells. Rab2 expression was efficiently depleted by siRNA transfection for 14 days without interrupting cell differentiation. Apolipoprotein (Apo) B100 secretion was significantly lower in Rab2 siRNA transfected cells RKIP dimers are responsible for GRK2 inhibition under basal condition. However, when the cells were oil-loaded, Rab2 disruption did not cause decrease in ApoB100 secretion. In the same way, triglyceride secretion was slightly Deiss K., Lohse M. J., Lorenz K. lower in Rab2 siRNA transfected cells under basal condition, but it was not different Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 between scrambled and Rab2 siRNA transfected cells by oil-load. Expression of Würzburg, Germany microsomal triglyceride transport protein (MTTP) was not affected by Rab2 disruption. Interestingly, Rab6 expression was significantly increased in Rab2 siRNA transfected cells. Expression and activity of G protein coupled receptor kinase 2 (GRK2) are elevated in Conclusion: These data suggest that Rab2 and Rab6 are involved in chylomicron several conditions of compromised heart function. Although GRK2 inhibition has been secretion in Caco-2 cells, and might compensate for each other. characterized as a promising therapeutic strategy in heart failure, a specific GRK2- inhibitor is not available. Raf kinase inhibitor protein (RKIP) inhibits Raf1 but it also acts as a physiological inhibitor of GRK2 upon phosphorylation by PKC at serine153. A detailed understanding of the RKIP/GRK2 interaction may help to identify inhibitory compounds for GRK2. 080 Since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching RKIP from a Raf1- into a GRK2-inhibitor. Co-immunoprecipitation assays showed that RKIP self-association was substantially Para-phenylenediamine induces cytochrome P450 1 and cyclooxygenase increased after PKC-mediated phosphorylation of RKIP. RKIP mutants either lacking or expression in human keratinocytes mimicking S153 phosphorylation confirmed that this phosphorylation is indeed a Clemens J., Hennen J., Blömeke B. prerequisite for RKIP/RKIP association. Cross-linking experiments with myc-tagged Universität Trier Ökotoxikologie/Toxikologie, Universitätsring 15, 54296 Trier, Germany RKIP in living cells or with purified RKIP revealed that RKIP phosphorylation by PKC promotes RKIP dimers – not oligomers. Keratinocytes may be exposed to para-phenylenediamine (PPD) via skin paintings and To test whether dimerization is a critical step for the association of RKIP with GRK2, we application of hair dyes. Keratinocytes have high level of N-acetyltransferase 1 (NAT1) generated a peptide to inhibit RKIP dimerization. Intriguingly, the peptide did not only and are able to enhance cytochrome P450 family 1 (CYP1) and cyclooxygenases prevent RKIP dimerization but also attenuated RKIP/GRK2 association. This implicates, (COX). Although PPD is acetylated in keratinocytes, we found that concentrations which that dimerization of RKIP is essential to bind GRK2. To determine whether RKIP dimers saturate N-acetylation capacities are associated with induction of COX-1, COX-2 and consequently inhibit GRK2 activity, we established RKIP mutants with high tendency to cPLA2 in HaCaT cells (Moeller, 2008). In this study we focused on arylhydrocarbon form dimers. Subsequent functional analyses demonstrated that enhanced dimerization receptor (AhR) translocation, CYP1A1 and 1B1 mRNA, and CYP1A1 enzyme activity of RKIP indeed translates into increased GRK2 inhibition. dose- and time-dependently. We found a clear induction of CYP1A1 (primary We conclude that PKC-mediated phosphorylation of RKIP is important for dimerization keratinocytes: 23-fold) and CYP1B1 (12-fold) mRNA expression already after treatment and that these dimers are essential for GRK2 binding and inhibition. Our results reveal with 50 µM PPD. The involvement of the AhR in this induction was demonstrated by new insights in the molecular mechanism of RKIP/GRK2 interaction and will help to AhR translocation in HaCaTs and the usage of siAhR knockdown cells. Significant develop specific GRK2 inhibitors. increase in ethoxyresorufin-O-deethylase (EROD) activity was detected in treated cells after 24h both in the presence and absence of serum. The maximal values were comparable to the effect of the positive control benzo[a]pyrene (1 µM). Interestingly, treatment of HaCaTs with 400 µM PPD did not result in an augmentation of EROD 083 activity in the absence of serum, while we previously demonstrated that only this concentration led to significantly increased levels of prostaglandin secretion (PGE2 and PGF2a). Knowing that PGE2 is able to inhibit CYP1A1 activity we speculate that the Expression and function of TRPM3 ion channels in epithelial MDCK2 cells reduced EROD activity in the presence of 400 µM is the result of this PGE -mediated 2 Dembla S., Meiser J., Philipp S. CYP1 inhibition. Remarkably, the observed induction of phase I enzymes by PPD were clearly restricted to those concentrations that saturated N-acetylation capacities of the University of Saarland Institute for experimental and clinical pharmacology and keratinocytes. Overall the results show that PPD induces CYP1 at lower concentrations, toxicology, Kirrberger Str. 1, 66421 Homburg, Germany while COX-2-dependent effects were restricted to higher concentrations. 2+ Federal Office of Public Health, Switzerland (11004203); Deutsche TRPM3 proteins build Ca permeable cation channels [1] activated by steroids [2] and Forschungsgemeinschaft, Germany (GRK 1319) sensitive to increased temperatures [3]. TRPM3 channels are expressed in pancreatic ß- J.C. and J.H. contributed equally to this work. cells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3]. However, Northern References blots and in situ hybridization experiments revealed that TRPM3 is also expressed in Moeller, R., Lichter, J. and Blömeke B. (2008). Impact of para-Phenylenediamine on epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. cyclooxygenases expression and prostaglandin formation in human immortalized PCR analysis of different epithelial cell lines indicated that TRPM3 is also expressed in keratinocytes (HaCaT). Toxicology 249:167-175 Madin-Darby canine kidney2 (MDCK) cells. Quantitative analysis of TRPM3 expression by qRT-PCR revealed a ~ 5 fold upregulation in MDCK2 cells grown in confluency compared to well separated, proliferating cells. In contrast the level of expression of TRPM7, a related ion channel described as regulator of proliferation in other cell types, remained constant. HEK293 cells overexpressing TRPM3 channels did not proliferate in 081 the presence of the TRPM3 agonist pregnenolone sulphate. However, as indicated by impedance analysis, the proliferation of MDCK2 cells in the presence PregS was only slightly affected. When we analysed the transepithelial resistance (TER) of MDCK2 Activation of Poly(ADP-ribose) Polymerase-1 by exposure to sulfur and nitrogen epithelial cells in transwells as a measure for the formation of tight junctions, we found mustards that the TER of cells grown in the presence of PregS was reduced. Interestingly, Ca2+ Debiak M., Lex K., Lutz G., Brenneisen A., Bürkle A. imaging experiments using Fura2 revealed that pregnenolone sulphate induces Ca2+- University of Konstanz Molecular Toxicology Group, Jacob-Burckhardt-Str. 31, 78457 entry in well separated MDCK2 cells but not in cells growing in confluency. We Konstanz, Germany hypothesize that TRPM3 might act as a regulator of cell proliferation and/or the formation of tight junctions in MDCK2 cells. Sulfur mustard (2,2’-dichlorodiethylsulfide; SM) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. As soon as SM was first used as a References warfare agent, research aimed at the development of an effective antidote was [1] Oberwinkler, J. et al. (2005) Alternative splicing switches the divalent cation launched. Early studies with first-generation inhibitors of poly(ADP-ribose) polymerases selectivity of TRPM3 Channels. J Biol Chem. 280, 22540-22548. (PARP) have revealed promising therapeutic potential in SM-induced skin injury, but the [2] Wagner, T.F. et al. (2008) Transient receptor potential M3 channels are ionotropic underlying mechanism remains elusive. The current renaissance of PARP inhibitors in steroid receptors in pancreatic beta cells. Nat Cell Biol. 12, 1421-1430. cancer chemotherapy has revived the discussion on their use for treatment of SM injury. [3] Vriens, J. et al. (2011) TRPM3 is a nociceptor channel involved in the detection of Thus we established a comprehensive study aiming the elucidation of the role of PARP noxious heat. Neuron 70, 1-13. in SM pathology based on model substance 2-chloroethyl ethyl sulfide (CEES), which is not classified as warfare agent. We have recently demonstrated that PARP becomes rapidly activated in living human keratinocytes (HaCaT) after treatment with CEES. The maximal PARP activity was 084 observed 10 minutes after treatment with 3 mM CEES. The activation was transient and dose dependent. To our knowledge this is the first demonstration of PARP activation after treatment with mustards in the context of live cells. Inhibition of GRK2 by RKIP improves cardiac contractility and structure in a An important question is how PARP-1 becomes activated upon treatment with mustards. transgenic mouse model of heart failure PARP-1 is a first-line protein involved in the cellular response to DNA strand breaks. However, mustards do not directly induce large numbers of such lesions. One possibility Denzinger S., Schmitt J. P., Lohse M. J., Lorenz K. is that PARP-1 is activated by DNA breaks incorporated as base excision repair (BER) Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 or nucleotide excision repair (NER) intermediates. Thus, we performed knockdown Würzburg, Germany experiments of APE1 and ERCC1, i.e. endonucleases involved in BER and NER, respectively. The reduction of APE1 expression had no effect on PARP activity. The Raf kinase inhibitor protein (RKIP) has been identified as a physiological inhibitor of Surprisingly, the knockdown of ERCC1 almost completely abolished the cellular PAR G-protein coupled receptor kinase 2 (GRK2). GRK2 initiates G protein coupled receptor production after CEES treatment. The functional consequence of the ERRC1-PARP (GPCR) desensitization. Since expression and activity of GRK2 are upregulated in cross-talk with regards to adduct removal is under investigation. However, our present human heart failure, it has been proposed that GRK2 inhibition may resensitize b- data indicate that PARP activity is not obligatory for the survival of cells upon CEES adrenergic receptor activity in heart failure patients. In this study, we evaluated chronic treatment, as revealed by the lack of effect of the potent PARP inhibitor ABT-888. GRK2 inhibition by RKIP as a potential strategy to improve cardiac function in heart failure. This work is supported by the German Armed Forces Grant E/ UR3G/AG001/9A804 To analyse the effect of RKIP on heart failure, RKIP transgenic mice were crossed with mice carrying a mutation in phospholamban (PLNR9C). PLNR9C causes severe heart S21
failure and premature death in humans and transgenic mice. Cardiac function was sensitive soluble guanylyl cyclases. Interestingly, activation of cGMP/cGKIalpha signal significantly improved in the presence of RKIP as shown by left ventricular transduction was associated with an increase in ERK1/2 and p38 phosphorylation, catheterization and echocardiography. Expression of heart failure marker genes ANF growth and migration of B16 melanoma cells. Similar results were obtained with and BNP was indistinguishable between wild-type mice and mice co-expressing RKIP WM1205 human melanoma cells. In conclusion, we have identified a GC-A/GC- and PLNR9C. In line with these findings, the life span of double transgenic mice was B/cGMP/cGKIalpha pathway in melanoma cells, which stimulates tumor cell growth and significantly prolonged compared to PLNR9C transgenic mice. Slow calcium transport into migration in vitro. Pharmacologic inhibition of cGMP signaling may offer a promising the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy strategy for the treatment of melanoma. of PLNR9C transgenic mice. Since Western blot analyses of RKIP transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the RKIP mediated rescue. In the presence of RKIP, calcium 087 reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. Furthermore, coexpression of RKIP significantly attenuated pathological cardiac remodelling. Interstitual fibrosis and apoptotic cells were quantified 2+ Synergy of Cav2.3 and Cav3.2 Voltage-gated Ca Channels in Experimentally in histological sections after Sirius Red- and TUNEL-staining. Induced Epilepsy This study revealed a protective function of RKIP in a genetic mouse model of human Dibué M. A. H.1,2, Alpdogan S.1, Tevoufouet E. E.1, Chen C. - C.3, Campbell K. P.4, dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial 1 1 fibrosis and apoptosis. A detailed understanding of this rescue may help to find a new Hescheler J. , Schneider T. 1 therapeutic strategy to improve cardiac contractility in heart failure. University of Cologne Institute of Neurophysiology, Robert-Koch-Str. 39, 50931 Köln, Germany 2University of Cologne Center for Molecular Medicine Cologne (CMMC), Robert-Koch- Str. 21, 50931 Köln, Germany 3Academia Sinica Institute of Biomedical Sciences, 128 Sec. 2, Academia Rd. Nankang, 085 Taipei 11529, Taiwan 4Departments of Molecular Physiology and Biophysics, Neurology, and Internal Medicine, and the Howard Hughes Medical Institute, University of Iowa Roy J. and Different roles for Gαi2 and Gαi3 in myocardial and cerebral ischemia Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building 285 1 2 3 4 4 5 5 Devanathan V. , Hagedorn I. , Köhler D. , Pexa K. , Piekorz R. , Kraft P. , Stoll G. , Newton Road, Iowa City, Iowa 52242-1101, United States 6 7 2 1 Birnbaumer L. , Rosenberger P. , Nieswandt B. , Nürnberg B. 1Universität Tübingen Institut für Pharmakologie, Wilhelmstr. 56, 72074 Tübingen, An increasing body of evidence supports important roles for voltage-gated calcium Germany channels in idiopathic generalized epilepsies (IGEs), however which calcium channels 2Universität Würzburg Rudolf-Virchow-Zentrum für Experimentelle Biomedizin, Josef- participate in IGE pathogenesis and how has yet to be fully understood. Recently, it has Schneider-Straße 2, 97080 Würzburg, Germany been proposed that Cav2.3 (R-type) and T-type calcium channels jointly contribute to 3Universität Tübingen Zentrum für Hypoxie und Inflammation, Waldhörnlestraße 22, oscillatory bursting in the reticular thalamus (RT)1, which is associated with absence 72074 Tübingen, Germany epilepsy. Cav3.2 is one of the two T-type calcium channels known to be expressed in the 4 2 Universität Düsseldorf Institut für Biochemie und Molekulare Biologie II, RT . It has been demonstrated that ablation of either Cav2.3 or Cav3.2 reduces Universitätsstraße 1, 40225 Düsseldorf, Germany susceptibility to experimentally induced epilepsy3;4 and in addition that both channels 5Universität Würzburg Neurologische Klinik und Poliklinik, Josef-Schneider-Straße 11, share several pharmacological properties5-7. To gain further insight into interacting 97080 Würzburg, Germany mechanisms of these two channels in epilepsy, we tested Cav2.3(-|-), Cav3.2(-|-) and 6 National Institute of Health Transmembrane Signaling Group, Research Triangle Park, Cav3.2(-|-)xCav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. We North Carolina, 27709, United States provide first in vivo data supporting a synergistic mode of action for Cav2.3 and Cav3.2 7Universität Frankfurt Klinik für Anästhesiologie, Theodor-Stern-Kai 7, 60590 Frankfurt, calcium channels in epileptogenesis. Germany References Gαi-proteins comprise a group of three highly related members characterized by specific 1. Zaman T, Lee K, Park C et al. Cav2.3 channels are critical for oscillatory burst expression patterns. Based on previous work of Gi-mediated signaling pathways in discharges in the reticular thalamus and absence epilepsy. Neuron 2011;70:95-108. cardiomyocytes and platelets, we checked Gαi expression in mouse heart and platelets. 2. Talley EM, Cribbs LL, Lee JH, Daud A, Perez-Reyes E, Bayliss DA. Differential The analysis revealed the presence of Gαi2 and Gαi3 with Gαi2 as the predominant distribution of three members of a gene family encoding low voltage-activated (T-type) isoform. Gene-targeted mice lacking either Gαi2 or Gαi3 were analyzed to unravel the calcium channels. J Neurosci 1999;19:1895-911. physiological role of Gαi-proteins in the cardiovascular system. 3. Weiergräber M, Henry M, Radhakrishnan K, Hescheler J, Schneider T. Hippocampal Extraordinarily prolonged bleeding times in Gαi2-deficient animals were an obvious seizure resistance and reduced neuronal excitotoxicity in mice lacking the Cav 2.3 E/R-type phenomenon. Detailed analysis using isolated platelets Gαi2-deficient mice exhibited voltage-gated calcium channel. J Neurophysiology 2007;97:3660-9. reduced platelet activation and attenuated aggregation in response to stimulation by 4. Becker AJ, Pitsch J, Sochivko D et al. Transcriptional upregulation of Cav3.2 various agonists accompanied by reduced thrombus formation and diminished stability mediates epileptogenesis in the pilocarpine model of epilepsy. J Neurosci on a collagen-coated surface. Employing in vivo injury/thrombosis models revealed 2008;28:13341-53. abrogated thrombus formation selectively in Gαi2-deficient mice. Comparable results 5. Soong TW, Stea A, Hodson CD, Dubel SJ, Vincent SR, Snutch TP. Structure and were obtained in experiments using mice with megakaryocyte/platelet-specific Gαi2– functional expression of a member of the low voltage-activated calcium channel family. deficiency. To assess the pathophysiological consequences of platelet Gαi2 function, we Science 1993;260:1133-6. challenged these mice in experimental models of myocardial and cerebral ischemia. The 6. Kang HW, Park JY, Jeong SW et al. A molecular determinant of nickel inhibition in results clearly show that platelet-Gαi2–deficient mice were protected from both, Cav3.2 T-type calcium channels. J Biol Chem 2006;281:4823-30. myocardial and cerebral ischemia. In contrast, conventional Gαi2-deficient mice 7. Kang HW, Moon HJ, Joo SH, Lee JH. Histidine residues in the IS3-IS4 loop are subjected to the heart ischemia/reperfusion model exhibited a significantly increased critical for nickel-sensitive inhibition of the Cav2.3 calcium channel. FEBS Lett susceptibility to ischemic injury as compared to wild type controls. In contrast, Gαi3- 2007;581:5774-80. deficient mice were strongly protected from injury. Thus we suggest that Gαi2 and Gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related Gαi isoforms. 088
086 The deubiquitinase CYLD regulates mechanisms of RIP1/RIP3-dependent necroptosis in neuronal cells Diemert S.1, Krieg S.2, Kim S. W.2, Plesnila N.2,3, Culmsee C.1 A cGMP/cGKI signaling pathway in melanoma cells 1Philipps Universität Marburg Institut für Pharmakologie und Klinische Pharmazie, Karl- 1 1 2 3 3 1 Dhayade S. , Feil S. , Griessinger C. , Kneilling M. , Schittek B. , Feil R. von-Frischstraße 1, 35032 Marburg, Germany 1Eberhard-Karls-University Tübingen Interfaculty Institute of Biochemistry, Hoppe- 2Ludwig-Maximilians-Universität München Institut für Schlaganfall- und Seyler-Str. 4, 72076 Tübingen, Germany Demenzforschung, 81377 München, Germany 2Eberhard-Karls-University Tübingen Department of Preclinical Imaging and 3Royal College of Surgeons in Ireland Department of Neurodegeneration & Department Radiopharmacy, Laboratory for Preclinical Imaging and Imaging Technology of the of Physiology, Dublin Ireland Werner Siemens-Foundation, Röntgenweg 13, 72076 Tübingen, Germany 3Eberhard-Karls-University Tübingen Department of Dermatology, Liebermeisterstraße The deubiquitinating enzyme CYLD is a well-known regulatory component of the Nf-kB 25, 72076 Tübingen, Germany pathway. Dysfunctional CYLD has been implicated in tumor genesis, altered immune responses and the regulation of microtubule dynamics. More recently CYLD has been The cGMP signaling pathway is activated by nitric oxide (NO), natriuretic peptides (ANP, identified as a key player in TNF-induced necrotic cell death. Though increasing BNP & CNP), and cGMP-elevating drugs. It regulates several physiological functions evidence emerges on the significance of necrosis in neurodegeneration, the particular such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. impact of CYLD on neuronal viability remains elusive. Recent studies indicate that cGMP signaling might also play a role in tumorigenesis, but In this study we used a model of glutamate toxicity in neuronal HT-22 cells to investigate the cellular and molecular mechanisms of cGMP’s potential pro- and/or anti-tumor the role of CYLD. In these neurons, glutamate induces glutathione depletion leading to activities are not well understood. This study has examined the expression and function increased ROS production. In response to glutamate HT-22 cells show increased levels of components of the cGMP pathway in melanoma cells of murine and human origin. We of lipidperoxidation and mitochondrial fragmentation, accompanied by an increase of have found that mouse B16 melanoma cells specifically express the alpha isoform of the RIP1-RIP3 complex formation. Repressing CYLD by siRNA attenuated lipidperoxidation, cGMP-dependent protein kinase type I (cGKIalpha) but not the beta isoform. Treatment deterred formation of the RIP1-RIP3 complex and prevented mitochondrial of intact cells with the membrane-permeable cGMP analog 8-Br-cGMP induced the fragmentation, thus promoting neuronal survival. Further, targeting RIP1 and RIP3 by phosphorylation of cGKI substrates, vasodilator stimulated phosphoprotein and siRNA significantly decreased glutamate induced cell death in HT-22 cells. In primary phosphodiesterase 5. ANP and CNP, ligands of the membrane-bound guanylyl cyclase neuronal cultures, glutamate induced excitotoxicity could not be prevented by CYLD GC-A and GC-B, respectively, did also activate the endogenous cGMP/cGKI pathway. depletion, however blocking RIP1 kinase by necrostatins was neuroprotective. In an in- However, B16 melanoma cells did not respond to DEA-NO, which stimulates NO- S22
vivo model of cerebral ischemia, we found, that CYLD -/- mice exhibit significantly 091 reduced infarction volume compared to control littermates. Overall, these data reveal a role for CYLD in RIP1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests Mitochondrial SK2/K 2.2 channels mediate neuroprotective effects CYLD-mediated mechanisms of neuronal cell death as a potential therapeutic target Ca Dolga A.1, Perocchi F.2, Doti N.3, Meissner L.4, Tobaben S.1, Grohm J.1, Zischka H.5, after acute brain injury in vivo. 6 1 Plesnila N. , Culmsee C. 1Philipps-Universität Marburg Inst. für Pharmakologie und Klinische Pharmazie, Karl von Frisch Strasse, 35043 Marburg, Germany 2Harvard Medical School Harvard Medical School and Massachusetts General Hospital, 089 Boston, United States 3Institute of Biostructures and Bioimaging–CNR, Napoli, Italy 4University of Munich Medical School Institute of Stroke and Dementia Research, Cyanamide-mediated inhibition of N-acetyltransferase 1 Munich, Germany Dierolf D., Bonifas J., Blömeke B. 5Helmholtz Zentrum München–German Research Center for Environmental Health, University Trier Department of Environmental Toxicology, Universitätsring 15, Bldg. N, Munich, Germany 54286 Trier, Germany 6University of Munich Medical School Institute of Stroke and Dementia Research, Munich, Germany Cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking In neurons, small-conductance calcium-activated potassium (KCNN/SK/KCa2) channels agent. Its therapeutic effect is mediated by reversible inhibition of aldehyde counteract NMDA receptor-mediated deregulation of intracellular calcium homeostasis dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme A and may therefore serve as promising therapeutic targets for a variety of neurological dependent N-acetylation by N-acetyltransferases. disorders. In this study, we investigated the potential role of KCa2.2 channels in a model Reported to be a substrate for N-acetyltransferases, cyanamide has a different of glutamate toxicity in immortalized mouse hippocampal HT-22 neurons. molecular structure to arylamines and hydrazines, the preferred substrates for N- We characterized the effects of KCa2.2 channel activation on HT-22 cell survival after the acetyltransferases. Therefore a more detailed investigation of its interrelations with N- glutamate challenge using the MTT assay and the real time xCELLigence impedance- acetyltransferases was performed. We analysed NAT1 enzyme activities after incubation based system. Further endpoints included measurements of lipid peroxidation, of THP-1 cells with cyanamide for 24h, and found that the metabolic conversion of the mitochondrial morphology and mitochondrial membrane potential. Analysis of mRNA classic substrate para-aminobenzoic acid was significantly reduced at physiologically levels revealed expression of KCa2.1 and KCa2.2 channel subtypes, while KCa2.3 relevant concentrations. In detail a significant dose- and time-dependent NAT1 protein channels were not detected. Positive pharmacological modulation of KCa2.2 channels by inhibition was observed for 100 and 1000 µM cyanamide using over-expressed human CyPPA significantly reduced glutamate-induced cellular death, as observed by both, recombinant NAT1 (insect cell cytosol containing recombinant human NAT1*4). impedance measurements and MTT assays. Further, CyPPA prevented mitochondrial However, no inhibition was found in the presence of recombinant NAT2*4. As we also fragmentation and preserved the mitochondrial membrane potential in glutamate- provide evidence that cyanamide is not metabolised via coenzyme A dependent N- exposed cells, pointing at a novel role of KCa2.2 channels for mitoprotection. This acetylation in vitro by human NAT1 or NAT2 cytosol, by THP-1 cells or by human liver conclusion was further confirmed by protective effects of CyPPA in the absence of cytosol, we can conclude that this inhibition is not based on substrate-dependent down- extracellular calcium and the inhibition of late phase increases in lipid peroxidation, regulation of NAT1. Further mechanistic and kinetic studies indicated that cyanamide which was previously attributed to mitochondrial demise. Although various potassium reacts with the active site cysteine residue of NAT1, leading to its rapid inhibition. Since channels have been identified in mitochondria, the presence of KCa2 channels at the presence of the reduction agent dithiothreitol did not reverse the results it could be intracellular locations has not yet been reported. Here, we demonstrate KCa2.2 channel that it is possibly not caused by oxidative processes. In sum these data indicate that expression at the inner mitochondrial membrane as detected by fluorescence confocal cyanamide is able to interact with cysteine residues of human NAT1, which causes its microscopy and Western blot analysis of isolated mitochondria and mitoplasts. Notably, inhibition but not by a substrate-dependent mode of action. Taken together our results only the inhibitory peptides specific to KCa2.2 channels reduced CyPPA mediated show, that cyanamide is not N-acetylated by human NATs, but might modulate NAT1 protective effects. Further, activation of KCa2.2 channels prevented tBid toxicity and dependent detoxification and activation of arylamines. translocation of mitochondrial AIF protein to the nucleus. In summary, we demonstrate for the first time that KCa2.2 channels are expressed in mitochondria, where they mediate mitoprotection and thus may serve as promising targets for novel strategies of neuroprotection. 090 References Dolga AM, Terpolilli N, Kepura F, Nijholt IM, Knaus HG, D'Orsi B, Prehn JH, Eisel UL, 2+ Dissecting the signal transduction pathway of acute hypoxic vasoconstriction Plant T, Plesnila N, Culmsee C (2011) KCa2 channels activation prevents [Ca ]i (HPV) in precapillary pulmonary arterial smooth muscle cells (PASMC) deregulation and reduces neuronal death following glutamate toxicity and cerebral 1 2 3 2 1 1 ischemia. Cell Death Dis. 2, e147 Mayer T. , Fuchs B. , Kalwa H. , Weißmann N. , Gudermann T. , Dietrich A. 1Ludwig-Maximilians-Universität München Walther-Straub-Institut für Pharmakologie und Toxikologie, Nußbaumstr. 26, 80336 München, Germany 2 Justus-Liebig-Universität, Gießen Excellence Cluster Cardio-Pulmonary System, 092 Aulweg 130, 35392 Gießen, Germany 3Harvard Medical School, Boston Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, United States Characterisation of a novel TRPV5 interacting protein Low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial Dörr J., Wissenbach U., Fecher-Trost C. vasoconstriction (acute HPV) redirecting blood flow to normoxic areas of the lung to Universität des Saarlandes Experimentelle und klinische Pharmakologie und assure optimal uptake of oxygen during ventilation. Acute HPV lasting several minutes Toxikologie, Kirrberger Str./Gebäude 46, 66421 Homburg, Germany occurs predominantly in the precapillary region of the pulmonary vascular tree [1]. Therefore, precapillary pulmonary arterial smooth muscle cells (PASMC) have been TRPV5 is a highly selective calcium channel expressed in various tissues amongst suggested as sensor as well as effector cells and TRPC6 a member of the classical others in placenta. The channel may be involved in transcellular calcium transport in transient receptor potential (TRPC) ion channel family was identified to be essential for epithelial tissues thereby playing some role in calcium homeostasis of the body. In the the initiation of Ca2+ influx and the subsequent contraction of PASMC [2]. However, the placenta TRPV5 is assumed to contribute to the maternal-fetal calcium transport. Most underlying oxygen sensor and the exact signal transduction pathway(s) in PASMC have probably TRPV5 is part of a multiprotein channel complex but most of the components not been fully elucidated yet. By using gene-deficient mouse models as well as down- of this complex are unknown so far. Our aim is to find interaction partners of the TRPV5 regulation of potential candidate proteins by specific small interfering RNAs (siRNAs), protein in the placenta that might contribute to the regulation of the TRPV5 protein we aim to dissect signaling cascades of TRPC6 channel activation in acute HPV. For function. Therefore we expressed the intracellularly located N- and C-terminal parts of PASMC isolation and culture from mice we use a technique based on magnetic TRPV5 (aa 1-330 and 571-723) as TRPV5-GST (glutathione-S-transferase)-fusion separation of intrapulmonary arteries originally developed in rats [3]. TRPC-expression proteins and used the purified recombinant proteins for pulling down proteins from in freshly isolated and passaged PASMC cultured in low (5%) and high fetal bovine human placenta cell extracts. The proteins pulled down by this approach were analysed serum (25%) was analyzed. Interestingly higher passage numbers resulted in a by mass spectrometry. We identified several potential TRPV5 interacting proteins which significant down-regulation of TRPC1 and TRPC6 the most predominantly expressed were not associated with the GST protein only. One of the proteins which was highly channel monomers in PASMC, while different serum concentrations resulted in no enriched with the N-terminal part of the TRPV5 protein is Calpain 6. In contrast to the significant changes in their expression rates. SiRNAs were designed, transfected and classical calpains (calcium activated cystein proteases), Calpain 6 is unique in that it successfully tested in HEK293 cells and PASMC. Initial results of the dissection of the lacks the cysteine residue in the active site. Calpain 6 is mainly expressed during signal transduction pathway activating TRPC6 and inducing acute HPV in PASMC will embryogenesis and is reported to be involved in cytoskeleton stabilisation but with be presented. unknown function in placenta. The interaction between TRPV5 and Calpain 6 was confirmed in reciprocal pulldown experiments and the TRPV5 binding region for Calpain References 6 was narrowed down by using overlapping N-terminal TRPV5-GST fusion proteins. [1] Staub, N. C. (1985). Site of hypoxic pulmonary vasoconstriction, Chest 88, 240S- After injection of TRPV5 cRNA into Xenopus laevis oocytes calcium uptake into the 245S. oocytes was measured; this uptake was largely reduced after co-injection of the Calpain [2] Weissmann, N. et al. (2006). Classical transient receptor potential channel 6 6 cRNA. In further experiments we want to study potential regulatory effects of the (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, TRPV5 protein on the Calpain 6 function in cell culture models. Proc. Natl. Acad. Sci. U.S.A. 103, 19093-19098. [3] Waypa G. B. et al. (2002) Mitochondrial reactive oxygen species trigger calcium increases during hypoxia in pulmonary arterial myocytes, Circ. Res. 91, 719-726.
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Methemoglobinemia can cause life-threatening situations – Traditional treatment Mitochondrial dysfunction in models of Alzheimer´s disease and further aspects Eckert A. 1 2 3 1 3 Durner J. , Leu C. , Boris B. , Reichl F. - X. , Höcherl E. Universitäre Psychiatrische Kliniken Basel Neurobiology Laboratory, Wilhelm Klein 1Institut für Pharmkologie und Toxikologie der Ludwig-Maximilians-Universität München, Strasse 27, 4012 Basel, Switzerland Nussbaumstraße 26, 80336 München, Germany 2Universitätsklinikum München Großhadern Transfusionmedizin, Marchioninistraße 15, The histopathological characteristics of Alzheimer’s disease (AD) are amyloid-ß (Aß) 81377 München, Germany containing plaques and neurofibrillary tangles (NFTs) as well as neuronal and synaptic 3Städtisches Klinikum München Schwabing Unfallchirurgie, Kölner Platz 1, 80804 loss. Until today, the underlying mechanisms of the interplay of plaques and tangles München, Germany remained unresolved. There is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several APP and tau transgenic mouse Background models. Recently, we examined mitochondrial function in a novel triple transgenic Amyl nitrite was first synthesised at the Sorbonne in 1844 by A-J Balard, who also mouse model (pR5/APP/PS2) – tripleAD mice – that combines both pathologic features reported that its vapor has given him a severe headache. In 1876 the english surgeon of the disease in brain. Using comparative, quantitative proteomics (iTRAQ) and mass Sir Brunton discovered that the inhalation of amyl nitrite relieved the pain of angina. It spectroscopy we found a massive deregulation of 24 proteins, of which one-third were worked immediately and the effect lasted up to one hour. Nowadays, amyl nitrite has mitochondrial proteins mainly related to complexes I and IV of the oxidative been used illegally by individuals as mood-altering substance or aphrodisiac to enhance phosphorylation system (OXPHOS). Remarkably, deregulation of complex I was related sexual pleasure or to get high for several minutes. Nitrite can induce to tau, whereas deregulation of complex IV was Aß dependent, both at the protein and methemoglobinemia and in rare cases methemoglobinemia can be life-threatening. activity levels. The tripleAD mice showed synergistic effects of Aß and tau already at the Objectives age of 8 months, resulting in a depolarized mitochondrial membrane potential. At 12 Rare life-threatening situations need a tight patient supply to save precious time. This months, the strongest defects on OXPHOS, synthesis of ATP and reactive oxygen can be realized with a treatment algorithm. species were exhibited in the tripleAD mice, again emphasizing synergistic, age- A 26 year old man was found unconscious with a Glasgow Coma Score of 3. In hospital associated effects of Aß and tau in impairing mitochondria. a methemoglobin (MetHb) level of 89 % was measured. Methemoglobinemia was Evidences from AD post-mortem brain as well as cellular and animal AD models indicate treated with methylene blue (MB). The patient died a few days later. that Aß and tau protein trigger mitochondrial dysfunction through a number of pathways, Conclusion such as impairment of oxidative phosphorylation, elevation of reactive oxygen species Methemoglobinemia can be induced by congenital deficiencies of enzymes, alterations production, alteration of mitochondrial dynamics, and interaction with mitochondrial in the Hb molecule or most commonly by (lethal) poisoning due to incorporation of proteins. Moreover, recent reports indicate that Aß may also interact directly with domestic or industrial chemicals as well as therapeutic agents. Toluidine blue and MB intracellular proteins such as the mitochondrial enzyme ABAD (Aß binding alcohol are treatments of choice in methemoglobinemia. Levels of MetHb exceeding 60 – 70 % dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in can cause coma and death. The amount of MetHb can be determined by blood gas AD, and Aß’s toxicity seems to be in part mediated by inhibition of ABAD. In total, a analysis. After administration of toluidine blue or MB, a MetHb monitoring with arterial vicious cycle as well as several vicious circles within the cycle, each accelerating the blood samples is impossible because of its discoloration. For monitoring the therapeutic other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau effects myocardial ischaemia (electrocardiography, Troponin), clinical indicators (level of and Aß. consciousness, dyspnoea) or laboratory parameters (creatinine kinase, neuron specific enolase) can be used. The regular treatment was not sufficient in our case. Therefore References some new (algorhythmic) aspects were elaborated. 1. Schmitt K, Grimm A, Kazmierczak A, Strosznajder JB, Götz J, Eckert A. Insights into By summarizing the case, the intake of a MetHb former was verified by measuring of Mitochondrial Dysfunction: Aging, Amyloid-β and Tau - a deleterious trio. Antioxid Redox MetHb in blood gas sample. The main problem was the life-threatening Signal. 2011 Nov 27. [Epub ahead of print] unconsciousness by unknown A-I-FDA: anamnesis was unknown or questionable; 2. Rhein V, Song X, Wiesner A, Ittner LM, Baysang G, Meier F, Ozmen L, Bluethmann intake of MetHb former was verified, but name, amount, composition, and way of H, Dröse S, Brandt U, Savaskan E, Czech C, Götz J, Eckert A. Amyloid-beta and tau incorporation were unknown; further drug abuse was unknown. synergistically impair the oxidative phosphorylation system in triple transgenic In cases with a life-threatening unconsciousness from first patient contact, a verified Alzheimer's disease mice. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):20057-62. MetHb formation and unknown A-I-FDA, an exchange transfusion should be considered Epub 2009 Nov 6. in an early stage of treatment to save the patient’s life.
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Olesoxime - potential therapeutic approaches for mitochondrial dysfunction Comparative studies on the effects of the human carcinogen inorganic arsenite Eckmann J.1, Eckert S. H.1, Kolesova N.1, Mikusev V.1, Michaud M.2, Bordet T.2, Pruss V and its recently identified thiolated metabolite thio-DMA on human urothelial R.2, Leuner K.1,3, Müller W. E.1, Eckert G. P.1 cells 1Goethe-Universität Pharmakologisches Institut für Naturwissenschaftler, Max-von-Laue Ebert F., Leffers L., Unterberg M., Schwerdtle T. Str. 9, 60438 Frankfurt, Germany Universität Münster Institut für Lebensmittelchemie, Corrensstr. 45, 48149 Münster, 2Trophos S.A. In vivo pharmacology, Luminy Enterprises, Case 931, 13288 Marseille, Germany France 3Friedrich-Alexander-University Dept. Pharmaceutical Technology, Cauerstrasse 4, It has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is 91058 Erlangen, Germany associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. In this context, in Olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the last five years one main focus has been given to the role of human inorganic arsenic the blood brain barrier. The cholesterol-oxime targets proteins of the outer mitochondrial metabolism and nowadays it is generally accepted that human biomethylation membrane and represents a promising drug candidate for neurodegenerative diseases1. contributes to inorganic arsenic induced carcinogenicity. Due to further improvements in We evaluated Olexoxime’s neuroprotective effects against mitochondrial dysfunction in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio an animal model for Alzheimer`s disease (AD). Dissociated brain cells (DBC) and V analogue of the well known metabolite dimethylarsinic acid (DMA ), the so called mitochondria were isolated from brains of C57/BJ6-Thy1-APPSL (AD-mice) mice that thiodimethylarsinic acid (thio-DMAV), has been discovered in human biological samples. were fed with 600 mg Olesoxime/kg feed for 3 months. Drug plasma levels reached After synthesizing and analytically characterizing this metabolite (Bartel et al. 2011, J approx. 600 ng/ml. Respiration of isolated mitochondria were significantly diminished in Toxicol. 2011:373141) we investigated its toxic effects in direct comparison with iAsIII in AD-mice due to reduced complex I, I+II and COX activities. Consequently, mitochondrial human urothelial cells. Thereby cell cycle studies revealed a G2/M- and S-phase arrest membrane potential (MMP) was significantly reduced in DBC from AD-mice. Olesoxime as well as subG1 peak formation in case of thio-DMAV. Moreover, thio-DMAV induced normalized respiration chain complex activities and the MPP. To further evaluate the apoptosis (subG1, caspase 3 activity) at lower concentrations and earlier time points as beneficial effects of Olesoxime on complex I activity, we challenged DBC with rotenone compared to iAsIII. Most likely this is partly due to the higher cellular bioavailability of ex vivo and observed that Olesoxime treatment was protective. To further clarify the thio-DMAV (AAS/ICP-MS). Regarding genotoxicity, a generation of DNA single strand mode of action, we analyzed the ability of Olesoxime to prevent opening of the breaks (alkaline unwinding technique) as well as an increased formation of reactive mitochondrial permeability transition pore (mPTP) in vitro using energized brain oxygen species (ROS, DCFH-DA-fluorescence) occurred only at high cytotoxic mitochondria by measuring Ca2+-and atractyloside (ATR) induced swelling. The opening V concentrations. However, thio-DMA strongly increased H2O2 induced ROS formation at of mPTP precedes apoptosis and can be induced by mitochondrial dysfunction due to very low nanomolar concentrations, which might result in cogenotoxic effects. Since our calcium overload, oxidative stress, elevated phosphate concentrations or adenine 2+ earlier studies have shown a strong inhibition of H2O2 induced poly(ADP-ribosyl)ation nucleotide depletion. Olesoxime prevented Ca - as well as ATR induced mitochondrial especially by trivalent methylated arsenic metabolites, actual studies investigate the swelling. ATR inhibits the adenine nucleotide translocase (ANT) that requires impact of thio-DMAV on cellular poly(ADP-ribosyl)ation, PARP-1 gene expression, appropriate membrane properties to mediate mitochondrial permeability transition protein level and cellular cleavage, which might hopefully give further hints regarding the (MPT). Since cholesterol (CHO) and its derivates represent potent modulators of mode of action behind thio-DMAV induced apoptosis. membrane viscosity, we related the effects of CHO and Olesoxime on mPTP opening to membrane properties. Both, CHO and Olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented ATR induced mPTP opening. However, CHO didn`t prevent Ca2+-induced mPTP opening, indicating a different mode of action for Olesoxime. Our data confirm Olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. The work was supported by the European Union under the 7th Framework Program for RTD - Project MitoTarget - Grant Agreement HEALTH-F2- 2008-223388.
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References Ligand-binding studies represent essential tools for pharmacological research on G 1Bordet T, Berna P, Abitbol J, Pruss RM. Olesoxime (TRO19622): A Novel protein-coupled receptors. In recent years, time-resolved fluorescence gained significant Mitochondrial-Targeted Neuroprotective Compound; Pharmaceuticals (2010) vol. 3 pp. relevance as readout for ligand binding studies. However, ligand-binding assays for 345 - 268. parathyroid hormone receptors (PTHRs) utilizing fluorescent parathyroid hormone (PTH) were missing. Therefore, we generated various fluorescent PTH analogues which exhibit properties of native PTH in terms of affinity, potency and internalization. For the purposes of academic and commercial research, we utilized labeled PTH to set up three 097 time-resolved fluorescence assay formats: (I) Classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (II) Homogeneous time- resolved fluorescence resonance energy transfer (HTRF) based on tag-lite technology PDGF-BB induces signaling of protein kinase A in a cAMP-independent, but in a for high through-put screening; (III) HTRF based on antibodies, a synergistic approach redox-dependent manner in rat renal mesangial cells using HTRF with minimized receptor modification. This work will facilitate the development of new drugs directed to the PTHR as well as fundamental research on the Eisel F., Boosen M., Beck M., Pfeilschifter J., Beck K. - F. PTHR. Klinikum der Johann Wolfgang Goethe Universität Institut für Allgemeine Pharmakologie und Toxikologie, Theodor Stern Kai 7, 60590 Frankfurt, Germany
Several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ROS) that may attack the glomerular filtration 100 barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. Otherwise, ROS also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. To define such redox-driven Anandamide Production in Eosinophilic Granulocytes is Independent of IL-5 and signaling devices, we performed a non hypothesis-driven proteomic approach, to identify Eotaxin Stimulation homo- or heteromeric protein complexes induced by ROS. To this end, protein lysates of Engeli S., Reinke J., Zörner A., Tsikas D., Jordan J. human podocytes were treated with or without hydrogen peroxide (250 µM) for 10 min. Medizinische Hochschule Hannover Institut für Klinische Pharmakologie, Carl-Neuberg- Thereafter, the cell lysates were subjected to diagonal 2D gel electrophoresis and Straße 1, 30625 Hannover, Germany putative redox-affected proteins were analyzed by MS/MS-analysis. By this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redox- Introduction: Some animal and in vitro studies suggest that endocannabinoids exert dependent manner. One of those proteins could be characterized as the regulatory anti-inflammatory effects. Specifically, inhaled anandamide reduced the obstructive subunit of protein kinase A (R-subunit of PKA), which belongs to the family of effect of leukotrien D4 in airways, and a specific cannabinoid receptor agonist serine/threonine kinases. To evaluate whether ROS is capable to activate PKA also in a significantly reduced pulmonary inflammation in guinea pigs. We have recently shown more physiological setting, we treated rat mesangial cells with PDGF-BB to induce ROS that segmental bronchial allergen challenge is associated with significant increases of formation and we could demonstrate that PDGF-BB induces dimerization of R-subunits anandamide concentrations in bronchoalveolar fluid of patients with asthma. The in a redox-dependent manner. To demonstrate whether PDGF-BB induces PKA- concomitant increase in eosinophilic counts, eotaxin and IL-5 concentrations in dependent pathways, we analyzed the effects of PDGF-BB on phosphorylation of serine bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic 157 of vasodilater stimulated protein (VASP) a classical target of PKA. In fact, PDGF-BB granulocytes in response to chemotactic stimuli. induced VASP phosphorylation independently of intracellular cAMP levels. Moreover, elevating cAMP levels via activation of adenylate cyclase with forskolin did not change Methods: Peripheral eosinophilic granulocytes were isolated from whole blood by the dimerization state of R-subunits. PDGF-BB-induced dimerization of the R-subunits means of Percoll gradient centrifugation and magnetic separation employing CD16- and subsequent phosphorylation of VASP was blocked by diphenyljodonium (DPI), antibodies conjugated to magnetic beads. Isolated cells were counted and anandamide indicating activation of a NADPH oxidase is essential for PKA activity. Taken together, measurements were typically performed in whole cell lysates of 1.5x106 eosinophils. We we demonstrate a redox-dependent activation of PKA by PDGF-BB and this may hint stimulated eosinophils with varying concentrations of IL-5, eotaxin-1 (CCL11), eotaxin-2 also for a probably protective role of ROS in rat mesangial cells. (CCL24), and eotaxin-3 (CCL26). To prevent anandamide degradation, a specific fatty acid amide hydrolase (FAAH) inhibitor (oloxa) was employed. Anandamide concentrations and FAAH activity were determined by stable isotope dilution using LC- MS/MS protocols. 098 Results: First, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. However, cellular anandamide content could only be measured when LuSens: a stable antioxidant response element dependent reporter gene cell line FAAH was effectively blocked with oloxa, and strong FAAH activity was demonstrated in for detection of skin sensitizers eosinophils. With oloxa, typical anandamide concentrations were in the range of 2-4 pM/1.5x106 eosinophils. Neither IL5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml Eltze T.1, Bauch C.1,2, Ramirez-Hernandez T.1, Kolle S.1, Mehling A.3, Wruck C. J.4, van 1 1 either alone or in varying combinations) did stimulate anandamide production after Ravenzwaay B. , Landsiedel R. 30min of incubation. 1 BASF SE Experimental Toxicology and Ecology, Ludwigshafen, Germany 2 University of Manchester Faculty of Life Sciences, Manchester, Great Britain Conclusion: Our results suggest that chemotactic molecules like eotaxin and IL-5 are 3 BASF Personal Care and Nutrition GmbH, Düsseldorf, Germany not responsible for increased anandamide formation in eosinophils during allergen 4 RWTH Aachen Universitätsklinikum, Aachen, Germany challenge. In a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. The suprisingly high FAAH activity in eosinophils Testing the potential sensitizing capacity of chemicals is currently done by using the may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis murine local lymph node assay (LLNA). Animal welfare and EU cosmetics directive by production of arachidonic acid. demands alternative methods to animal tests. The purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. Based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the Nrf2-Keap1-antioxidant response element (ARE) regulatory pathway. 101 Here, we report the results obtained from the LuSens assay that detects electrophilic chemicals using the Nrf2 pathway. The cell line LuSens was derived from immortalized keratinocyte HaCaT cells and carries a luciferase reporter gene under the control of an Screening Methodology for estimatation of dermal absorption in vitro ARE-element from the rat NADPH quinone reductase NQ1. The LuSens assay was in Fabian E., Goth C., Guth K., Mehling A., van Ravenzwaay B., Landsiedel R. house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 BASF SE Experimentelle Toxikologie, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, performance standard substances of the local lymph node assay. Germany The predictivity of this assay was compared to the predictivity of the murine LLNA and to human patch test data and can be considered as reliable screening approach (accuracy Dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or of 83% compared to human data). However, in order to cope with the complex multi-step cosmetic formulations, but often dermal absorption is a critical parameter in risk mechanism of skin sensitization, an integrated approach of in vitro assays mimicking assessment of pesticides or chemicals. Therefore, knowledge of dermal absorption is several steps was designed; thereof, the ARE-dependent gene activation represents e.g. helpful in formulation development. Skin absorption is routinely measured in vivo or one module. in vitro following OECD TG 427 or 428. However, these tests are complex, time consuming and expensive. Therefore, a method was developed to allow simple and rapid screening. The experiment uses dermatomized skin in modified Franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. 099 After 6h, the skin is washed and the amount of penetrated substance is quantified. The receptor fluid and the washing solutions are optimized for subsequent analyses by LC- MS. We performed dermal absorption screenings in parallel to our routine guideline Time-resolved fluorescence ligand-binding assays for parathyroid hormone studies and demonstrated a good correlation of the results of both study types: the total receptors recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. The efficacy of the Emami-Nemini A.1,2, Roux T.3, Leblay M.3, Bourrier E.3, Lamarque L.3, Trinquet E.3, 1,2 skin washing procedure is lower than under routine conditions, most probably due to the Lohse M. J. change to an LC-MS-compatible washing solution. 1 Julius-Maximilians-University of Würzburg Department of Pharmacology and Toxicology, Versbacherstraße 9, 97078 Würzburg, Germany Overall, the dermal absorption screening is an easy, fast and cost-effective screening 2 Julius-Maximilians-University of Würzburg Rudolf Virchow Center for Experimental method for the estimation of dermal absorption of a wide variety of test substances and Biomedicine, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany formulations. 3 Cisbio Bioassays, Parc Technologique Marcel Boiteux BP 84175, 30200 Codolet, France
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Internalization of biotin-labeled p53 tumor suppressor protein into p53-deficient Do histamine H2 and H4 receptors affect noradrenaline release in the human osteosarcoma cells by a novel streptavidin nanocarrier impairs cell viability and cerebral cortex? induces caspase activation Feliszek M.1, Schacht D.1, Schulte K.1, Jergas B.1, von Lehe M.2, Schlicker E.1 1 2 2 2 2 1 Fahrer J. , Ng D. Y. W. , Eisele K. , Kuan S. L. , Weil T. , Barth H. 1Universität Bonn Institut für Pharmakologie und Toxikologie, Sigmund-Freud-Str. 25, 1Universitätsklinikum Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein- 53105 Bonn, Germany Allee 11, 89081 Ulm, Germany 2Universität Bonn Neurochirurgische Universitätsklinik, Sigmund-Freud-Str. 25, 53105 2Universität Ulm Institut für Organische Chemie III, Albert-Einstein-Allee 11, 89081 Ulm, Bonn, Germany Germany The practical relevance of histamine H1 and H3 receptors in the brain can be easily The p53 tumor suppressor protein is frequently inactivated in human cancers by diverse deduced since H1 receptor antagonists of the first generation have a sedative effect and mechanisms. Owing to its fundamental role in the maintenance of genomic stability and an inverse H3 agonist, pitolisant (close to its introduction to the market), is active against cancer prevention, p53 is an attractive target in cancer therapy and several approaches excessive daytime sleepiness associated with narcolepsy. In this context the question were pursued to restore p53 function in tumor cells. Polyamidoamine (PAMAM) arises whether also H2 and H4 receptors possess a practical relevance in the brain. To dendrons are positively charged molecules with a systematically branched structure that this end, we examined whether the electrically evoked 3H-noradrenaline release in interact with the negatively charged cell membrane, inducing cellular uptake via superfused human cerebral cortex slices is affected by agonists at the above receptors. endocytosis. The H2 agonist impromidine 10 µM failed to affect noradrenaline release in human In the present study, biotin-labeled p53 protein was attached to a dendronized cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. maximum extent of facilitation was 50 %, the pEC50 was 6.9 and the pA2 of the H2 First, biotin-substituted PAMAM dendrons were conjugated with streptavidin to allow antagonist ranitidine against impromidine was 7.2. With respect to H4 receptors there is formation of the dendronized streptavidin nanocarrier. The nanocarrier displayed uptake some controversy in the literature whether they occur in the brain at all. However, we into HeLa cervix carcinoma and A549 lung adenocarcinoma cells without detectable were able to detect H4 receptor mRNA in the human and mouse cortex by the reverse cytotoxicity. Biotin-labeled p53 was then conjugated to the dendronized streptavidin, transcriptase polymerase chain reaction. In cortex slices of either species, noradrenaline preserving its specific DNA-binding in vitro. Immunoblot analysis revealed efficient release was not affected by the H4 agonist 4-methylhistamine 10 – 30 µM but inhibited internalization of biotin-p53 into HeLa cells in the presence of dendronized streptavidin. by histamine 10 µM via H3 receptors by 28 and 55 %, respectively. In mouse cortex In line with this finding, specific cellular uptake of biotin-p53 was observed by confocal membranes, 4-methylhistamine 30 µM also failed to affect 35S-GTPγS binding although microscopy, which showed a cytoplasmic and peri-nuclear localization in HeLa, A549 R-α-methylhistamine 3 µM, acting via H3 receptors, increased it by 20 %. In conclusion, and SaOS osteosarcoma cells. The internalized biotin-p53 also partially co-localized H2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig with early endosomes. Importantly, the delivery of biotin-p53 into p53-deficient SaOS but not human cortex. Despite the presence of H4 mRNA in the brain, functional cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and demonstrating its biological functionality. modulation of 35S-GTPγS binding (mice), could not be shown. This study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. 105
Murine Cx40 promoter activity is dependent on the transcription factor CREB 103 Fels B., Nunes F., Schmitz W., Müller F. U. Westfälische Wilhelms-Universität Münster Institut für Pharmakologie und Toxikologie, Domagkstraße 12, 48149 Münster, Germany Identification of novel AhR target genes in rat liver oval cells 1 2 3 3 1 Faust D. , Vondracek J. , Hruba E. , Machala M. , Dietrich C. Connexin 40 (Cx40) is a gap junction protein expressed in atrial myocytes and the 1Institut für Toxikologie, Universitätsmedizin Mainz, Obere Zahlbacherstr. 67, 55131 ventricular conduction system, mediating the electrical intercellular communication in the Mainz, Germany myocardium. Alterations in Cx40 function were linked to the pathophysiology of atrial 2Institute of Biophysics, Department of Cytokinetics, Kralovopolska 135, 61137 Brno, fibrillation and heart failure. In the heart, cAMP dependent gene transcription is Czech Republic regulated by members of the CREB/CREM/ATF family which bind to cAMP responsive 3Veterinary Research Institute, Department of Toxicology, Pharmacology, and elements (CREs). Similar to the human Cx40 gene promoter, the murine promoter Immunopharmacology, Kralovopolska 135, 61137 Brno, Czech Republic contains one CRE. Cardiomyocyte-specific overexpression of a CREM-isoform (CREM- Ib∆C-X) led to Cx40 down-regulation, suggesting that CREB related transcription factors The aryl hydrocarbon receptor (AhR) is a transcription factor involved in physiological are involved in Cx40 gene regulation. In order to study the functional role of CREB in the processes, but also mediates most, if not all, toxic responses to 2,3,7,8- regulation of the Cx40 promoter we have generated a luciferase based murine Cx40 tetrachlorodibenzo-p-dioxin (TCDD), some polycyclic aromatic hydrocarbons (PAHs) promoter reporter gene construct and monitored its activity in a permanent cell line upon and dioxin-like polychlorinated biphenyls (PCBs), such as PCB126. Activation of the overexpression of constitutive-active CREB (caCREB) and a non-phosphorylatable AhR by these ligands leads to its dimerization with ARNT and transcriptional activation dominant-negative CREB (dnCREB) isoform. Surprisingly, overexpression of caCREB of several phase I and II metabolising enzymes. While it is generally accepted that many and dnCREB both lead to a reduction of Cx40 promoter activity (caCREB 46% ± 5% vs PAHs are thereby transformed to genotoxic metabolites, this classical signalling pathway control 100% ± 3%; p<0.05 vs control , n=15), dnCREB 45% ± 2% vs control 100% ± so far failed to explain the tumour promoting effects of the nongenotoxic compounds 3%; p<0.05 vs control, n=18). TCDD and PCB126. Thus, there is an urgent need to define genetic programmes Conclusion: The activity of the murine connexin 40 promoter is modulated by CREB. orchestrated by AhR to unravel its role in physiology and toxicology. We have recently Both caCREB and dnCREB led to Cx40 down-regulation, which could be explained by shown that treatment of rat liver oval cells with TCDD or PCB126 leads to a release from induction of inhibitory transcription factors CREB/CREM/ATF1 transcription factor family, contact-inhibition involving activation of the AhR, elevation of JunD protein levels and which in turn could suppress Cx40 promoter activity. (Supported by the DFG) transcriptional activation of cyclin A (1, 2). Loss of contact-inhibition is one hallmark of tumour promotion. To better understand AhR-driven pathways we identified the transcriptional programme using high density microarrays in response to PCB126. Already 6 h after treatment, 69 genes were found to be upregulated and 76 genes 106 downregulated indicating that these are direct AhR-dependent target genes. DAVID analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, TGF-b signalling and cell-cell communication. Ten of the Redox-dependent regulation of human protease-activated receptor-2 by activated 69 genes were selected for confirmation by semi-quantitative RT-PCR. Using the AhR factor X inhibitor CH-223191 and siRNA directed against AhR and ARNT, we further Jobi K.1, Rauch B. H.2, Doller A.3, Eberhardt W.3, Fischer J. W.1, Schrör K.1, Fender A. demonstrated that AhR- and ARNT-function is required for transcriptional activation of 1 the selected genes. Finally, we identified the transcription factor Foxq1as a novel AhR C. 1 target protein in rat liver oval cells. Although the function of Foxq1 is poorly understood, Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der it has been shown very recently that Foxq1 is overexpressed in colorectal cancer and is Heinrich-Heine Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany 2 involved in epithelial-mesenchymal transition in breast cancer cells. Its function in AhR- Ernst-Moritz-Arndt-Universität Greifswald Institut für Pharmakologie, Friedrich-Loeffler- mediated tumour promotion, however, remains to be determined. Str. 23d, 17487 Greifswald, Germany 3 pharmazentrum Frankfurt, Klinikum der J.W. Goethe Universität Institut für Allgemeine References Pharmakologie und Toxikologie, Theodor Stern Kai 7, 60590 Frankfurt a.M., Germany 1. Vondracek et al., (2005) Toxicol. Sci., 83, 53-63 2. Weiss et al. (2008) Oncogene, 27, 2198-2207 Aim: Activated factor X (FXa) exerts coagulation-independent actions such as proliferation of vascular smooth muscle cells (SMC) through protease-activated receptors PAR-1 and PAR-2. Both receptors are upregulated upon vascular injury but the underlying mechanisms have not been defined. We examined if FXa regulates PAR- 1 and PAR-2 expression. Methods: Human saphenous vein SMC were serum-deprived prior to study. Gene expression was determined by realtime PCR, total protein and cell-surface expression by western blot and flow cytometry respectively. DNA synthesis was assessed by [3H] thymidine incorporation, oxidative stress by dichlorofluorescein diacetate fluorescence and 8-isoprostane formation. Gene silencing utilised commercially available siRNA. Transcription factor/DNA binding was determined by chromatin immunoprecipitation assay, binding of mRNA stabilising factors to target mRNA by immunoprecipitation (“pull-down”) PCR. S26
Results: FXa increased PAR-2 mRNA, protein and cell-surface expression and and dorsal longitudinal anastomatic vessel formation compared to control injected fish. augmented PAR-2-mediated mitogenesis. PAR-1 expression was not influenced. The This phenotype could be rescued by early re-expression of NDPK B. Secondly, ischemia regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a driven angiogenesis was studied in NDPK B-depleted mice and wildtype littermates after PAR-2 selective activating peptide. The thrombin inhibitor argatroban or PAR-1 gene excision of the left femoral artery. Hind limb blood flow was assessed by laser Doppler silencing did not influence FXa-stimulated PAR-2 expression. FXa increased oxidative perfusion imaging immediately before and after ligation (day 0) and on postoperative stress and expression of the NADPH oxidase subunit NOX-1 in SMC. NOX-1 gene days 3, 7, 14, 21, 28, and 35. A significant reduction of recovery was observed in the silencing prevented FXa-stimulated PAR-2 regulation, as did ebselen and catalase. NDPK B depleted mice at days 3 and 7. Thirdly, in vitro-sprouting angiogenesis was Exogenous hydrogen peroxide increased PAR-2 expression and mitogenic activity. FXa analyzed in human umbilical vein endothelial cell (HUVEC) spheroids with and without induced nuclear translocation and PAR-2 DNA binding of nuclear factor kB (NFkB). siRNA-mediated NDPK B knockdown. Vascular endothelial growth factor (VEGF) - Inhibition of NFkB prevented FXa-stimulated PAR-2 expression. In separate studies, induced sprouting was significantly attenuated by NDPK B knock down by more than FXa promoted PAR-2 mRNA stabilisation through increased human antigen R 50% in comparison with control transfected HUVEC. We conclude from these results (HuR)/PAR-2 mRNA binding and cytoplasmic shuttling. HuR gene silencing abolished that NDPK B is an essential regulator of angiogenesis. The loss of NDPK B may FXa-stimulated PAR-2 expression. specifically interfere with the VEGF-induced migration and proliferation during Conclusion: Expression and mitogenic activity of vascular PAR-2, but not PAR-1, is endothelial sprouting. upregulated by FXa. This action involves transcriptional and post-transcriptional mechanisms mediated through NOX-1-containing NADPH oxidase and its downstream effectors hydrogen peroxide, NFkB and the mRNA stabilising protein HuR. Continued generation of FXa by the mural thrombus, and the autoregulatory feedback control of 109 PAR-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. Ethylene oxide in blood of ethylene-exposed volunteers Kessler W.1, Numtip W.1, Pütz C.1, Klein D.1,2, Filser J. G.1,2 1Helmholtz Zentrum München Institut für Toxikologie, Ingolstädter Landstr. 1, 85764 107 Neuherberg, Germany 2Technische Universität München Institut für Toxikologie und Umwelthygiene, Biedersteiner Str. 29, 80802 München, Germany The mRNA stabilising factor HuR is a critical regulator of human protease- activated receptor 4 Ethylene (ET) is a commercially important high volume industrial chemical. Inhaled and 1 2 3 3 1 1 Pavic G. , Rauch B. H. , Doller A. , Eberhardt W. , Fischer J. W. , Schrör K. , Fender A. endogenous ET is metabolized in mammals to ethylene oxide (EO), which is 1 C. carcinogenic in rats and mice. Until now, no data on the oxidation of ET in ET-exposed 1Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der humans has been published. Heinrich-Heine Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany In the present study, we investigated the formation of EO in four male adult volunteers 2Ernst-Moritz-Arndt-Universität Greifswald Institut für Pharmakologie, Friedrich-Loeffler- exposed for 4 hours to constant atmospheric ET concentrations of 5, 20, or 50 ppm by Str. 23d, 17487 Greifswald, Germany means of a breathing mask. During exposure, ET concentrations were measured in 3pharmazentrum Frankfurt, Klinikum der J.W. Goethe Universität Institut für Allgemeine inhaled and exhaled air by GC/FID and EO concentrations in venous blood by GC/MS. Pharmakologie und Toxikologie, Theodor Stern Kai 7, 60590 Frankfurt a.M., Germany Rates of ET metabolism were obtained from the product of the differences in the ET concentrations with the pulmonary ventilation. In each subject, linear correlations were Aim: We recently reported that functional expression of PAR-4 thrombin receptors is found between the ET exposure concentration and the rate of ET metabolism or the EO induced in human saphenous vein SMC exposed to high glucose. This effect could be concentration in blood. Mean rate of ET metabolism was attributed in part to transcriptional mechanisms mediated through NFkB but the 5.5 ± 1.4 nmol/h/ppm/kg body weight. Steady-state concentrations of EO in blood contribution of post-transcriptional effects such as mRNA stabilisation is not known. This differed by a factor of 2 between the volunteers. These inter-individual differences likely study explored the potential role of the mRNA stabilising factor human antigen R (HuR) reflect the polymorphism of glutathione S-transferase theta, the main EO metabolizing in the regulation of PAR-4. enzyme in human liver. Mean EO concentration in blood at steady state was Methods: Human saphenous vein SMC were serum-deprived prior to study. Gene 1.5 ± 0.08 nmol/l blood per ppm of ET. expression was determined by realtime PCR, protein expression by western blotting. The data will be used for validating a physiological toxicokinetic model which will Gene silencing utilized commercially available siRNA. HuR binding to PAR-4 mRNA was describe the ET related EO tissue burdens in rodents and humans. The model determined by immunoprecipitation (“pull-down”) PCR. predictions will support risk evaluations of ET. Results: High glucose (25 mM vs 5.5 mM) slowed PAR-4 mRNA degradation in the Financially supported by the Lower Olefins Sector Group of Cefic. presence of actinomycin D. PAR-1 mRNA decay was not affected. HuR binding to PAR- 4 mRNA and nucleo-cytosolic shuttling was enhanced by high glucose, total HuR protein expression was not affected. HuR siRNA abolished the high glucose-stimulated induction of PAR-4 mRNA. Hydrogen peroxide (H2O2) also induced cytosolic HuR 110 shuttling and increased PAR-4 mRNA and total protein expression. The role of endogenously generated H2O2 in the regulatory effect of high glucose on PAR-4 expression was investigated with the NADPH oxidase inhibitors In vitro effect of STW11 on human dendritic cells apocynin/diphenyliodonium (to prevent H2O2 generation) and cell-permeant catalase (to 1 2 1 1 2 degrade cellular H O ). Both approaches prevented the stimulatory effect of high Fink C. , Bonaterra G. A. , Kelber O. , Weiser D. , Kinscherf R. 2 2 1 glucose on PAR-4 expression. Cyclic AMP has been reported to suppress HuR Steigerwald Arzneimittelwerk GmbH Scientific Department, Havelstr. 5, 64295 activation and in the present study, the cyclic AMP stimuli forskolin and cicaprost Darmstadt, Germany 2 (prostacyclin analog) suppressed basal HuR shuttling and PAR-4 transcript stability. Philipps-Universität Marburg Department of Medical Cell Biology, Robert-Koch-Str. 8, Cicaprost also attenuated high glucose-induced HuR binding to PAR-4 mRNA and as a 35032 Marburg, Germany consequence normalised PAR-4 expression and inflammatory signalling in high glucose- treated cell. Extracts of Echinacea (purple coneflower) are used in the prevention and therapy of Conclusion: The regulation of PAR-4 thrombin receptors in human vascular SMC is infectious diseases. The medicinal product STW 11 contains the extract from purple critically dependent on the mRNA stabilising actions of HuR. Through activation of HuR, coneflower, and in addition, extracts of monkshood, venom of honey bee and high glucose and other HuR stimuli such as Ang II and exogenous H2O2, increase PAR- bushmaster snake in homeopathic dilutions. Previous studies showed a stimulation of 4 expression, while cyclic AMP agonists such as prostacyclin oppose this effect. Such the cellular and humoral immune response. interactions could potentially represent a fine-tuning mechanism to control PAR-4 Dendritic cells (DCs) are antigen presenting cells that act at the interface of the innate expression and ultimately also the mitogenic and inflammatory actions of thrombin in the and adaptive branches of the immune system. During stages of DC differentiation, the vessel wall. ability to internalize antigens varies and decreases during maturation. In this study, we determined the influence of STW11 on the expression of maturation related genes (CD1a, CD83), cytokines (TNFα, Il-4, IL-12), chemokines (CCR7), major histocompatibility complex II(MHC-II) and Toll-like receptors (TLR2, TLR4). In mature (mDC) and immature DC (iDC) using real-time RT-PCR. Peripheral blood mononuclear 108 cells (PBMCs) were isolated from buffy coats of human volunteers by densitygradient (Ficoll®) and seeded in 6 well plates. Non-adherent cells were eliminated. To induce iDC development, 50 ng/ml IL-4 and 50 ng/ml granulocyte macrophagecolony stimulating Nucleoside Diphosphate Kinase B is an Essential Regulator of Angiogenesis factor (GM-CSF) were added. At day 6, maturation was induced by addition of 1 1 1 1 1 2 2 Feng Y. , Butenschön V. M. , Qiu Y. , Gross S. , Wolf N. M. , Völker K. , Kuhn M. , lipopolysaccharide (LPS) at a final concentration of 1µg/ml and cultured for additional 3 3 1 Skolnik E. , Wieland T. days. 1Institut für experimentelle und klinische Pharmakologie, Klinikum Mannheim, After incubation with different concentrations (0.1-5%) of STW11 a significant increase Maybachstr. 14, 68169 Mannheim, Germany of CD1a (1.6-2.1 fold), CD83 (1.2-1.3 fold), TNFα (1.2-1.4fold), CD40 (1.3-1.5 fold), 2Physiologisches Institut Lehrstuhl I, Röntgenring 9, 97070 Würzburg, Germany MHC-II(1.8-2.3 fold) and CCR7 (1.3-1.4 fold) mRNA expression was measured in mDC 3Division of Nephrology, New York University Langone Medical Center, New York, treated with STW 11 compared to control. However, in mDC concentrations of 0.1-5% 10016, United States STW11 did not increase the expression of IL-4, IL12, ADAM19, CD11c, TLR2 and TLR4 genes. Nucleoside diphosphate kinase B (NDPK B) is a member of a family of ubiquitously In iDC, compared to control, we found a significantly increased expression of CD83 (2.1- expressed enzymes required for nucleoside triphosphate synthesis. Thus, they are 2.6 fold) and TNFα (2.6-3.3-fold) genes after treatment with 0.1-5% STW11, involved in the regulation of a variety of cellular processes, e. g. G protein mediated respectively, but no effect was found on the expression of CD1a, IL-4, IL12, ADAM19, signal transduction. However, whether NDPK B has a specific role in the regulation of CD11c, CD40,TLR2, TLR4, MHC-II and CCR7. angiogenic processes in endothelial cells is unknown. Therefore, we studied the function In summary, these data demonstrate a stimulatory effect of STW 11 in iDC and of NDPK B in the vasculature in a developmental, an ischemia-induced and an in vitro especially in mDC, concerning an increase of various genes related to maturation model of angiogenesis. Firstly, depletion of NDPK B expression was achieved by (CD83), immunomodulation (TNFα, CD40), adhesion (CCR7) and antigen presentation morpholino-mediated knockdown in zebrafish embryos in which the developing (MHC-II) and are in accordance with the therapeutic use in infectious diseases. vasculature can be visualized by EGFP expression in the endothelium. 72h post fertilization, NDPK B knockdown larvae showed a dramatic inhibition of intersegmental S27
111 Objective: Sphingosine-1-phosphate (S1P) is a cellular signaling lipid generated by sphingosine kinase-1 (SPHK-1). This study investigated the potential regulation of Estimation of the consumer inhalation risk of waterproofing sprays SPHK-1 in thrombin-stimulated human vascular smooth muscle cells (SMC) and in mice treated with the direct thrombin inhibitor dabigatran. Fischer M.1, Koch W.2, Windt H.2, Dasenbrock C.1 1 Methods: SPHK-1 expression was determined by Taqman® real-time PCR and Fraunhofer Institut für Toxikologie und Experimentelle Medizin Toxikologie und Western blotting in human vascular SMC and ApoE-deficient mice. Cell proliferation was Umwelthygiene, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany 2 determined via DNA synthesis and cell counting. Binding of the mRNA stabilising protein Fraunhofer Institut für Toxikologie und Experimentelle Medizin Aerosoltechnik und human antigen R (HuR) to target mRNA was measured by immunoprecipitation Chemische Analytik, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany (pulldown) PCR. ApoE mice were treated with dabigatran etixilate (DE)-supplemented chow (10 mg DE/g chow) or matching placebo for 6 months. Plaque area was Waterproofing sprays are widely used consumer products containing for example determined by oil red O staining. fluorinated polymers or silicon based compounds dissolved in alcohols or volatile Results: Thrombin induced a time- and concentration-dependent (1-100 nmol/L) petroleum distillates. There have been repeated reports on cases of severe acute increase in SPHK-1 mRNA and protein expression in human saphenous vein SMC, n=6- respiratory disorders especially when using products that newly entered the market. It is 7. This was mimicked by a synthetic PAR-1 ligand. Inhibition of SPHK-1 attenuated hypothesized that impairment of the pulmonary surfactant by deposition of inhaled thrombin-induced SMC proliferation but not SMC migration (n=5). The regulatory action respirable particles of the active compound is one of the main causes of the acute lung of thrombin on SPHK-1 expression was suppressed by siRNA against the mRNA injury. Since the inhalation toxicity cannot be predicted a priori based on the physical stabilisiserHuR. In thrombin-stimulated SMC, HuR binding to SPHK-1 mRNA and and chemical properties of the formulation, proper test strategies are required to ensure subsequent nucleo-cytosolic shuttling was enhanced. Accordingly, thrombin induced consumer safety. SPHK-1 mRNA stabilisation in SMC in the presence of actinomycin D. In ApoE-deficient We propose to combine screening tests addressing both, exposure and acute lung mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly toxicity. The exposure potential of the spray product is characterized by determining the reduced aortic SPHK-1 expression by 50% (n=5) and plaque size by 35% compared to release fraction of the active compound in the respirable particle size range under control animals (n=10). conditions relevant for the product application. This is carried out by spraying defined Conclusions: Thrombin induces SPHK-1 expression and S1P synthesis in vascular quantities of the product into a control volume and measuring the concentration of health SMC via the mRNA stabilising protein HuR. This leads to increased SMC proliferation. related size fractions. This procedure takes into account spray ageing, especially size Inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques reduction of the droplets due to solvent evaporation. possibly by reducing SPHK-1 expression. The isolated perfused lung is used as a model for testing acute toxicity. Ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm MMAD generated from the liquid spray formulation. Lung compliance and lung resistance are continuously monitored during exposure. Dose dependent deviations from the normal values (without exposure) are used as read-out parameters. 114 Using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. In its current stage Mycotoxin contamination and cytotoxicity of grain mill products 1 1 2 2 1 of development the simple test method is recommended for screening of substances Föllmann W. , Behm C. , Zühlke S. , Spiteller M. , Degen G. H. only. 1Leibniz Research Centre for Working Environment and Human Factors, Ardeystr. 67, 44139 Dortmund, Germany 2Institute of Environmental Research, TU Dortmund, Otto-Hahn-Strasse 6, 44221 Dortmund, Germany 112 Typical grain mill products from North-Rhine Westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced Induction of oxidative damage in calf thymus DNA by the Fusarium mycotoxin fractions) from the milling process were analysed for their mycotoxin content by LC- zearalenone after metabolic activation with liver microsomes MS/MS. The cytotoxicity of sample extracts with known mycotoxin composition was then assessed in V79 cell cultures by means of the neutral red uptake assay, in parallel with Fleck S. C., Pfeiffer E., Metzler M. pure reference mycotoxin mixtures. KIT - Institute of Applied Biosciences Chair of Food Chemistry, Adenauerring 20a, Extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and 76131 Karlsruhe, Germany enniatin B (from not detectable to 0.4 µg/g DON and 0.5 µg/g EnnB) induced no measurable cytotoxicity. On the other hand, although mycotoxin contamination levels Zearalenone (ZEN) is an estrogenic mycotoxin produced by Fusarium species. The were also rather low in bran, these samples induced strong cytotoxicity: Extracts of bran adverse effects of ZEN and its reductive metabolite zearalenol (ZEL) are often derived from rye and spelt were more cytotoxic than those of wheat bran. The cytotoxic compared to those of 17-beta-estradiol (E2) and estrone (E1). These endogenous effects of the bran samples cannot be related to their mycotoxin content as comparable estrogens are associated with an increased risk for cancer, which may be mediated by concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by P450-catalyzed not cytotoxic. metabolic activation to catechols (Wang et al., Chem Res Toxicol 23, 1365, 2010). Like By-products from certain stages of the milling process (sorting and waste fractions) were E2 and E1, ZEN and ZEL exhibit marked estrogenicity and also undergo aromatic found to contain mycotoxins at rather high levels: Enniatin B was detected in nearly all hydroxylation to catechol metabolites (Pfeiffer et al., Mol Nutr Food Res 53, 1123, 2009). samples, and also T-2 toxin, HT-2 toxin, ergotamine, ergocornin and deoxynivalenol The aim of the present study was to examine the formation of catechol metabolites of were present. Waste sample extracts with notable mycotoxin levels (up to 6 µg/g DON, 7 ZEN by liver microsomes of various species and their potential for redox cycling. µg/g EnnB, 16 µg/g ergotamine, 50 ng/g HT-2 toxin) exert pronounced cytotoxicity in Catechol metabolites are frequently associated with the generation of reactive oxygen V79 cells. The cytotoxicity of these samples was somewhat stronger than expected species and subsequent oxidative damage of DNA, for which 8-oxo-7,8-dihydro-2’- when compared with mixtures of reference mycotoxins tested in parallel. deoxyguanosine (8-oxo-dG) is a common biomarker. The propensity of the catechol In summary, the tested flour and wholemeal flour extracts contained only low levels of metabolites of ZEN and ZEL to cause the formation of 8-oxo-dG in isolated calf thymus mycotoxins and were not cytotoxic. In contrast, bran samples showed cytotoxicity which DNA was determined using a LC-ESI-MS/MS method. To this end, ZEN was incubated cannot be explained solely by their mycotoxin content. This unexpected observation in with microsomes from human, rat, mouse, bovine and porcine liver as well as with real samples and combination effects of mycotoxin mixtures require further studies. human CYP1A2, and the incubations were extracted with ethyl acetate. The extract was analyzed with LC-MS and then added to a solution of calf thymus DNA in the presence Acknowledgement: The study was supported by a grant from the Land North-Rhine of copper(II) ions and NADPH. Westphalia within EU Ziel 2 Programm NRW 2007 – 2013 (EFRE) for the project The formation of 8-oxo-dG could be demonstrated with each extract. The levels of 8-oxo-dG “Mykotoxine in Lebens- und Futtermitteln – ein ungelöstes Problem in der correlated directly with the extent of catechol formation, which increased from steer to swine Qualitätssicherung” to human to mouse to rat microsomes. 15-Hydroxylated ZEN/ZEL, which is the major catechol, was more reactive than 13-hydroxylated ZEN/ZEL to form 8-oxo-dG. In conclusion, our study has shown that the catechol metabolites of ZEN are highly reactive and give rise to oxidative DNA damage in vitro. In addition, recent research from our laboratory revealed that the catechols of ZEN are less efficiently inactivated by 115 catechol-O-methyl transferase than the catechols of E2 and E1. The genotoxic potential of ZEN may constitute another biological activity in addition to the well-known estrogenicity. The ultraviolet B (UVB) activated aryl hydrocarbon receptor (AhR) regulates proliferation and apoptosis of human keratinocytes Supported by Deutsche Forschungsgemeinschaft (Grant ME 574/32-1) and “Food and Frauenstein K., Abel J., Fritsche E., Krutmann J., Haarmann-Stemmann T. Health” of KIT. Leibniz Institut für umweltmedizinische Forschung, Auf'm Hennekamp 50, 40225 Düsseldorf, Germany
The AhR is a ligand-activated transcription factor that mediates the toxicity of dioxins 113 and related compounds. Upon ligand binding the AhR translocates into the nucleus and dimerizes with ARNT to modulate gene expression, e.g. of CYP1A1. Recently, we have shown that UVB irradiation of human keratinocytes results in activation of the AhR and Thrombin regulates expression of sphingosine kinase-1 (SPHK-1) in human associated EGFR signaling leading to an enhanced expression of CYP1A1 and vascular smooth muscle cells - inhibition by dabigatran reduces vascular SPHK-1 proinflammatory COX-2, respectively. The initial step is the UVB induced intracellular expression and atherosclerotic burden in vivo formation of the tryptophan photoproduct 6-formylindolo[3,2b]carbazole (FICZ), a high 1 1 2 2 2 1 affinity AhR ligand. Thus, the FICZ activated AhR is an important mediator of the DNA Flößer A. , Böhm A. , Ermler S. , Rosenkranz A. C. , Schrör K. , Kroemer H. K. , Rauch damage independent part of the UVB response. Our current study aims to identify B. H.1 1 further aspects of AhR mediated UVB responses. Therefore, we analysed changes in Universitätsmedizin Greifswald Institut für Pharmakologie, Felix-Hausdorff-Str. 3, 17487 protein expression, proliferation and apoptosis in AhR+/+ and AhR-/- keratinocytes Greifswald, Germany (NCTC 2544) by western blot, flow cytometry and BrdU incorporation. 2Universitätsklinikum Düsseldorf Institut für Pharmakologie und Klinische Pharmakologie, Universitätsstraße 1, 40225 Düsseldorf, Germany S28
UVB exposure of NCTC cells led to a dose-dependent increase in apoptosis. Compared 118 to AhR+/+ cells, AhR-/- cultures exhibited an increased amount of apoptotic cells. This finding was confirmed in irradiated AhR+/+ cells, pretreated with the AhR antagonist 3’- methoxy-4’-nitroflavone. Moreover, the proliferation of sham as well as UVB irradiated Skin sensitization potential of unsaturated compounds in the loose-fit coculture- AhR-/- cells was significantly decreased. In AhR-/- cells we found a reduced expression based sensitization assay (LCSA) of checkpoint kinase 1 (Chk1), an important cell cycle regulator that arrests the cell in Frohwein T.1, Sonnenburg A.1, Schreiner M.2, Stahlmann R.1 G2/M upon DNA damage. Interestingly, UVB exposure led to a higher net 1 phosphorylation of Chk1 in AhR-/- cells, indicating that this pathway is responsible for Charité Universitätsmedizin Berlin Institut für Klinische Pharmakologie und Toxikologie, Luisenstr. 7, 10117 Berlin, Germany the observed AhR-dependent differences in proliferation and apoptosis. Further 2 expression analyses of Chk1 client proteins emphasize our hypothesis. Bundeswehrkrankenhaus Berlin Innere Medizin, Scharnhorststraße 13, 10115 Berlin, In conclusion our study identifies the AhR as an anti-apoptotic player in UVB irradiated Germany human NCTC cells. Therefore, we propose that the AhR is a suitable target to prevent UVB induced skin diseases. The murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT) have been used to study the sensitization potential of a series of unsaturated compounds by Kreiling et al. (2008). We have examined the same substances in the loose-fit coculture-based sensitization assay (LCSA), developed by our working group (Schreiner et al., 2007). Eight unsaturated compounds [oleic acid (OA), linoleic acid 116 (LA), linolenic acid (LnA), undecylenic acid (UA), fumaric acid (FA), maleic acid (MA), squalene (SQ), 1-octyn-3-ol (OC)] and succinic acid (SuA) were investigated using a coculture of keratinocytes and dendritic cell-related cells (DCrc). Sensitization potential Synthetic progestins exert divergent effects on thrombosis in a murine model of was quantified by flow cytometry measuring the increase of CD86 expressed on DCrc atherothrombosis (EC50 = half maximal effective concentration). A pronounced induction of CD86 at low 1 2 2 1 Freudenberger T. , Heim H. - K. , Mayer P. , Fischer J. W. concentrations was seen with LA, LnA and OA (EC50: 3, 5 and 5 µmol/L, respectively). 1Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der UA exhibited an intermediate response (EC50: 307 µmol/L). With OC and MA, we Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany observed effects at higher concentrations only (EC50: 1340 and 2293 µmol/L). No 2Bundesinstitut für Arzneimittel und Medizinprodukte, Kurt-Georg-Kiesinger-Allee 3, significant increase of CD86 was observed with FA, SuA and SQ. Because of poor 53175 Bonn, Germany solubility, SQ could not be studied adequately. Induction of CD86 was generally observed at concentrations which did not cause a major impairment of cell viability. Our Background: Medroxyprogesterone acetate (MPA), a synthetic progestin often used in results show a high degree of concordance with those obtained by the GPMT, except for postmenopausal hormone replacement therapy, has previously been described to be OA. In comparison with the results of the LLNA, those compounds which showed a pro-thrombotic in a murine model of accelerated atherosclerosis. However, nothing is so strong effect in the LLNA (OA, LA, LnA) also induced an increase of CD86 at low far known about effects of progestins with receptor profiles different from MPA (i.e. concentrations, whereas those with low stimulation indices in the LLNA induced no agonism or antagonism of mineralocorticoid- or androgen-receptors), such as significant increase of CD86 (FA, SuA) or only at higher concentrations (UA). We drospirenone, levonorgestrel and norethisterone acetate. Methods: Apo-/- mice were observed a discrepancy between the tests with MA and OC, causing a strong stimulation bilaterally ovariectomized (OVX) and substituted subcutaneously with MPA, of the murine lymph nodes, while the expression of CD86 was increased at high drospirenone, levonorgestrel and norethisterone acetate as well as the respective concentrations only. We assume that MA and OC might be false-positives in the LLNA, placebo pellets for 90 days on a Western-type diet. Subsequently, thrombosis was because they were also negative in the GPMT. induced by photochemical injury to the right carotid artery using Rose Bengal and a green light laser. Results: Compared to placebo, animals substituted with MPA showed References significantly shortened times to occlusion of the right carotid artery (placebo MPA: 50.0 ± Kreilin R, et al. Comparison of the skin sensitizing potential of unsaturated compounds 5.8 min. vs. MPA: 33.4 ± 4.2 min., n = 6 – 9, p < 0.05). In contrast, drospirenone, as assessed by the murine local lymph node assay (LLNA) and the guinea pig levonorgestrel or norethisterone acetate did not alter thrombotic responses. However, at maximization test (GPMT). Food and Chemical Toxicology 2008; 46: 1896-1904 least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 Schreiner M, et al. A loose-fit coculture of activated keratinocytes and dendritic cell- min., n = 7 – 8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. related cells for prediction of sensitizing potential. Allergy 2007; 62: 1419-1428. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. Furthermore, analysis of aortic gene expression revealed that in aortas of MPA-treated mice expression of matrix-metalloproteinase 9 (MMP-9) was induced as compared to placebo-treated mice. Conclusion: MPA, a progestin with glucocorticoid 119 effects, exerts a pro-thrombotic effect that is either progesterone- or glucocorticoid- receptor-dependent while progestins with receptor profiles different from MPA do not show a significant pro-thrombotic effect. Furthermore, the pro-thrombotic effect exerted Inhibition of the organic cation transporters OCT1 and OCT2 by drugs frequently by MPA may be associated with increased expression of MMP-9, a metalloproteinase used in psychiatry being known to destabilize atherosclerotic plaques and make them more prone to rupture. Münch K., Maas R., Zolk O., Fromm M. F. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich- Alexander Universität Erlangen-Nürnberg, Fahrstraße 17, 91054 Erlangen, Germany
Background: The first step in elimination of many cationic drugs is their uptake from the 117 blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (OCT1) and OCT2, respectively. The pivotal role of OCTs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces OCT- Rapid Screening for Mitochondrial Toxicity In Vitro Using an Oxygen-sensitive mediated elimination of a second drug. Although many psychoactive drugs are cationic Phosphorescent Probe at pH 7.4 and some of these have already been recognized as OCT inhibitors, a Freyberger A. systematic screen of this class of compounds is missing. Bayer HealthCare BPH GDD GED Toxikologie - P & CP, Aprather Weg 18, 42096 Methods: We screened a drug library of 50 most frequently prescribed psychoactive Wuppertal, Germany drugs (inpatient prescriptions in Germany, at least 4 million DDD each) for their inhibitory interaction with OCT1 and OCT2. Human embryonic kidney (HEK) cells stably Impaired mitochondrial function has been implicated with disease, aging, and drug- overexpressing OCT1 or OCT2 and the prototypical OCT substrate 1-methyl-4- induced toxicities. Analyzing mitochondrial respiration (MR) rates is one of the most phenylpyridinium (MPP+) were used as a test system. Cells transfected with the empty informative ways to assess mitochondrial function as it provides information on the the vector were used as controls. bioenergetic capacity of a tissue, however, previously measurements using Results: At 20 µM, 59% and 51%, respectively, of the tested compounds significantly polarography (Clark electrode) were cumbersome with only low throughput. In this work decreased OCT1- and OCT2-mediated uptake of MPP+. The most potent inhibitors we explored Luxcel’s water-soluble phosphorescent oxygen-sensitive probe (inhibition >75%) of OCT1 were chlorprothixen and clomipramine, whereas olanzapine, MitoXpressTM for the assessment of mitochondrial toxicity in freshly isolated male rat clomipramine and doxepin were the most potent inhibitors of OCT2. In contrast, neither liver mitochondria (RLM) in a 96-well plate format using glutamate/malate (20 mM/0.5 at 20 µM nor at 200 µM carbamazepin, haloperidol, lithium, moclobemide and valproic mM) and succinate (25 mM) as respiratory substrates. Inhibition of mitochondrial acid did significantly inhibit MPP+ uptake into HEK-OCT1 or HEK-OCT2 cells. There complexes I to IV, adenosine triphosphate synthetase and the adenosine diphosphate was a significant correlation between the degree of OCT1 and OCT2 inhibition (p (ADP) / adenosine triphosphate (ATP) antiporter by rotenone, thenoyltrifluoracetone Conclusions: Our results demonstrate that inhibition of OCT function by psychoactive (TTFA), antimycin A, potassium cyanide, oligomycin, and atractyloside was readily drugs has to be considered as a potential mechanism underlying drug-drug interactions. detected in ADP-stimulated RLM. Use of the two different substrates in parallel allowed Considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and to discriminate complex I inhibition by rotenone from complex II inhibition by TTFA, clomipramine between 30 and 80 µM in humans, inhibitory interactions of these whereas downstream of these complexes inhibition by the other model inhibitors compounds with hepatic OCT1 have to be taken in account. Our data will help to create occurred independent of the substrate used. Decoupling of MR from oxidative a chemoinformatic model to predict potential OCT-dependent interactions of phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (FCCP) was psychoactive drugs with the hepatic or renal elimination of coadministered drugs. detected best in the absence of ADP. Compared to polarographic measurement, the use This project is supported by the German Federal Ministry of Education and Research of an oxygen-sensitive probe is superior with regard to assay cycle time and sample (BMBF), project grant No. 01 EX1015B. throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new References chemical entities. Bachmakov I, Glaeser H, Fromm MF, König J: Interaction of Oral Antidiabetic Drugs with Hepatic Uptake Transporters: focus on OATPs and OCT1. Diabetes 57: 1463-1469, 2008 Ito K, Iwatsubo T, Kanamitsu S, Ueda K, Suzuki H, Sugiyama Y: Prediction of pharmacokinetic alterations caused by drug-drug interactions: metabolic interaction in the liver. Pharmacol Rev 50:387– 412, 1998 Kido Y, Matsson P, Giacomini KM: Profiling of a prescreption drug library for potential renal drug-drug interactions mediated by the organic cation transporter 2. J Med Chem 54(13): 4548-58, 2011 S29
Koepsell H: Substrate recognition and translocation by polyspecific organic cation Based on these data purine-derivatives might inhibit proliferation and induce apoptosis transporters. Biol Chem 392: 95-101, 2011 in glioma cells. As a result we hypothesize that these compounds could be potentially Lohse MJ, Müller-Oerlinghausen B: Psychopharmaka. In: Schwabe U, Paffrath D, eds. interesting for the drug-development of GBM therapy and therefore a clue for chemical Arzneiverordnungs-Report 2009. Berlin Heidelberg: Springer Verlag. pp 767-811, 2009 modifications. Further studies are required to identify the exact underlying mechanism of Nies AT, Hofmann U, Resch C, Schaeffeler E, Rius M, Schwab M: Proton Pump action of the tested purine-analogues. Inhibitors inhibit Metformin Uptake by Organic Cation Transporters (OCTs). PloS One 6(7):e22163, 2011 Zolk O, Solbach TF, König J, Fromm MF: Structural determinants of inhibitor interaction with the human organic cation transporter OCT2 (SLC22A2). Naunyn Schmiedebergs 122 Arch Pharmacol. 379(4):337-48, 2008 Zolk O, Solbach TF, König J, Fromm MF: Functional Characterization of the Human Organic Cation Transporter 2 Variant p.270Ala>Ser. Drug Metab Dispos. 37(6):1312-8, The Biological Role of Adenosine Receptors in Brown Adipose Tissue 2009 1 1 2 1 Gnad T. , Mutlu S. , Müller C. E. , Pfeifer A. 1PharmaCenter Bonn, Universität Bonn Institut für Pharmakologie & Toxikologie, Sigmund-Freud-Straße, 53105 Bonn, Germany 2PharmaCenter Bonn, Universität Bonn Pharmazeutisches Institut, Pharmazeutische 120 Chemie I, An der Immenburg 4, 53121 Bonn, Germany
Brown adipose tissue (BAT) is responsible for basal and inducible energy expenditure in Genome-wide DNA methylation in chronic heart disease mammals. BAT contains large amounts of mitochondria and is highly vascularized. BAT 1 1 2 1 3 3,4 2 Gilsbach R. , Preißl S. , Rühle F. , Weiss S. , Mühle A. , Doenst T. , Stoll M. , Hein lipolysis and thermogenesis are stimulated by sympathetic neurons. Importantly, recent 1 L. findings indicate that adult humans possess metabolically active BAT1. 1University of Freiburg Institute of Experimental and Clinical Pharmacology and Here, we analyzed the expression and function of adenosine receptors in BAT. Toxicology, Albertstr. 25, 79104 Freiburg, Germany Adenosine receptors (AdoR) are members of the superfamily of G protein-coupled 2 University of Münster Leibniz Institute for Arteriosclerosis Research, Domagkstr. 3, receptors. There are four subtypes of AdoRs in humans referred to as AdoRA1, A2A, A2B 48149 Münster, Germany and A3. They are widely expressed in tissues and mediate a variety of cellular functions, 3University of Leipzig Heart Center Department of Cardiac Surgery, Strümpellstr. 39, mostly due to their regulation of cAMP levels within cells. Interestingly, it has been 04289 Leipzig, Germany shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through 4 2 University of Jena Department of Cardiac and Thoracic Surgery, Erlanger Allee 101, AdoRA1 or A2A, respectively . However, the role of adenosine in the differentiation of 07747 Jena, Germany brown preadipocytes to adipocytes and in BAT function is not clear. To analyze the role of AdoRs in BAT, we use preadipocytes isolated from BAT of newborn mice and Background subjected them to a differentiation protocol.3 Cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. The molecular mechanisms controlling gene Abundance of AdoRA1, A2A, A2B and A3 mRNA was measured using qPCR. All four expression in cardiac failure are only partially known. DNA methylation is one epigenetic receptor subtypes are present in preadipocytes with AdoRA2B being the most abundant. mechanism that regulates long-term changes in gene-expression. To elucidate whether AdoRA1, AdoRA2A and AdoRA3 are significantly transcriptionally upregulated - albeit at DNA methylation is altered during the development and progression of chronic heart varying degree - during differentiation. AdoRA1 is upregulated 4.4 fold (+/- 0.3 fold) and failure, genome-wide DNA methylation profiles were determined in myocardial biopsies 11 fold (+/- 0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes from control patients and patients with cardiac hypertrophy or failure. (n=5). AdoRA2A is 23 fold (+/- 1.96 fold) upregulated at day 4 and 57 fold upregulated Methods and Results (+/- 2.48 fold) at day 7, respectively (n=4). AdoRA3 was found upregulated 3.6 fold (+/- Cardiac biopsies were obtained from patients with aortic aneurysm who served as 0.46 fold) at day 7 (n=4). In contrast to this, AdoR2B was downregulated to 0.87 fold (+/- control and did not show clinical signs of chronic heart disease (EF: 58±3 %, n=3) and 0.09 fold) at day 4 and to 0.69 fold (+/- 0.04) day 7 compared to preadipocytes (n=5). from patients with aortic stenosis. The latter group was subdivided according to the ejection fraction into hypertrophic (EF: 63±7 %, n=3) and failing patients (EF: 28±1 %, To investigate the functional role of AdoR in BATi cells, we analyzed lipolysis in mature n=3). After bisulfite conversion of extracted DNA, the methylation status of genomic DNA cells after acute treatment with specific agonists and antagonists. We observed that was quantified using the Infinium® HumanMethylation450 BeadChip (Illumina). This AdoRA2A activation by CGS21680 significantly increased lipolysis by 88% (+/- 0.33%) microarray allows analysis of more than 485,000 methylation-sites throughout the whole compared to untreated control. Moreover, AdoRA1 antagonist PSB36 increased lipolysis genome at single-base-pair resolution. by 35% (+/- 0.05%) (n=4). These experiments identified 1280 CpG sites in hypertrophic samples and 1365 CpG In conclusion, AdoR are highly regulated during brown fat cell differentiation. Lipolysis of sites in failing samples which were differentially methylated compared to control mature brown fat cells is significantly increased by AdoRA2A agonist or AdoRA1 specimens (delta >15%; p<0.05). 523 CpG sites were significantly altered in both aortic antagonist, respectively. stenosis groups compared with control hearts. From these CpGs, 496 sites were altered concordantly in hypertrophic and failing samples. Analysis of regions harbouring distinct References CpG densities revealed that most changes occured in shelf regions of CpG islands Literature whereas the methylation status in the CpG islands was more stable. Further analysis 1. Virtanen, K.A. et al. Functional brown adipose tissue in healthy adults. N Engl J Med showed that differences in methylation were most frequent in gene body, enhancer and 360 (2009). 3`UTR regions. Specifically 9 probes spanning a CpG-island at the promotor region of 2. Borglum, J. D. et al. Changes in adenosine A1- and A2-receptor expression during the muscle-specific serine kinase 1 (SRPK3) showed diminished CpG-methylation in adipose cell differentiation. Mol Cell Endocrinol 117 (1996). hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. 3. Haas, B. et al. Protein kinase G controls brown fat cell differentiation and Remarkably, no alterations of DNA-methylation were observed in loci of classic marker mitochondrial biogenesis. Sci Signal 2 (99); (2009) genes of chronic heart failure like NPPA, SERCA, CTGF, MYH6 or MYH7. Conclusions These results indicate that DNA methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. 123
Identification of novel targets of β-adrenergic signaling through 121 phosphoproteomics of the heart in vivo Göbel P.1, Rochais F.2, Zahedi R.3, Sickmann A.3, Laggerbauer B.1, Engelhardt S.1,4 1TU München Institut für Pharmakologie und Toxikologie, Biedersteiner Str. 29, 80802 Purine-derivatives influence the proliferation of cancer cells – A new approach on München, Germany treatment of Glioblastoma multiforme 2Universität Würzburg Rudolf-Virchow-Zentrum DFG-Forschungszentrum für 1 2 3 4 1 Gliesche D. , Iaroshenko V. O. , Volochnyuk D. M. , Tolmachev A. O. , Bien S. , Experimentelle Biomedizin, Josef-Schneider-Str. 2, 97080 Würzburg, Germany 5 5 1 1 Sternberg K. , Schmitz K. - P. , Kroemer H. K. , Meyer zu Schwabedissen H. 3Leibniz-Institut für Analytische Wissenschaften Bioanalytik, Otto-Hahn-Str.6b, 44227 1Institut für Pharmakologie Ernst-Moritz-Arndt Universität, Greifswald, Germany Dortmund, Germany 2Institut für Chemie Universität Rostock, Rostock, Germany 4Munich Heart Alliance, München, Germany 3Enamine Ltd., Kiev, Ukraine 4Institut für Chemie National Taras Shevchenko Universität, Kiev, Ukraine Activation of the sympathetic nervous system and the subsequent activation of β- 5Institut für Biomedizinische Technik Universität Rostock, Rostock, Germany adrenergic receptors (βARs) through catecholamines represents the strongest mechanism to increase cardiac function. However, long-term activation of cardiac βARs Gliomas are the most abundant type of primary brain tumor in the central nervous is clearly detrimental and β-blockers have been introduced as an effective treatment system in adults. The current standard of Glioblastoma multiforme (GBM) therapy is modality in cardiac failure. Despite their central role in cardiac physiology and disease, surgery followed by radiotherapy and chemotherapy. However the morbidity and our knowledge about the intracellular mechanism of βAR stimulation is confined to a few mortality of GBM remain very high and the median survival period is only 15 months targets and is likely incomplete. even with treatment. Therefore it is important to identify novel drugs to reduce GBM cell Here, we report a functional proteomics approach to directly assess the entire proliferation. phosphoproteome of βAR-stimulated mouse hearts in vivo. Purine-analogues (PA) are well known for their anti-proliferative effects on eukaryotic To identify proteins that are phosphorylated in response to β-adrenergic stimulation in cells. In this study novel PA were synthesized and the library of substance-derivatives vivo, we treated mice with isoproterenol or, as a control, with propranolol. After lysis of was tested using different GBM cell lines namely LN18, U87-MG and GL261. The effect hearts and tryptic digest, phosphopeptides were enriched by TiO2 or immobilized metal on proliferation and viability was assessed by using BrdU and Resazurin assays. Using ion affinity chromatography (IMAC). Subsequent analysis of eluated peptides by tandem these in vitro methods we were able to identify several compounds with cytotoxic and mass spectrometry (MS/MS) mapped several phosphopeptides to cardiac proteins, anti-proliferative effects in vitro showing IC50 values in the deeper µM range. among which known mediators of βAR signaling such as phospholamban, troponin I and Cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 myosin binding protein C. We then employed multiple reaction monitoring (MRM) as a and propidium iodide based cell cycle FACS analysis to discriminate between apoptosis quantitative approach to assess changes of phosphorylation after βAR stimulation. and cell cycle arrest. Using this combination of MS approaches, we identified 39 peptides with PKA S30
consensus phosphosites that were more abundantly detected under βAR stimulation. 126 Among those, we found myozenin-2 (MYOZ2) and G protein signaling modulator 1 (GPSM1, also termed AGS3) as proteins previously not related to βAR signaling. We validated the βAR-dependence of phosphorylation at these sites in isolated Identification of the amino acid transporter, responsible for Fluoroethyltyrosine cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. Current (FET) uptake in glioblastoma efforts aim at the functional characterization of these novel candidate mediators of βAR 1 2 1 2 1 signaling in the heart. Graf J. , Benedikt S. , Habermeier A. , Roesch F. , Closs E. I. 1Institut für Pharmakologie, Obere Zahlbacher Straße 67, 55101 Mainz, Germany Taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in 2 vivo. We have identified several new targets of βAR signaling that may represent Institut für Kernchemie, Fritz Strassmann Weg 2, 55128 Mainz, Germany essential factors in cardiac physiology and disease. 18 2-(O-[ F]Fluoroethyl)-l-tyrosine (FET) is an important tracer for diagnosis of glioblastoma via positron emission tomography (PET). PET detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. FET, a modified amino acid, is not incorporated in proteins but accumulates in 124 glioblastomas. One pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. We investigated in more detail (a) which individual, cloned amino acid transporters accept FET as substrate Distribution of Metamizole metabolite MAA in autopsy material with regard to and (b) which transporter is responsible for the major FET transport into glioblastoma renal and hepatic dysfunction cells. Studies with Xenopus laevis oocytes, expressing individual human amino acid 1 1 2 1 Goltz L. , Oertel R. , Pietsch J. , Kirch W. transporters, revealed that system L, y+L and b0+ amino acid transporters recognize FET 1Institut für Klinische Pharmakologie, TU Dresden, Fiedlerstr. 27, 01307 Dresden, as substrate (LAT1 and 2, y+LAT2, and b0+AT, respectively). In contrast, y+LAT1 and Germany ATB0,+ did not transport FET. RNA expression studies using qRT/PCR revealed that 2Institut für Rechtsmedizin, TU Dresden, Fetscherstr. 74, 01307 Dresden, Germany LAT1 is the dominant amino acid transporter in all glioblastoma cells investigated (LN229/U373/U87MG/U251/A172/T98G). A strong LAT1 expression was also shown on Background: Drug measurement in autopsy material is normally used to investigate the the protein level. To find out whether LAT1 is the main transporter responsible for FET cause of death. In our study it was possible to measure concentrations of drugs that accumulation, we first studied transport of the parent amino acid L-tyrosine in LN229 were part of a regular treatment without connection to the cause of death. Metamizole is glioblastoma cells. [3H]Tyr uptake was completely Na+-independent and inhibited by used as an analgetic and spasmolytic agent. The active metabolite MAA (4-Methyl- Leu, Phe and Trp, but not by Arg, Pro or Ser. siRNA-mediated down-regulation of LAT1 Aminoantipyrin) is metabolized by the liver and eliminated by the kidney. Hepatic and in LN229 cells led to a concomitant decrease of LAT1 mRNA and Tyr transport (down to renal dysfunction can therefore influence MAA clearance. 3% and 20%, respectively). These results indicate that Tyr is exclusively transported by Methods: MAA concentrations were measured in different samples of the autopsy LAT1 in LN229 cells. We are currently performing transport studies using [18F]FET to material (heart blood, venous blood, urine, liver, kidney and brain) using an HPLC- investigate whether FET transport is also exclusively mediated by LAT1 in glioblastoma MS/MS method. Information about the dosage and time of drug application as well as cells. A further question is if LAT1, a sodium-independent transporter, can be information about existing renal or hepatic disorders were taken from the corresponding responsible for the accumulation of FET observed in glioblastoma cells. If true, other patient records. Because of the low number of cases an explorative single-case study amino acid derivatives that are LAT1 substrates might also proof useful in cancer was necessary. diagnosis. Results: 10 cases with oral intake of Metamizole in a customary continuous dosage could be indentified. The MAA distribution into body liquids and organs depended on the time between last oral intake and death. In two cases without renal or hepatic diseases MAA blood levels were below 10 µg/ml. Five cases with combined renal and hepatic 127 disorders showed either increased blood levels of 40-50 µg/ml or prolonged MAA elimination half-life of up to 12 hours. In one case with manifest hepatic insufficiency an MAA concentration of more than 200 µg/ml was measured in venous blood. Two cases Telmisartan reduces adipose tissue inflammation and biglycan accumulation in with renal insufficiency alone had MAA venous blood levels of less than 10 µg/ml. diabetogenic LDL-receptor knockout mice Discussion: MAA steady state levels of around 10 µg/ml after continuous oral Metamizole intake in healthy subjects have been described in literature. It is also known, Grandoch M., Nagy N., Fischer J. W. that even moderate hepatic disorders can cause a prolonged MAA elimination half-life. Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der Continuous Metamizole treatment in patients with hepatic disorders has however not Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany been investigated before. Our results show that patients with combined hepatic and renal dysfunction had much higher MAA blood levels after treatment with similar In addition to lowering blood pressure some of the angiotensin II AT1 receptor Metamizole doses than those without such diseases. The MAA concentration in the antagonists (ARB) such as telmisartan have additional beneficial effects on the onset of patient with manifest hepatic insufficiency was in the toxic range. Renal insufficiency type 2 diabetes mellitus and obesity. This was contributed mainly to peroxisome alone did not cause elevated MAA concentrations. proliferator activated receptor (PPAR)γ modulating activity. Hyaluronan (HA), a high Conclusion: Our results suggest that Metamizole should be applied with care in molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and patients with hepatic dysfunction. biglycan, are known to be involved in atheroprogression. Mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. The aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an ARB without PPARγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the 125 interrelationship with adipose tissue inflammation using the LDLR-/- model of accelerated atherosclerosis. Male LDLR-/- mice were fed either a diabetogenic diet alone or in combination with Cell-specific binding of Clostridium difficile Toxin A telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of Goy S., Olling A., Tatge H., Just I., Gerhard R. age for 17 weeks. All treatment groups except of the lower valsartan dose showed Medizinische Hochschule Hannover Toxikologie, Carl-Neuberg-Straße 1, 30625 significant effects on reducing the aortic plaque score. The content of HA, collagen and Hannover, Germany decorin in the aortic root were not changed. However, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. Toxin A is one of two toxins secreted by Clostridium difficile leading to changes in cell In addition, a trend towards decreased mac2-positive macrophages in abdominal morphology and apoptosis in mucosal cells of the intestine. It functions as a adipose tissue was detectable after telmisartan treatment as well as a strong reduction glucosyltransferase glucosylating small GTPases such as Rho, Rac1 and Cdc42. The in the adipose tissue mRNA expression of biglycan. Finally, telmisartan reduced the major domains of this 308 kDa single-chain protein are the glucosyltransferase domain, expression of hyaluronan catabolizing enzymes potentially leading to an increase of high cysteinprotease domain, translocation domain and receptor-binding domain. Previous molecular weight HA in the adipose tissue, which is thought to be homeostatic and anti- studies showed the importance of the complete receptor-binding domain for endocytosis inflammatory. of Toxin A. An EGFP fusion protein of Toxin A1875-2710 consisting of the putative receptor- In summary, the results of this study underline the pronounced anti-inflammatory binding domain (EGFP-RBD) was utilized to investigate cell-specific differences in Toxin capacity of telmisartan on atherosclerosis and adipose tissue inflammation in A binding via flow cytometry. There were vast differences in binding of EGFP-RBD by comparison to valsartan and strongly suggest that biglycan might be an additional target human colonocitic cell lines HT29 and CACO-2, which displayed strong binding, of telmisartan not only concerning matrix composition of atherosclerotic lesions but also compared to Chinese hamster ovary cells CHO-C6 that did not bind EGFP-RBD. The concerning the structure of adipose tissue and metabolic effects of the compound. different binding properties were also observed in different cell types isolated from periperal human blood. Neutrophils and monocytes bound EGFP-RBD whereas lymphocytes did not. Binding characteristics of the isolated receptor-binding domain were confirmed by a fusion protein of mCherry and the entire Toxin A (TcdA-mCherry) 128 better reflecting the complex multidomain structure of Toxin A. Additionally we compared cell-binding and cytotoxic properties of Toxin A (TcdA) and Toxin A1-1874 (TcdA1-1874), which lacks the receptor binding domain, using western blot analysis. Data obtained by Within the orchestra of DNA repair – who plays first violin? flow cytometry reflects cell-binding properties of TcdA and TcdA1-1874 in western blot. Cytotoxicity assay using TcdA and TcdA1-1874 revealed that those cell lines binding Griewel E., Schonn I., Dartsch D. C. EGFP-RBD in flow cytometry are approximately 10-fold more susceptible towards Toxin Hamburg University, Institute of Pharmacy klinische Pharmazie, Bundesstraße 45, A. Cells that do not bind EGFP-RBD were less susceptible towards Toxin A with some 20146 Hamburg, Germany cells being equally susceptible towards both toxins. A better understanding of Toxin A targets and identification of novel binding sites within Toxin A can provide insights in the Avoiding or overcoming resistance against topoisomerase IIα (topoIIα) inhibitors like toxin structure and may help to identify therapeutic targets. etoposide and doxorubicin is crucial for an effective cancer therapy. TopoIIα inhibitors induce DNA double strand breaks (DSB), but cells have developed several ways of DNA repair, which may contribute to a resistant phenotype. Elucidation of these pathways is important to understand DNA repair-based mechanisms of resistance and to define inherent pharmacological targets. To this end, we investigate DNA repair and selected key players in human colorectal cancer cells after treatment with topoIIα inhibitors. Doxorubicin-induced DSB were quickly and effectively repaired in HT-29 cells and S31
human primary malignant cancer cells derived from peritoneal effusions of a patient with SM-GCKO mice. To our surprise, the GCKO animals were fertile and produced offspring colorectal carcinoma, as assessed by comet assay. The primary cancer cells were more albeit at a reduced rate compared to WT animals. efficient in DSB repair than HT-29 cells, and their doxorubicin IC50 was four times higher. Our data show that interruption of NO/cGMP signaling results in complete absence of Comparative protein expression levels showed that the primary cells had less Rad 51 NO-induced relaxation of penile corpus cavernosum in mice and reduces the ability to and 52 as well as less topoIIα, while Ku70 and 80 levels were similar. Another very produce offspring but does not abolish fertility. interesting protein is the MRN (Mre11-Rad50-Nbs1) complex that initializes the phosphorylation of ATM and thereby starts the signalling cascade. The newly described MRN-ATM pathway inhibitor mirin interrupts MRN activity by inhibiting the exonuclease activity of Mre11. The toxicity of mirin in HT-29 cells was measured using a 131 luminescence-based assay detecting the amount of ATP, which is correlated with cellular viability. Mirin did not show any toxic effects up to a concentration of 100 µM and incubation times of 24 hours, indicating that mirin can be used under these conditions Novel modes of invasive cell motility regulated by the formin class of actin without detrimental effects. We are currently investigating the effect of mirin on the nucleators toxicity of topoIIα inhibitors. The inhibition of DNA repair may be a valuable strategy to enhance the effect of DNA- Khan J., Grosse R. damaging anticancer drugs. Since tumours (even of the same entity) are not only Philipps-Universität Marburg, Pharmakologisches Institut, Karl-von-Frisch-Str. 1, 35034 heterogeneous, but also polyclonal, a broad selection of response modifiers of Marburg, Germany anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. Pathological invasive cell migration essentially reqires actin polymerization. Formins are the largest group of Rho-GTPase effectors involved in actin nucleation and assembly as well as microtubule dynamics. Here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. We identified the actin-dependent steps and structures involved for this process. Using live cell analysis we characterize 129 the distinct actin dynamics controlled by formin-like 2 and Rho function. The specific involvement of this signaling module will be discussed.
Dietary polyphenols affect SUMO and HDAC expression in human colon carcinoma cells Groh I., Chen C., Esselen M. 132 TU Kaiserslautern Fachbereich Chemie Fachrichtung Lebensmittelchemie und Toxikologie, Erwin-Schrödinger-Straße, 67663 Kaiserslautern, Germany Formin-driven Nuclear Actin Assembly Controls MAL/SRF Activity Introduction Evidence has been provided that diet and environmental factors directly influence Baarlink C., Wang H., Grosse R. epigenetic mechanisms associated with cancer development in humans. Epigenetics Philipps-Universität Marburg, Pharmakologisches Institut, Karl-von-Frisch-Str. 1, 35034 play an important role in the control of gene expression. Epigenetic mechanisms Marburg, Germany comprise modulation in DNA methylation, histone modification and non-coding RNA. Several polyphenols have been reported to possess histondeacetylase (HDAC) Polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of inhibitory properties [1]. Histone deacetylation is generally linked to transcription the SRF cofactor MAL (also known as MRTF-A) through release of actin/MAL repression. Furthermore, HDAC belongs to the group of small ubiquitin-related modifier interactions and subsequent nuclear accumulation of MAL. Formins directly promote (SUMO) substrate proteins. SUMOylation of HDAC is associated with a modulation of its assembly of actin filaments thereby efficiently regulating MAL-dependent transcription biological activity [2]. Little is known so far about the mechanism by which HDAC for cell shape, adhesion and motility. Here we show that formins assemble F-actin and SUMOylation mediates inhibition of gene transcription. We addressed the question promote MAL activation inside the mammalian nucleus. The Rho-GTPase effector whether SUMO E1 and HDAC 1 expression and whether potential HDAC-SUMOylation mDia2 rapidly enters the nucleus in a signal-dependent fashion and an active mDia will be affected by polyphenols such as chlorogenic acid, genistein and (-)- confined to the nucleus potently promotes release of G-actin from MAL to specifically epigallocatechin-3-gallate (EGCG). activate SRF. Live cell imaging reveals formin-mediated nuclear actin dynamics. Methods and Results Moreover, using actin assembly assays we find that inhibition of endogenous mDia Chlorogenic acid, genistein and EGCG decreased SUMO E1 protein level in the human formins controls F-actin turnover in isolated nuclear extracts. Thus, formin activity is colon carcinoma cell line HT29 after 24h of incubation measured with Western Blot dynamically compartmentalized to the mammalian nucleus to potently regulate actin- analysis. EGCG exhibited the most pronounced effect at concentrations ≥ 50 µM. HDAC dependent MRTF function. 1 expression was also affected by these polyphenols. The direct impact of polyphenols on the HDAC SUMOylation is detected by co-immunoprecipitation experiments with the respective antibodies against HDAC-1 and SUMO E1. These experiments are still under investigation. 133 Summary In conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the SUMO and HDAC expression in vitro. In further studies the direct impact on Role of the sodium-dependent organic anion transporter (SOAT) for placental subtract-SUMOylation will be investigated. These studies contribute to a better estrogen synthesis understanding of potential chemopreventive effects of dietary polyphenols on specific Grosser G.1, Schweigmann H.1, Galuska C. E.2, Hartmann M. F.2, Alber J.1, Funk K.1, epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. Ugele B.3, Wudy S. A.2, Geyer J.1, Petzinger E.1 1 References Institute of Pharmacology and Toxicology, JLU Giessen, Frankfurter Str. 107, 35392 Giessen, Germany [1] Link et al. (2010) Cancer chemoprevention by dietary polyphenols: promising role for 2 epigenetics. Biochem Pharmacol. 80, 1771-92. Division of Paediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, JLU Giessen, Feulgenstr. 12, 35392 Giessen, Germany [2] David et al. (2002) SUMO-1 modification of histone deacetylase 1 (HDAC1) 3 modulates its biological activities, J. Biol. Chem. 277, 23658–23663. University Hospital, Department of Gynaecology and Obstetrics, LMU Munich, Maistr. 11, 80337 Munich, Germany
In women the placenta becomes the main source of maternal estrogens during pregnancy. Placental estrogen biosynthesis is located in the syncytiotrophoblast, a 130 syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. Since the human placenta is unable to convert cholesterol into 17-OH-pregnenolone, the placenta tissue highly Fertility in male mice lacking NO-sensitive guanylyl cyclase depends on the supply of C-19 steroids for their conversion into C-18 estrogens. In Groneberg D.1, Lies B.1, Jäger R.1, König P.2, Friebe A.1 contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively 1Universität Würzburg Physiologie, Röntgenring 9, 97072 Würzburg, Germany via diffusion, circulating sulfated steroid hormones are delivered to the placenta via 2Universität Lübeck Anatomie, 23538 Lübeck, Germany carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (StS). DHEAS of maternal and fetal origin contributes about equally to The NO/cGMP cascade is thought to be essential for penile erection. Within the smooth the placental formation of estrone (E1) and estradiol (E2), while 16αOH-DHEAS supplied muscle of corpus cavernosum, nitric oxide activates the NO-sensitive guanylyl cyclase by the fetus contributes to over 90% of placental estriol (E3) synthesis. SOAT, a member (NO-GC) which raises the intracellular concentration of cGMP. This second messenger of the SLC10 family with highest expression in hormone-responsive tissues such as activates the cGMP-dependent protein kinase I (PKGI) and subsequent phosphorylation testis, placenta, and mammary gland has been shown to transport the sulfoconjugated of target proteins leads to relaxation of cavernosal smooth muscle. steroid hormones dehydroepiandrosterone sulfate (DHEAS), estrone sulfate (E1S), and Knock out of key enzymes of the NO/cGMP cascade has led to discrepant results: The pregnenolone sulfate (PREGS) [1]. Aim of this project is to investigate the role of SOAT deletion of PKGI in the mouse has been shown to lead to erectile dysfunction whereas for placental estrogen synthesis by means of the choriocarcinoma cell line JEG-3 as in mice lacking neuronal NO synthase are fertile. vitro model for the human syncytiotrophoblast. To investigate the role of the NO receptor in fertility we have generated mice lacking Therefore, we characterized a JEG-3 cell line that transformed DHEA into E2 and NO-GC (GCKO), a bottleneck enzyme of the NO/cGMP cascade. We have shown that 16αOH-DHEA into E3. By qRT-PCR we found expression of StS and aromatase, both lack of NO-GC resulted in arterial hypertension concomitant with a totally abolished NO essential for estrogen synthesis in these cells. Upon transient transfection of SOAT the responsiveness of vascular and gastrointestinal smooth muscle. In addition, we carrier was located in the cell membrane of transfected JEG-3 cells. Currently we generated a mouse line in which NO-GC was specifically deleted in smooth muscle cells investigate the transformation of DHEAS of these SOAT-JEG-3 cells by LC-MS-MS. (SM-GCKO). We could demonstrate transport of 16αOH-DHEAS for stably transfected SOAT-HEK293 Using these KO strains we here examined the role of NO/cGMP signaling with regards cells. In situ hybridization and immunohistochemistry showed coloured to the smooth muscle relaxation of corpus cavernosum. NO failed to affect corpus syncytiotrophoblasts and vascular endothelial cells in late term placenta. cavernosum from GCKO in organ bath experiments: neither exogenously produced NO In conclusion, SOAT-mediated transport of sulfated steroids could play a pivotal role for by NO donors nor endogenous NO release from neurons induced by electrical field placental estrogen synthesis from sulfated steroid hormones. stimulation led to relaxation. Similar results were observed in the corpus cavernosum of S32
Supported by DFG Research Group 1369 "Sulfated Steroids in Reproduction”, Project 4 1996). With the basis on NOAELs, the TTC concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no References adverse effects of the substance can be detected. It is assumed that exposures below [1] Geyer J, Döring B, Meerkamp K, Ugele B, Bakhiya N, Fernandes CF, Godoy JR, this level will not result in health risks. Three separate TTCs were derived (Munroe et al., Glatt H, Petzinger E (2007) Journal of Biological Chemistry 282:19728-19741. 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (Cramer et al., 1978). The TTC values are derived from empirical distribution of the NOELs/NOAELs in the class taking the 95th percentiles and dividing 134 them by the default uncertainty factor of 100. It is assumed that the probability is very low that the unknown NOAEL of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known NOELs/NOAELs. Hence, at exposures The area vasculosa of chicken yolk sac as alternative method for polymer toxicity below the TTC values, the probability of adverse effects on human health is considered testing to be very low. 1 2 3 1,4 Grund S. , Bräuer R. , Nowak G. , Fischer D. References 1 Friedrich-Schiller-Universität Jena Lehrstuhl für Pharmazeutische Technologie, Otto- Cramer et al. Estimation of toxic hazard - a decision tree approach. Food and Cosmetic Schott-Strasse 41, 07745 Jena, Germany Toxicology;16: 255-276 (1978) 2 Universitätsklinikum Jena Institut für Pathologie, Ziegelmuehlenweg 1, 07743 Jena, Munro et al. Correlation of structural class with no-observed effect levels: a proposal for Germany establishing a threshold of concern. Food and Chemical Toxicology 34: 829-867(1996) 3 Universitätsklinikum Jena, Erlanger Allee 101, 07747 Jena, Germany 4Friedrich-Schiller-Universität Jena Jena Center for Soft Matter (JCSM), Humboldtstraße 10, 07743 Jena, Germany
Developing non-animal test systems for evaluation of toxicity was important in the past 136 and will remain essential in the future. Here we present a toxicity test using the chicken yolk sac area vasculosa (CAV) of fertilized white leghorn chicken eggs [1, 2] and compare it to Hen’s egg test on chorioallantoic membrane (HET-CAM) [3] for polymer Tissue-specific regulation of KIBRA gene expression in renal and neuronal cell- toxicity testing. types 1 1 2 1 1 1 Fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile Guske K. , Herrmann M. , Schmitz B. , Schelleckes M. , Duning K. , Kremerskothen J. , 2 1 petri dishes. Test substances were applied on the CAV and the appearance of different Brand S. - M. , Brand E. effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was 1University Hospital Muenster Internal Medicine D, Albert-Schweitzer-Campus 1, determined by light microscopy after 1 - 48 h (Fig.1). These effects were combined to a Gebäude A1, 48149 Münster, Germany CAV test score based on the irritation score calculation used for HET-CAM evaluation. 2Medical Faculty of the Westfalian Wilhelms-University Muenster Department of Different polymers like poly(ethylene glycol) (PEG; neutral), poly(ethylene imine) (PEI; Molecular Genetics of Cardiovascular Disease, Albert-Schweitzer-Campus 1, Gebäude cationic) and dextran sulphate (DS; anionic), as well as guideline-conform D3, 48149 Münster, Germany (recommended HET-CAM protocol from the Interagency Coordination Committee on the Validation of Alternative Methods) negative (0.9 % NaCl) and positive controls (1 % Introduction: KIBRA, mainly expressed in kidney and brain tissue, is involved in brain sodium dodecyl sulphate (SDS) and 0.1 N NaOH) were investigated. Additionally LD50 development and memory formation as a postsynaptic scaffold protein. In podocytes, values for different cationic polymers have been determined. KIBRA is proposed to regulate cell motility as a linker between components of the Within the selected incubation times (1 - 48 h), effects such as vessel lysis and blood cytoskeleton and polarity protein complexes (Duning et al, JASN 2008). Furthermore, component aggregation could be detected. Additionally to HET-CAM, lethality as well as KIBRA has been identified as key regulator of the Hippo pathway, which is involved in recovery of the CAV could be observed. Differences between neutral, positively and organ size control and tumorigenesis. In the current study, we focused on the negatively charged polymers were obtained. PEI showed strong vessel lysis and identification of KIBRA gene expression regulation and functional promoter aggregation of blood components whereas DS and PEG showed none of these effects. characterization. Lethality was found to increase from PEG < DS < PEI and is concentration and time dependent. The results demonstrate that differences, regarding the toxicity of the used Methods: Serial promoter deletion constructs were generated by cloning 3627 bp of the polymers, can be shown with this test. These findings in the CAV test can be well 5'flanking region of KIBRA into the pGL3-vector system. Deletion constructs were correlated with already existing data. transiently transfected into human neuroblastoma cells (SH-SY5Y) and immortalized In summary, the CAV test provides same data (testing control substances) and more human kidney epithelial (IHKE) cells. Potential transcription factors (TFs) were information (recovery and lethality) compared to HET-CAM and could be a suitable investigated in cotransfection experiments. Transcriptional start sites (TSS) were model for toxicity testing of polymers. determined by rapid amplification of 5'cDNA ends (5'RACE). TSS utilization between cell lines was assessed by semiquantitative PCR. References 1. Nowak, G. (2000) Int Angiol 19 (Suppl I): P149 Results: Transcriptional activity (TA) of the KIBRA promoter P1 was separated by 2. Rosenbruch, M., Holst, A. (1990) Toxicol Vitro 4: 327-331. ~1060 bp into two distinct regions, promoter P1a and P1b. Deletion constructs 3. Luepke, N.P. (1985) Food Chem Toxicol 23: 287-291. harbouring promoter P1b were transcriptionally active only in IHKE cells. 5'RACE revealed two alternative TSS in both cell lines upstream of the annotated TSS (NM_015238). Exclusively in IHKE cells, two additional TSS were detected in intron 1, resulting in two alternative exons. Deletion constructs harbouring the putative regulatory regions (P2 and P3) of both exons were transcriptionally active only in IHKE cells. Overexpression of full length TCF7L2 (transcription factor 7-like 2 [T-cell specific, HMG- box]) resulted in a ~3-fold increase of promoter P1a and intron promoter P2 TA.
Conclusion: KIBRA gene expression is driven by a complex alternative promoter system comprising the constitutional promoter P1 and three alternative promoters P1b, P2 and P3. The TSS utilization is cell type-specific. Subsequent usage of an alternative translation start site within exon 3 could result in truncated KIBRA protein isoforms. TCF7L2 is involved in the differential KIBRA gene expression regulation. Resulting KIBRA protein isoform and their cellular function will be assessed in further studies.
Figure 1: Scheme of experimental design 137
Comparative investigation of different skin integrity tests for dermal penetration studies in vitro 135 Guth K.1, Schäfer-Korting M.2, Fabian E.1, Landsiedel R.1, van Ravenzwaay B.1 1BASF SE Experimental Toxicology and Ecology, Carl-Bosch-Str. 38, 67056 Ludwigshafen, Germany The Threshold of Toxicological Concern - Conceptual Approach 2Freie Universiät Berlin Institut für Pharmazie, Königin-Luise-Str. 2+4, 14195 Berlin, Gundert-Remy U. Germany Institute of Clinical Pharmacology and Toxicology, Berlin, Germany Skin Absorption in vitro based on the study of human/animal skin ex vivo or Risk characterisation of chemicals consists of three steps (1) hazard identification and reconstructed human epidermis, respectively, is an alternative method which is accepted characterisation, based on substance-specific toxicological hazard data, (2) estimates of by the OECD. Guideline TG 428 and a corresponding technical guidance document (GD the level of exposure toward the substance and (3) the comparison between the 28) give technical guidance how to perform valid experiments1,2. The requirements toxicologically safe level and the exposure level. include integrity evaluation tests for the skin samples. Different tests are proposed to In contrast to the classical risk assessment approach, the threshold of toxicological ensure an exclusively use of undamaged skin. To decide which test suites best to our concern (TTC) approach is developed as a tool to assess the risk of substances without routine test strategy, we investigated the correlation between integrity test results and toxicity data. Its application requires (1) information on human exposure, for which it is absorption profiles of various penetrants (logP range: -0.07 – 6.2). essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the TTC concept. Finite dose experiments using rat and human skin were performed with 14C-labeled Instead of chemical specific no observed (adverse) effect levels (NOELs/NOAELs), the testosterone, caffeine, MCPA (4-chloro-2-methylphenoxyacetic acid) and its 2-ethyl- TTC approach utilises knowledge on the empirical distribution of several hundreds of hexyl-ester MCPA-2EHE. For each experiment at least three of the five following NOELs/NOAELs, originally 613, based on toxicological testing in animals (Munroe et al., integrity tests were conducted: transepidermal electrical resistance (TEER), S33
transepidermal water loss (TEWL), transepidermal tritiated water flux (³H2O), linked. Similar results were also obtained with B cells from DBA/2, CBA or BALB/c mice. transepidermal absorption of methylene blue (BLUE) , transepidermal absorption and In vivo, infection with either influenza virus or murine γ-herpesvirus induced the flux of a ³H-labeled internal standard (ISTD); ³H-Testosterone or ³H-Mannitol was used expected expression of GrB in cytotoxic T lymphocytes, but not in B cell populations. We as ISTD. The applied radioactivity of the ³H-ISTD was selected to show no analytical also investigated a possible role of GrB on the humoral immune response to NP-KLH, 14 interference with the C-penetrants. TEER, TEWL and ³H2O represent pre-study, ISTD but GrB-deficient mice produced normal amounts of antibody with typical affinity concurrent and BLUE post-study tests. Calculated maximal permeability constants (Kp) maturation and heightened secondary response, demonstrating conclusively the and absorbed doses (AD) of the penetrants were compared to the results of the integrity redundancy of GrB for antibody responses. Our results highlight the complex tests. Individual linear regression analysis was used to evaluate the correlation evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune The correlations varied over a wide range for all five methods and four penetrants. The responses. best correlations in average were achieved with the ISTD. No inverse correlations were obtained for the ISTD, but partly for TEWL, TEER, ³H2O and BLUE.
In conclusion, the ISTD represents best the achieved absorption profiles of the test 140 compounds and is based on that the most suitable integrity test for our dermal absorption studies. We will further confirm its effectiveness and generate a sufficient historical database in order to include the ISTD in our routine test protocol. Investigations of the cholinergic neurotransmitter system in DYT1 mice 1 1 2 2 1 References Hamann M. , Kuschka J. , Gioioso V. , Sharma N. , Richter A. 1 1OECD Guideline 428 for testing of chemicals – skin absorption in vitro method, OECD, Freie Universität Berlin, Dept. of Veterinary Medicine Institute of Pharmacology and 2004. Toxicology, Koserstraße 20, 14195 Berlin, Germany 2 2OECD GD 28, Guidance document for the conduct of skin absorption studies, OECD Harvard Medical School, Massachusetts General Hospital Dept. of Neurology, Boston, series on testing and assessment Number 28, 2004. Massachusetts United States
Early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the DYT1 gene defect with deletion of a glutamic acid residue in the protein torsinA. Despite the gene defect, the pathophysiology is poorly understood. 138 Animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. Sharma et al. (2005, J. Neurosci. 25 [22], 5351- 5355) initially described a transgenic mouse model (DYT1 mice) with overexpression of Investigation of miRNA expression and DNA Methylation in focal and non-focal mutant torsinA. Previous studies in these mice pointed to alterations in the cholinergic brain tissue of therapy-resistant epilepsy patients system. To investigate the functional relevance of these in-vitro findings, we carried out 1,2 2 1 2 Haenisch S. , Chhibber A. , Cascorbi I. , Kroetz D. pharmacological in-vivo experiments and determined the density of striatal cholinergic 1Universitätsklinikum Schleswig-Holstein Institut für Experimentelle und Klinische interneurons as well as the expression of choline acetyltransferase in different brain Pharmakologie, Arnold-Heller-Str.3, D-24105 Kiel, Germany regions. The acute intraperitoneal administration of the cholinomimetic drug pilocarpine 2University of California, San Francisco, USA School of Pharmacy, Department of (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) Bioengineering and Therapeutic Sciences, 1550 4th St. RH584E, San Francisco, CA did not induce pronounced effects in DYT1 mice compared to wildtype controls. The 94158, United States higher incidence of epileptic seizures in DYT1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an Background: Resistance to anticonvulsants affects one third of all epilepsy patients. altered synaptic plasticity in DYT1 mice. The immunohistochemical investigations Limited bioavailability of the drug at the target site caused by increased expression of revealed a moderately reduced density of striatal cholinergic interneurons in the efflux transporters on the blood brain barrier or alterations of target genes are potential dorsomedial subregion of DYT1 mice compared to wildtype controls, while significant mechanisms for therapy resistance. However, these mechanisms alone cannot differences in other striatal subregions were not detected. Western Blot analysis did not completely explain the observed resistance and it is likely that multifactorial alterations show clear differences in the expression of choline acetyltransferase between DYT1 and lead to pharmacoresistance. There is increasing evidence that expression of microRNAs wildtype control mice. These results indicate that the cholinergic system seems not to probably caused by DNA modifications is deregulated in many neuronal diseases. We play a key role in this line of DYT1 mice. Ongoing receptor autoradiographic analysis of hypothesize that miRNA regulation of target genes is involved in drug resistance in binding to different muscarinic receptors subtypes have to further clarify the existence of epilepsy. possible alterations within the cholinergic system of these DYT1 mice. Methods: Hippocampal focal and cortical non-focal brain tissue samples from 13 Supported by the Dystonia Medical Research Foundation. patients diagnosed with MTS (mesial temporal sclerosis) who underwent neurosurgery have been screened for miRNA expression using TaqMan low density arrays. In silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target 141 genes. Using the program R (Bioconductor) a Mann-Whitney-U test was performed to compare miRNA expression between brain regions. Pyrosequencing was performed to investigate methylation status 5’-upstream of DNA regions encoding for selected Cyclin-dependent kinase 6 (CDK6) is required for IL-1- or TNF-induced candidate miRNAs. inflammatory gene expression Results: Out of 754 miRNAs, 150 were detected in both tissue types. The expression of one miRNA was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the Handschick K., Beuerlein K., Weber A., Jurida L., Müller H., Kracht M. hippocampus relative to the cortex. Evidence could be found that down-regulation of the Justus-Liebig-University Giessen Rudolf-Buchheim-Institute of Pharmacology, latter is possibly caused by hypermethylation of 5’-flanking region of its encoding DNA Frankfurter Strasse 107, 35392 Giessen, Germany locus. Bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. SOX11, MECP2, BSN), as well as one ABC efflux- Inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as transporter, as potential targets for these differentially regulated miRNAs. anti-proliferative agents. Thus, the ATP-competitive kinase inhibitor PD332991 which Conclusion: Differential regulation of two miRNAs could contribute to an altered function inhibits CDK4 and CDK6 is currently tested in patients with solid tumors such as glioma. of several genes resulting in an imbalance between neuronal excitation and inhibition We found that PD332991 suppressed IL-1-induced expression of IL-8 suggesting that that is independent from mechanisms presently targeted by anticonvulsants. This work CDK4 or CDK6 may have unknown anti-inflammatory properties. To study the effects of was supported by a fellowship from DFG and NIH grant GM61390. CDK6 on the IL-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of CDK6, CDK6 S178P, in asynchronized HeLa cells. CDK6-expressing cells were identified by GFP which was expressed from the same promoter, isolated by laser-microdissection and analysed for mRNA expression using a down-scaled RT-qPCR assay. Compared to the uninduced 139 state, CDK6 S178P enhanced IL-1-induced IL-8 and IL-6 mRNA expression. Moreover, shRNA-mediated suppression of endogenous CDK6 confirmed a role of this kinase in regulation of maximal IL-1-induced gene expression of IL-8. These data also revealed Lack of Granzyme B expression by mouse B cells reveals evolutionary unique that the contribution of CDK6 to inflammatory gene expression is highest in G1, when cytotoxic function of human B cells activity of endogenous CDK6 is activated by D-type cyclins. These findings were 1,2 3 3 4 1 3 Hagn M. , Belz G. T. , Kallies A. , Jahrsdörfer B. , Sutton V. R. , Tarlinton D. M. , corroborated in HeLa cells expressing fluorescent ubiquitin-dependent cell cycle 1 1,5 Hawkins E. D. , Trapani J. A. indicator (Fucci) proteins. HeLa-Fucci cells from G1, G1/S, G2 or mitotic states were 1Peter MacCallum Cancer Centre Cancer Immunology Program, St Andrew's Place, isolated by laser-microdissection and analyzed by RT-qPCR for TNF-inducible gene East Melbourne 3002, Australia expression. Stable knockdown of CDK6 in HeLa Fucci or inhibition by PD332991 2The University of Melbourne Department of Pathology, Parkville 3010, Australia suppressed inducible IL-8 expression. Microarray experiments identified many additional 3The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville genes that required active CDK6 for maximal IL-1-or TNF-inducible gene expression. 3052, Australia We also found that CDK6 co-immunoprecipitated with p65 NF-κB, colocalized with p65 4Ulm University Institute of Transfusion Medicine, Helmholtzstr.10, 89081 Ulm, Germany in the nucleus and was recruited together with the p65 subunit to the proximal IL-8 5The University of Melbourne Department of Microbiology and Immunology, Parkville promoter as assessed by ChIP and Re-ChIP experiments. Collectively, these results 3010, Australia suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. These results also imply that pharmacological targeting of Recently, it has been reported that human B cells express and secrete the cytotoxic CDKs may have effects and side-effects on the immune system in addition to inhibition protease Granzyme B (GrB) after the combined stimulation of the IL-21- and the B cell of cell cycle progression. receptors. GrB produced by B cells is enzymatically active and B cells deliver GrB to sensitive cancer cell lines, thereby inducing apoptosis. To date, there is little experimental evidence on the mechanisms involved in GrB expression, or its function in B cell biology. As experimental transgenic murine systems should enable us insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli, but were unable to detect either GrB expression or its proteolytic activity, even when antigen specific transgenic B cell receptors were cross-
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received T cells polarized in the presence of wild-type DCs, about 40% eosinophils were 142 –/– detected. In contrast, the transfer of T cells polarized in the presence of H4R DCs yielded only about 10-20% eosinophils in BAL fluids. Mutations in TRPC6 ion channels in patients suffering from familial focal In summery, the H4R on DCs plays an important role for T cell polarization and segmental glomerolusclerosis consequently affects the allergic reaction during sensitization. Since the lack of the H4R on DCs reduced their ability to stimulate proper Th2 polarization of CD4+ T cells, we Harteneck C.1, Weber S.2, Büscher A.2, Riehle M.1, Hoyer P.2, Jeruschke S.2, Kolatsie conclude that HA via the H R significantly affects the manifestation of asthmatic M.2, Nagel M.3, Winyard P.4, Gollasch M.5 4 1 inflammation. Universitätsklinikum Eberhard Karls Universität Institut für Pharmakologie und Toxikologie, Wilhelmstraße 56, 72072 Tübingen, Germany 2Universitäts-Kinderklinik Essen Pädiatrie II, Hufelandstr. 55, 45122 Essen, Germany 3Center of Nephrology and Metabolism, Albert-Schweitzer-Ring 32, 02943 Weißwasser, Germany 145 4UCL Institute of Child Health Nephro-Urology Unit, London, WC1N 1EH, Great Britain 5Charité Berlin Department of Nephrology, Lindenbergweg 80, 13125 Berlin, Germany Antioxidant polyphenols and their effects on Nrf2 (SKN-1) signalling in a cell TRP channels form a heterogeneous family of calcium-permeable channels, which play culture system and the model organism C. elegans major roles in physiological functions ranging from sensory reception to cellular signal Havermann S., Wätjen W. transduction. Members of the TRPC subfamily (classic transient receptor potential Heinrich-Heine-Universität Düsseldorf Institut für Toxikologie, P.O. Box 101007, 40225 channels) are downstream targets of hormone receptors. Of particular interest is the Düsseldorf, Germany biological role of TRPC6 channels. They are directly activated by diacylglycerol due to phospholipase C-driven signalling pathways which are involved in smooth muscle Oxidative stress has been connected with a variety of diseases, (e.g. Alzheimer`s and contractility, neuronal plasticity, keratinocyte differentiation and renal function. Their Parkinson´s disease), cancer and ageing over the last years. Certain polyphenols were impact in renal function became evident from analyzing patients suffering from familial shown to have an antioxidant capacity as well as being able to activate the protective forms of focal segmental glomerolusclerosis (FSGS) which could be linked to TRPC6 Nrf2 signalling pathway. Compared to direct radical scavengers modulators have the mutations. Since the first descriptions at least 17 different pathogenic mutations have advantage of building up a permanent defense against oxidative insults whereas been identified in humans to cause FSGS. In order to study the underlying scavengers do not protect any more after consumption or may even cause stress due to pathophysiological mechanisms of TRPC6 mutations, we have analysed all mutated redox cycling. TRPC6 channels known to date heterologously expressed cells. One set of mutations We have employed cell culture based assays (DCF, Western Blot, GFP reporters) to showed a gain-of-function phenotype which has been previously suggested to cause an analyse the effects of polyphenols. Further we tested the coumpounds in vivo in the increased intracellular calcium load and subsequent cell death. Hence, gain of function nematode C. elegans where SKN-1 is the Nrf2 homologue. mutations fit to the current paradigm of FSGS pathophysiology. However, another set of Baicalein and caffeic acid phenethylester (CAPE) protected cells and the nematode from mutations found in the patients showed a loss-of-function phenotype. Our results enable ROS accumulation after application of stress (shown by DCF assay). a change in the current paradigm for the role of TRPC6 in renal pathophysiology and Activation of Nrf2 signalling is correlated with translocation of the transcription factor into may provide a basis for our understanding of the pathophysiology of loss-of-function the nucleus. In both systems Nrf2::GFP accumulation in the nuclei could be observed mutations in familial focal segmental glomerolusclerosis. after incubation with baicalein (fluorescence microscopy). But while CAPE is a potent activator of Nrf2 in cells, it has no effect on SKN-1 localisation. Further the effect on the Nrf2 protein amount was investigated by western blot analysis. The expression of target genes can be investigated by differing means: While PCR methods and western blotting 143 are standard for in vitro studies, the vast number of available GFP reporter strains offers opportunities for research using C. elegans. We have performed congruent assays in a cell culture system and the model organism Towards science-based thresholds for genotoxic carcinogens? — An introduction C. elegans to compare antioxidative capacity and effects of polyphenols on Nrf2 signalling. Therefore, depending on the substance tested, C. elegans is a suitable model Hartwig A. system to investigate effects of natural compounds in an organism. Karlsruher Institut für Technologie (KIT) Institut für Angewandte Biowissenschaften, Abteilung Lebensmittelchemie und Toxikologie, Adenauerring 20a, 76131 Karlsruhe, Germany
Risk assessment for genotoxic carcinogens is an important challenge in toxicology. 146 Even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. Thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, Exposure of the German Population with Di-Ethyl-Hexyl-Phthalate 1 1 1 2 in the environment and in food. In order to deal with this situation and to set priorities for Heiland A. , Heinemeyer G. , Sommerfeld C. , Conrad A. risk management, different concepts have been established such as the ALARA 1Bundesinstitut für Risikobewertung Wissenschaftliche Querschnittsaufgaben, principle (As Low As Reasonably Achievable) and the Margin Of Exposure (MOE), Diederdorfer Weg 1, 12277 Berlin, Germany based on the ratio between concentrations being carcinogenic in experimental animals 2Umweltbundesamt, Corrensplatz 1, 14195 Berlin, Germany and the actual exposure of humans for example via foodstuff. While usually linear dose- response-relationships have been used as default assumption, analytical methods are Being associated with adverse health effects, the human exposure to DEHP is subject to now available to assess the induction and repair of DNA lesions on low exposure concern. Quantifying the population’s exposure and determining the contributions of conditions, including environmental background exposure, and to relate the extent of different exposure routes is a key task of environmental health risk assessment. exposure-induced DNA lesions to endogenous DNA damage. This may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. The study presented comprises a review of the available data on DEHP levels in foods, Within this workshop, different examples will be discussed and research need will be consumer products, and house dust. Extensive survey data, e.g. from the current identified. National Nutrition Survey II and the EU RAPEX system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in Germany. The study also included analytical analyses of DEHP levels in selected foods and consumer goods (incl. migration rates for mouthing). Probabilistic techniques allowed elucidating 144 the exposure’s variation and the relevance of different routes.
Mean exposure estimates for German children and adults to DEHP are 36 and 26 µg/(kg d), resp. For children, food accounts for 36% of the total exposure, followed by mouthing Dendritic cells from H4R-deficient mice lose their ability to properly stimulate T lymphocytes (30%) and house dust (19%). The adult exposure is almost entirely (82%) due to food. As dietary exposure is a result from concentration and consumption, foods exhibiting Hartwig C., Seifert R., Neumann D. high contamination e.g. Butter (8%) and dressings (mayonnaise) (14 %) as well as MHH Pharmakologie, Carl-Neuberg Str. 1, 30625 Hannover, Germany highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. The incidence of allergic airway diseases is increasing throughout the world, especially The mean estimate of children’s DEHP exposure via mouthing revealed 0,9 µg/(kg d). in Western countries. Although histamine (HA) is found at high concentrations in High exposures were estimated (95th percentile) up to 10,8 µg/(kg d). asthmatic lungs, a role for HA in bronchial asthma is still a neglected topic in clinical research. In particular, the capacity of HA to modulate the underlying immune reaction is On average people in Germany are exposed to DEHP below the current TDI of far from being understood. The histamine H4-receptor (H4R) is involved in acute 50 µg/(kg d). However, individual exposures exceeding the TDI still cannot be excluded. inflammation and Th2 cytokine production. Consequently, we intended to analyze the Current data on DEHP and other plasticizers in foods are scarce, which warrants role of H4R in a murine Th2 lymphocyte transfer-based model of asthma. Specifically the broader monitoring. Our findings highly facilitate further exposure modeling focusing on ability of H4R expressed on dendritic cells (DCs) to modulate T cell function was DEHP substitutes and risks of combined exposure. analyzed. OVA-specific CD4+ T cells were polarized in vitro under Th2-favoring –/– conditions with OVA peptide–pulsed DCs, obtained either from wild-type or H4R mice. This study was funded by the Federal Ministry for the Environment, Nature Conservation Analysis of the polarized T cells after in vitro restimulation revealed a marked decrease –/– and Nuclear Safety in the frame of the environmental research plan of IL-4 production in T cells polarized in the presence of H4R DCs compared to those (Umweltforschungsplan, Förderkennzeichen (Ufoplan) 3707 61 201). polarized in the presence of wild-type DCs. Thus, on DCs, the H4R is essential for proper stimulation of spleen T cells and for directing their polarization towards a Th2 phenotype. The transfer of in vitro polarized T cells into recipient mice and subsequent provocation elicited an asthma-like disease. The H4R on DCs not only affects in vitro polarization of T cells, but also the in vivo function of the obtained polarized T cells. A parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. When analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of T cells occurs. In BAL fluids of mice that S35
147 pharmacological intervention. Accordingly, P2X7 blockers are currently tested in phase II clinical trials. In an attempt to identify P2X7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate Scenario based sensitivity analysis of aggregated dietary exposure of DEHP in the compound library (Spectrum Collection) and a stably transfected HEK293hP2X7 cell line. German Population With IC50 values of 1-2 µM, the tricyclic antipsychotics prochlorperazine and 1 1 1 2 3 trifluoperazine showed a high potency and efficacy to block the ATP (1 mM)-triggered Heinemeyer G. , Heiland A. , Sommerfeld C. , Conrad A. , Zenie A. 2+ 2+ 1 increases in the intracellular Ca concentration ([Ca ]i) that was mediated by human Bundesinstitut für Risikobewertung Wissenschaftliche Querschnittsaufgaben, P2X7 (hP2X7). The closely related phenothiazine-class neuroleptic drugs, such as Diedersdorfer Weg 1, 12277 Berlin, Germany chlorpromazine or triflupromazine did not have an appreciable effect on hP2X7- 2Umweltbundesamt, Corrensplatz 1, 14195 Berlin, Germany 2+ 3 mediated Ca influx. Whole-cell inward currents, measured at -60 mV, were blocked by Joint Research Centre, Ispra, Italy more than 60% by 3-10 µM prochlorperazine. The inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the P2X7 On the basis of the available measurements of DEHP, the exposure assessment has protein. Prochlorperazine added intracellularly via the patch pipette did not substitute for been focused on 37 food categories characterising a selection of the most important the extracellularly applied drug, indicating that its binding site is accessible from the food groups covering all major food classes of the German population. Based on an extracellular side. In addition, both compounds blocked Yo-Pro-1 uptake when extensive literature survey, the analysis considered the available data of DEHP preincubated before P2X7 stimulation with 1 mM ATP or when applied subsequent to measurements in food, as well as the official German food control data taken from the the agonist. Interestingly, when added to a HEK293 cell line expressing the rat P2X7, National Food Consumption Survey. The high amount of considered data allowed the 2+ perazines potentiated the ATP-induced increase in [Ca ]i. Measurements in human consideration of several exposure assessment tiers (deterministic and probabilistic by monocyte-derived macrophages confirmed the ability of prochlorperazine and using Monte Carlo simulation). 2+ trifluoperazine to inhibit ATP-evoked increases in [Ca ]i, changes in Yo-Pro-1 permeability and whole cell currents. Taken together, we conclude that perazine-type A quantitative evaluation of the uncertainties of the estimate of the 37 food categories neuroleptics impede on P2X7 activity in a species-specific manner, presumably by groups has been performed by means of a sensitivity analysis by using the methodology binding to an extracellularly accessible binding site of recombinant or natively expressed proposed within the 2008 WHO IPCS guidance document of characterising and P2X7. communication uncertainty in exposure analysis. Qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of DEHP in all food categories. The probabilistic Monte Carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. Sensitivity 150 analyses were applied to prove the impact correlation of food groups for uncertainties. By a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. For Pre-clinical evidence for geno- and cytoprotective properties of lipid lowering this purpose, particular “meals” have been described as fixed combined scenarios and. drugs (statins) 1 2 1 The aggregation leads to a considerable higher exposure estimate which can be Henninger C. , Hülsenbeck J. , Firtz G. explained by the combination of high contaminated foods with others of high 1Heinrich Heine Universität Düsseldorf Institut für Toxikologie, Universitätsstrasse 1, consumption. The evaluation confirms the considerable role of possibly high 40225 Düsseldorf, Germany contaminated foods e.g. fats, or mayonnaise. 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz Institut für Toxikologie, The evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and Obere Zahlbacher Straße, 55131 Mainz, Germany pragmatic tool for uncertainty analysis in exposure assessment. HMG-CoA reductase inhibitors are common drugs for the therapy of This evaluation has been performed by the BfR on behalf of the Federal Environment hypercholesterolemia. While inhibiting the cholesterol biosynthesis they have additional, Agency (UBA) within the frame of the environmental research plan so called pleiotropic effects, which are mainly based on interference with the (Umweltforschungsplan, Förderkennzeichen (Ufoplan) 3707 61 201) isoprenylation of small regulatory Rho GTPases. Rho GTPases regulate proliferation, morphology, motility, differentiation, apoptosis and survival of cells. Pre-treatment of References human umbilical vein endothelial cells (HUVEC) with low doses of the statin derivative WHO/IPCS (2008) Uncertainty and data quality in exposure assessment. lovastatin (LOV) protected them from ionizing radiation- (IR), doxorubicin- (DOX) and (IPCS harmonization project document ; no. 6) Part 1: guidance document on etoposide- (ETO) induced cell death in vitro. LOV reduced IR-induced increase in characterizing and communicating uncertainty in exposure assessment. World Health p53/p21 protein levels and impaired the activation of NF-κB, CHEK1 and AKT kinase. Organization 2008 Similarly, pre-treatment with LOV also lowered DOX-induced stabilisation of p53 and phosphorylation of CHEK1 and SAPK/JNK. While LOV had no influence on IR-induced initial DNA damage formation in HUVEC and rat cardiomyoblasts (H9c2), it decreased DOX- and ETO-induced phosphorylation of histone H2AX, which is a surrogate marker 148 of DNA-double strand breaks. This indicates that LOV specifically protects against the genotoxicity of topoisomerase type II poisons. In an acute and subacute Balb/c mouse model LOV protected from IR-induced toxicity. The transcription factor CREB modulates NFATc3-dependent transcription This effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mRNA levels of Il6, Ctgf and Tnfα. The same was true for Heinick A., Nunes F., Freese C., Schmitz W., Müller F. U. DOX-induced toxicity, i.e. heart and liver damage. Similar to the in vitro experiments, Westfälische Wilhelms-Universität Münster Institut für Pharmakologie und Toxikologie, DOX-induced hepatic DNA-damage was attenuated by LOV treatment. Overall, liver and Domagkstrasse 12, 48149 Münster, Germany heart toxicity were reduced by LOV as mirrored by the serum levels of Gldh/Gpt and cTn-I, respectively. Both in liver and in heart we observed collagen rich perivascular The transcription factor cAMP response element (CRE)-binding protein (CREB) plays a areas following DOX treatment. Under situation of LOV-co-treatment these areas critical role in regulating gene expression in response to activation of the cAMP- occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. dependent signaling pathway, which is implicated in the pathophysiology of heart failure. PCR-array-based mRNA analyses showed inhibitory effects of LOV on DOX-triggered We observed CREB knock-out cardiomyocytes to be larger than wildtype 2 expression of genes involved in oxidative stress response, drug transport, DNA repair, cardiomyocytes (cell area in µm ; mean±SEM; CREB-KO 4871±258 vs. WT 3396±171; cell cycle progression and cell death. For instance, up-regulation of p21, Wee1, n=68/69 cells, N=4 mice, p<0.01 vs. ctr.). The Nuclear factor of activated T-cells c3, cJun/Fos and Hmox-1 following DOX administration was attenuated by LOV. Altogether, NFATc3, is another transcription factor involved in the development of heart failure and we suggest that including LOV in current cancer therapeutic regimen might widen the also a known positive regulator of hypertrophy. Hence, we investigated whether therapeutic window of anticancer therapeutics by lowering normal tissue damage. inhibition of the CRE-dependent transcriptional activation has an impact on the NFATc3 signaling pathway. We first studied the effects of an overexpression of a dominant negative CREB mutant (dnCREB) or of NFATc3 on the activity of a NFAT-dependent model promoter in a permanent cell line. Overexpression of dnCREB evoked an 8.0±1.5 fold increase of the NFAT model promoter activity (n=36; N=6 transfections), but had no 151 impact on Kv4.2 promoter activity which is known to be regulated by NFATc3 (1.2±0.1 fold; n=18; N=3; p<0.05 vs. ctr.). NFATc3 overexpression led to a 4.5±0.7 fold increase of the NFAT-dependent model promoter activity (n=12; N=2) and to an inhibition of Influence of Roux-en-Y gastric bypass surgery on the gene expression of Kv4.2 promoter activity (0.8±0.1 fold; n=18; N=3; p<0.05 vs. ctr.). We conclude that metabolism and transport proteins in the small intestine 1 2 3 4 2 1 CREB is a negative regulator of NFAT-mediated gene transcription and that activation of Henrike B. , Oswald S. , Häsler R. , Ludwig K. , Siegmund W. , Cascorbi I. NFATc3 might contribute to the observed hypertrophy of CREB-KO cardiomyocytes. 1Institute of Experimental and Clinical Pharmacology, Arnold Heller straße 3 Haus 30, 24105 Kiel, Germany 2Department of Clinical Pharmacology, Greifswald, Germany 3Institut for Clinical Molecular Biology, Kiel, Germany 149 4Department of Surgery, Clinic Südstadt, Rostock, Germany
Background: Roux-en-Y gastric bypass is an accepted procedure in weight loss surgery Neuroleptika der Perazin-Klasse sind potente Modulatoren des P2X7-Rezeptors - which significantly reduces body weight and obesity-related co-morbidity. A small sized Perazine-type neuroleptic drugs are potent modulators of P2X7 receptors pouch from the upper stomach is created and attached to the jejunum bypassing the lower half of the stomach and the duodenum. The aim of our study was to investigate Hempel C., Nörenberg W., Urban N., Sobottka H., Schaefer M. the gene expression in different parts of the small intestine, namely in duodenum and Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, jejunum and the possible change in the jejunum one year after by-pass surgery. Härtelstraße 16-18, 04107 Leipzig, Germany Methods: During surgery of twelve obese patients, one duodenal and one jejunal biopsy was obtained from the mucosa. A third jejunal biopsy was collected one year after P2X7 receptors belong to a family of ATP-gated, non-selective cation channels, which surgery. mRNA expression profile was analyzed by Affymetrix Human Gene 1.0 ST chip play an important role in immune cell activation, inflammatory hyperalgesia and annotating approximately 28,000 human genes. Significance of expression was neuropathic pain. They differ from other P2X family members by the low ATP affinity, calculated by Mann-Whitney U test and Benjamini Hochberg method for adjustment of and by the ability to form or recruit dilated pores in the sustained presence of ATP. Owing to its involvement in many diseased states, P2X7 is a promising target for S36
the p values. In addition, array data underwent cluster analysis for identification of critical SULTs, mutagenicity tests including bacteria expressing different SULT forms substantial differences of gene regulation among the three different types of biopsies. were conducted. (±)-1´-OHME (separated into its enantiomers) served as test Results: Of particular interest in our study was the expression of genes coding for compound. We could show that hSULT1A1, standing out due to its high expression level metabolism and transport proteins. Therefore 42 genes from the 156 significant in many tissues, can efficiently activate both enantiomers even at low concentrations. differentially regulated genes, were selected for the qRT-PCR analysis. Genes coding Furthermore, DNA adduct formation in hSULT1A1-proficient and SULT-deficient bacteria for ABCB1 and ABCG8 transport proteins showed higher expression in the jejunal tissue was examined after incubation with 7 µM of (+)- or (-)-1´-OHME. For selective detection one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). and quantification of ME-derived 2´-deoxyadenosine (dA) and 2´-deoxyguanosine (dG) Moreover, CYP7A1 mRNA involved in metabolic processes is higher expressed in adducts we developed a sensitive tandem mass spectrometry method including stable postoperative jejunum than in the jejunum tissue taken during the surgery (fold change isotope dilution analysis. Adduct formation was only observed in bacteria expressing 1.9). hSULT1A1. The concentration dependence of adduct formation in hSULT1A1-proficient In conclusion Roux-en-Y gastric bypass operation leeds a change of mucosal gene bacteria was examined for (+)-1´-OHME. Both adducts turned out to be concentration- expression profile in the jejunum during one year. There was also a significant dependent. To check the extent and organ specificity of adduct formation in vivo we differential gene expression between the original duodenum and jejunum one year after administered 54 mg/kg bw (±)-1´-OHME (i.p.) to mice carrying the hSULT1A1/1A2 gene surgery. These results give strong evidence that jejunum not exposed to pancreatic but cluster. Mice getting only the vehicle served as controls. Animals were sacrificed and only to gastric fluids may change its gene regulation. DNA from eight organs was extracted. By means of tandem mass spectrometry adducts were measured and quantified. dA and dG adduct formation was observed in all tissues studied, but not in untreated animals. Furthermore, adduct levels were higher than in experiments using wild-type mice. Altogether, we herein could show that sulfo 152 conjugation leads to bioactivation of ME to a DNA-binding intermediate in vitro and in vivo. This work was financially supported by Bundesinstitut für Risikobewertung. Regulation of gene expression of urate transporter SLC2A9 Hentschel K.1, Wolf S.1, Zimmermann U.2, Rimmbach C.1, Kroemer H. K.1, Rosskopf D.1, Meyer zu Schwabedissen H.1 1Universitätsmedizin Greifswald C_DAT Institut für Allgemeine Pharmakologie, Felix- 154 Hausdorff-Str.3, 17489 Greifswald, Germany 2Universitätsmedizin Greifswald Klinik und Poliklinik für Urologie, Fleischmannstr. 42-44, 17489 Greifswald, Germany Transcriptional regulation of soluble adenylyl cyclase (sAC) and other genes involved in aldosterone signalling 1 1 1,2 3,2 1 Background. Numerous genome-wide association studies (GWAS) identified Herrmann M. , Guske K. , Schmitz B. , Salomon A. , Roosterman D. , Brand S. - 2,3 1 polymorphisms located in transporter genes such as SLC2A9, ABCG2, NPT1, and M. , Brand E. URAT1 as predicitive for the serum levels of urate1. These genes encode membrane 1University Hospital Münster Internal Medicine D, Albert-Schweitzer-Campus 1, 48149 proteins expressed in the apical membrane of human kidney proximal tubule cells and Münster, Germany are assumed to facilitate tubular exchange of urate2,3,4,5. Importantly several single 22Medical Faculty of the Westfalian Wilhelms-UniversityMünster Department of nucleotide polymorphisms (SNP) located in vicinity of SLC2A9 have been identified as Molecular Genetics of Cardiovascular Disease, Domagkstr. 5, 48149 Münster, Germany highly associated with serum urate levels. 3Leibniz-Institute for Arteriosclerosis Research Molecular genetics of cardiovascular Aim. Little is known about the transcriptional regulation of SLC2A9. Therefore, the aim disease, Domagkstr. 5, 48149 Münster, Germany of our study was to investigate which sequences in the SLC2A9 gene harbour cis- elements and regulate its gene expression. We also asked whether intronic SNPs Objective: The soluble adenylyl cyclase (sAC) activates the Na+/K+-ATPase in renal influence gene expression at the transcriptional level. epithelial collecting duct cells. Nuclear sAC constitutes a functional complex with cAMP Methods and Results. Performing dual luciferase reporter gene assays we found gene response element binding protein (CREB), suggesting a more general role of sAC in regulating modules in the SLC2A9 gene. DNA from human kidney samples was then overall gene regulation. We determined the chromatin binding capacities of sAC at CRE genotyped for rs6449237 being part of this region. Next total SLC2A9 mRNA-expression sequences and its influence on genes, which play a role in aldosterone signalling. levels of the samples were determined using real-time quantitative RT-PCR assay. Male Furthermore, we functionally characterised expression relevant promoter portions of samples with two minor alleles of SNP rs6449237 showed lower SLC2A9 mRNA levels sAC and the influence of aldosterone and cAMP mediated signalling pathways on sAC than samples with the wild type alleles. The effect was not seen in females. Reporter gene regulation. gene constructs with either minor or major allele of rs6449237 were then used in Design and Methods: In vascular endothelial cells (EA.hy926) and in human kidney cell luciferase assays, however showing no significant difference in activity. Furthermore lines (HEK293T; IHKE), we performed Chromation Immunoprecipitation (ChIP) assay mRNA-expression levels of other urate transporter genes were determined in kidney with antibodies against sAC and CREB. We conducted transfection with a CRE samples. After linear regression a positive correlation of mRNA-expression of SLC2A9, luciferase reporter vector and sAC promoter constructs, following treatment with sAC URAT1, NPT1, and OAT10, respectively was observed. inhibitors and aldosterone. Total RNA of EA.hy926 cells, which were treated with sAC Conclusion. Our data suggest that the SLC2A9 SNPs rs6449237 and rs74794351 inhibitors and aldosterone, was isolated and subsequently analysed by real-time PCR might influence SLC2A9 mRNA-level without controlling the transcriptional activity. It for expression of genes involved in aldosterone signalling. needs to be elucidated whether those SNPs alter mRNA stability. However, the mRNA Results: coexpression of SLC2A9 and other urate transporter might be attributed to a common In vivo binding of sAC at CRE motifs was shown using CRE consensus sequences in gene regulating pathway of an “transportosome“ controlling urate homeostasis. ChIP experiments. Specific pharmacological inhibition of sAC led to a significant decrease of transcriptional activity of the CRE control vector in endothelial and kidney References cell lines. Furthermore, we were able to show the different effects of sAC on the 1. Kolz et al. Meta-analysis of 28,141 individuals identifies common variants within five expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid new loci that influence uric acid concentrations. PLoS Genet. 2009 Jun;5(6):e1000504. receptor and Na+/K+-ATPase alpha1 and beta1 and sAC itself. Regulation of sAC itself is 2.Vitart et al. SLC2A9 is a newly identified urate transporter influencing serum urate mediated by two different promoter portions, which are influenced by aldosterone and concentration, urate excretion and gout. Nat Genet. 2008 Apr;40(4):437-42. inhibition of sAC and differentially accessed in kidney and endothelial cells. 3. Woodward et al. Identification of a urate transporter, ABCG2, with a common Conclusions: functional polymorphism causing gout. Proc Natl Acad Sci U S A. 2009 Jun sAC has transcriptional trans-acting properties as it interacts with CRE sites and 23;106(25):10338-42 potentially influences the expression of genes, which play a role in aldosterone 4. Iharada et al. Type 1sodium-dependent phosphate transporter (SLC17A1 Protein) is signalling. Transcription of sAC is regulated via aldosterone and cAMP. The location of a Cl(-)-dependent urate exporter. J Biol Chem. 2010 Aug 20 promoter TA is cell type- and stimulation-specific. 5.Enomoto et al. Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature. 2002 May 23;417(6887):447-52.
155 153 The role of the sodium-calcium exchanger (NCX1) in cardiac pacemaking Herrmann S., Stieber J., Ludwig A. Sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate Friedrich-Alexander-Universität Erlangen-Nürnberg Institut für Experimentelle und ester binding to DNA in vitro and in vivo Klinische Pharmakologie und Toxikologie, Fahrstrasse 17, 91054 Erlangen, Germany 1 2 3 1 1 Herrmann K. , Engst W. , Appel K. E. , Monien B. H. , Glatt H. - R. 1Deutsches Institut für Ernährungsforschung Ernährungstoxikologie, Arthur-Scheunert- The mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. Allee 114-116, 14558 Nuthetal, Germany The unique feature of sinoatrial node (SN) cells is the ability to generate a spontaneous 2Deutsches Institut für Ernährungsforschung Analytik, Arthur-Scheunert-Allee 114-116, diastolic depolarization that periodically initiates action potentials which set the heart 14558 Nuthetal, Germany rhythm. The molecular origin of this cardiac pacemaker activity is still a matter of debate. 3Bundesinstitut für Risikobewertung Lebensmitteltoxikologie, Thielallee 88-92, 14195 Recent findings point to a coordinated interplay between intracellular Ca2+-cycling Berlin, Germany processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. In this study, we investigated Methyleugenol (ME) is a secondary metabolite occurring in many herbs and spices. the contribution of the cardiac sodium-calcium exchanger (NCX1) to pacemaking. NCX1 Although ME is hepatocarcinogenic in rodents, standard genotoxicity tests were is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. It negative. This may be due to the lack of critical activating enzymes responsible for the was speculated that the membrane depolarizing current INCX, whose activity is terminal bioactivation of ME to a genotoxicant. ME is initially hydroxylated by dependent on intracellular Ca2+-fluctuations, represents a main determinant of the cytochrome P450 enzymes yielding 1´-hydroxymethyleugenol (1´-OHME). This alcohol spontaneous diastolic depolarization. We used an inducible and sinoatrial node-specific can be further activated by sulfotransferases (SULTs) to an electrophilic sulfate ester Cre-transgene to delete NCX1 in the murine pacemaker system. The successful that can be easily attacked by DNA. The DNA adducts formed could lead to mutation creation of a cardiac pacemaking and conduction system specific NCX1 knockout and further carcinogenicity observed in animals. (cpNCX1KO) was demonstrated by transcript quantification as well as The aim of the present study was to clarify whether individual human (h) and murine immunofluorescence experiments. Telemetric ECG recordings of cpNCX1KO displayed a SULT forms are involved in the activation of ME to a genotoxicant. In order to identify distinct cardiac phenotype. Mutant animals were deeply bradycardic and lost their S37
capability of maintaining a stable heart beat as demonstrated by various ECG (CREs) in its target gene promoters. The Icer mRNA expression is regulated by an abnormalities like SN arrhythmia, SN pauses, AV block and ventricular tachycardia. intronic promoter of the Crem gene. In vascular smooth muscle cells (VSMCs) Analysis of the spontaneous activity of isolated SN preparations showed a slower and CREM/ICER is involved in the regulation of cell proliferation and apoptosis with arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia physiological consequences in vivo. For instance CREM-knockout mice, in which none and arrhythmia induced by deletion of NCX1 results from a slower and arrhythmic of the known isoforms can be expressed, exhibit an increased neointima formation after intrinsic pacemaker activity. A battery of experiments using different heart rate lowering carotid ligation as well as an increased atherosclerotic plaque formation after high fat as well as increasing drugs revealed an altered heart rate modulation in cpNCX1KO diet on an ApoE background. These observations were associated with an increased animals as compared to controls. In conclusion, these initial results establish NCX1 as a proliferation rate in isolated CREM deficient VSMCs. On this background we wanted to major contributor to cardiac pacemaking. clarify the specific role of ICER isoforms in the vasculature. In first experiments we examined the inducibility of Icer in primary VSMCs and smooth muscle cell lines. Reporter luciferase assays showed that the activity of the Icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (FSK) in 156 immortalized rat VSMCs (13.7 ± 0.93; n=12 from 3 isolations). In A7r5 rat smooth muscle cells the Icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of FSK stimulation (n=20 from 4 isolations). These pilot experiments showed Formation and excretion of glutathione conjugates of the ultimate carcinogen of that the Icer promoter is inducible in VSMCs by cAMP dependent pathways. Further benzo[a]pyrene in human Caco-2 cells experiments have to be carried out to elucidate the role of ICER in the vascular system 1 2 2 1 for example by stimulation of primary VSMCs and analysis of ICER knockout mice. Hessel S. , John A. , Seidel A. , Lampen A. (Supported by the DFG) 1 Bundesinstitut für Risikobewertung Lebensmittelsicherheit, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany 2Biochemisches Institut für Umweltcarcinogene, Prof. Dr. Gernot Grimmer-Stiftung, Lurup 4, 22927 Grosshansdorf, Germany 159 A wide variety of contaminants are ingested through food, among them the pro- carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) which is resorbed and partially metabolized in the enterocytes of the small intestine. Previous in vitro Overexpression of transmembrane channel-like proteins (TMCs) uncouples studies revealed that BP phenols are excreted as phase II metabolites including BP receptor-mediated calcium mobilisation glucuronides and BP sulfates. This export is mediated by the breast cancer resistance Hill K., Urban N., Straub I., Schaefer M. protein (BCRP/ABCG2). The ultimate carcinogenic phase I BP metabolite anti-BP-7,8- Universität Leipzig - Universitätsmedizin Rudolf Boehm-Institut für Pharmakologie und dihydrodiol-9,10-epoxide (BPDE) can be detoxified by glutathione conjugate formation Toxikologie, Härtelstr. 16-18, 04107 Leipzig, Germany catalyzed by various glutathione S-transferases. In the present study, differentiated human intestinal Caco-2 cells were used as a model The family of transmembrane channel-like proteins (TMCs) consist of 8 members for the human small intestine to investigate the detoxification of BPDE and the (TMC1-TMC8) All TMC genes are predicted to encode transmembrane proteins with at subsequent transport of the stereoisomeric glutathione conjugates in the presence of an least six membrane-spanning helices. Mutations of TMC1 cause deafness in human and inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase mice whereas TMC6 and TMC8 (also referred to as EVER 1 and 2) are linked to (gGT) at the surface of the cells. The results indicate that the glutathione conjugates of epidermodysplasia verruciformis (EV), a skin disorder, which is characterised by an BPDE are formed and excreted mainly to the apical and to a minor extent to the enhanced susceptibility towards cutanous infections by human papillomaviruses. The basolateral side of the polarized Caco-2 monolayer. To stimulate the transport rate cell biological and physiological functions of TMC proteins still remain elusive. several inducers known to enhance gene expression of xenobiotic-metabolizing We have overexpressed TMC6 and TMC8 in HEK293 cells to get insights into their enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). physiological function. All TMCs were located within the endoplasmic reticulum (ER) However, solely oltipraz substantially increased the efflux of BPDE glutathione after overexpression of YFP or CFP-tagged constructs. Ratiometric calcium imaging conjugates after inhibition of gGT. Inhibition studies revealed that the multidrug revealed that after overexpression of TMC6 or TMC8, stimulation of Gq-coupled resistance-associated proteins (MRPs/ABCCs) are involved in the transport of the BPDE receptors with carbachol and ATP resulted in a greatly reduced amount of calcium glutathione conjugates. Stable ABCC1, ABCC2 and ABCC3 knockdown cell lines were release from the ER. Moreover, challenging TMC6- or TMC8-expressing cells with the generated allowing to demonstrate that ABCC1 mediates the basolateral, ABCC2 the SERCA pump inhibitor thapsigargin was also not followed by a release of ER-based apical excretion of the BPDE glutathione conjugates. In conclusion, the ultimate calcium within the cell. To test whether the lack of calcium release was caused by a carcinogen BPDE is detoxified via glutathione conjugation and subsequently excreted by reduced calcium content within the ER, we investigated calcium dynamics within the ER Caco-2 cells in both apical and basolateral directions. .This finding is equivalent to a using an ER-targeted FRET-based calcium indicator (D1ER Cameleon). The transport into the feces as well as blood system in the in vivo situation. experiments revealed that the amount of calcium within the ER was reduced upon overexpression of TMC6 or TMC8. Recently, it has been reported that TMC6 and TMC8 might influence intracellular zinc distribution in human keratinocytes. We could confirm the presence of TMC6 and TMC8 mRNA in a human keratinocytes cell line (HaCat). 157 Upon overexpression of TMC8, HaCat cells revealed the same phenotype as described above for the HEK293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the ER calcium store. The mechanism by which Signaling via IRAG regulates Store operated Calcium Entry (SOCE) in aortic VSMC overexpression of TMC proteins causes a reduced calcium concentration within the ER remains unclear. Considering that the presumed topology of the TMC proteins distantly Hieke B., Hüttner J., Schlossmann J. resembles those of other ion channel superfamilies such as anoctamins, one might University of Regensburg Department of Pharmacology and Toxicology, Universitätsstr. speculate that a conductance through the TMC protein itself leads to a calcium leak from 31, 93053 Regensburg, Germany the ER.
The mechanisms involved in the activation of store operated calcium entry (SOCE) through depletion of intracellular stores and their regulation are not yet fully understood. We examined the effect of inositoltriphosphate-receptor associated cGMP-kinase substrate (IRAG) on SOCE. Aortic vascular smooth muscle cells (VSMC) from wild type 160 (WT) and IRAG-Knock-out (KO) mice were loaded with the calcium indicator Fura 2-AM and SOCE was measured as a change in the intracellular calcium concentration. In experiments with VSMC from WT mice SOCE was attenuated by the application of 8-Br- Terahertz radiation does not cause genomic damage 1 2 3 2 3 2 cGMP. This effect was not observed in VSMC isolated from IRAG-KO mice. These Hintzsche H. , Jastrow C. , Heinen B. , Kleine-Ostmann T. , Koch M. , Schrader T. , 1 differences in the strength of the SOCE-signal were abolished by the replacement of Stopper H. extracellular sodium with N-Methyl-D-Glucamine. The observed sodium dependence of 1Universität Würzburg Institut für Pharmakologie und Toxikologie, Versbacher Str. 9, the SOCE regulation via IRAG suggests, that an alternated sodium conductance might 97078 Würzburg, Germany be responsible to some extent for the differences detected in WT and IRAG-KO VSMC. 2Physikalisch-Technische Bundesanstalt, Braunschweig, Germany As a change in sodium conductance might result in a changed membrane potential we 3Universität Marburg Fachbereich Hochfrequenz und Felder, Marburg, Germany tried to track these changes with the FLIPR membrane potential assay kit while executing the SOCE protocol with and without 8-Br-cGMP. No significant differences in Terahertz radiation is defined as radiation between 0.1 THz and 10 THz. A number of membrane potential could be detected in the various stages of SOCE. In conclusion, our applications are currently being developed using radiation in this frequency range. results indicate that IRAG exhibits a dual action on calcium regulation as it inhibits not These applications will lead to exposure of the general public, making it very important only the intracellular calcium release but also the extracellular calcium influx through to study potential effects on biological systems. Historically, only a few studies on effects SOCE. caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. During the last decade, a number of studies on effects caused by radiation with frequencies around 100 GHz have been published. The present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 158 0.106 THz, 0.380 THz and 2.520 THz. Two skin cell types were used, primary human dermal fibroblasts (HDF) and a keratinocyte cell line (HaCaT). The cells were irradiated applying different exposure Induction of the ICER promoter in vascular smooth muscle cells times and different power intensities. Two genotoxicity tests were applied: the comet 1 1 1 2 2 1 1 assay quantifies DNA strand breaks as well as alkali-labile sites whereas the Hildebrandt I. , Seidl M. D. , Nunes F. , Endo S. , Kojima N. , Schmitz W. , Müller F. U. micronucleus test quantifies chromosomal damage. All experiments were performed and 1 Westfälische Wilhelms-Universität Institut für Pharmakologie und Toxikologie, evaluated under blinded conditions as three independent replicate experiments. Positive Domagkstr 12, 48149 Münster, Germany (MMS-treated) and negative (untreated, sham-exposed) controls were included. 2 Tokyo Metropolitan Institute of Gerontology, Tokyo Japan In the comet assay no DNA damage was observed as a consequence of the exposure under all experimental conditions. The same was true for the chromosomal damage Several transcription factor isoforms are encoded by the Crem (cAMP response element investigated with the micronucleus test. The latter finding was particularly interesting for modulator) gene. One prominent isoform is the inducible cAMP early repressor (ICER), the experiments at 0.106 THz, because this type of radiation had been reported to cause which acts as a transcriptional repressor on so called cAMP responsive elements mitotic disturbances. Therefore these experiments were extended, applying higher S38
power intensities and longer exposure periods. Also with these modifications, no 163 genomic damage was observed in the form of micronucleus formation. All in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. This result is in line with published findings on genotoxicity of Soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation low-frequency terahertz radiation around 0.1 THz. The question, why the reported Hoffmann L. S.1, Kipschull S.1, Jennissen K.1, Friebe A.2, Pfeifer A.1 mitotic disturbances do not lead to manifest genomic damage remains open and 1 requires further research. Universität Bonn Institut für Pharmakologie und Toxikologie, Sigmund-Freud-Str. 25, 53127 Bonn, Germany 2Universität Würzburg Physiologisches Institut I, Röntgenring 9, 97070 Würzburg, Germany
161 Brown adipose tissue (BAT) uses energy to produce heat by inducible thermogenesis. Recent studies show that active BAT is present in adults and involved in human energy balance, suggesting that the energy consuming property of BAT might be exploited to Down-regulation of inducible inflammatory genes of colon cancer cells by dietary fight obesity and related diseases. The NO/cGMP signaling pathway is a key player in tea-flavonoids diverse physiological processes. Recently, we have shown in BAT that cGMP signaling 1 2 3 Hoensch H. , Mueller D. , Richling E. is connected with insulin signaling and abrogation of cGMP signaling leads to impaired 1Marienhospital Private Praxis für Gastroenterologie, Martinspfad 72, 64285 Darmstadt, BAT differentiation and function (Haas, B. et al., Sci Signal, 2009). Germany Here we investigated the role of the cGMP generating enzyme soluble guanylate 2Universität Lebensmittelchemie, Erwin-Schrödinger-Str. 52, 67663 Kaiserslautern, cyclase (sGC) in BAT differentiation in vitro. -/- Germany Mesenchymal stem cells isolated from BAT of newborn sGCβ1 mice and wt littermates 3Universität Lebensmittelchemie, Erwin-Schrödinger-Str.52, 67663 Kaiserslautern, were differentiated in vitro into brown adipocytes in the presence or absence of cGMP. Germany Abundance of sGC isoforms was determined by qRT-PCR and Western Blotting. BAT differentiation was assessed by RedO staining of accumulated intracellular lipids, Introduction: Tea flavonoids derived from camomile and green tea such as apigenin and measurement of triglyceride (TG) content, determination of expression of BAT marker epigallocatechin gallate (EGCG) can inhibit intestinal neoplasia. Recurrences of proteins PPARγ, C/EBPα, aP2 and BAT marker genes UCP1, PGC1α, Cidea. adenomas and cancers were reduced in patients with resected colorectal cancer by The α2 and β1 isoforms of sGC were highly expressed in BAT whereas α1 sGC could not treatment with tea bioflavonoids after tumor operation [1]. be detected. RedO staining of wt brown adipocytes showed basal differentiation which - To clarify the biomolecular pathway for suppression of neoplasia we investigated the was increased upon addition of 8-pCPT-cGMP. In contrast, staining was lower in sGCβ1 anti-inflammatory effect of a nutritional supplement Flavo Natin® (FN) which had been /- cells compared to wt under control conditions and increased in the presence of cGMP. -/- used in the clinical study on tertiary tumor prevention and of EGCG in a colon tumor cell TG measurement showed that sGCβ1 brown adipocytes contain approximately 50% line. The aim of our study was to investigate if tea flavonoids are capable to suppress less lipids than wt cells under basal conditions. Addition of cGMP doubled TG content in the inflammatory markers produced by tumor cells after cytokine stimulation. both genotypes. Similar results were observed for marker protein expression. Deletion of Method: We studied the cytotoxicity of FN in the colon cancer cell line T-84 by resazurin sGC resulted in 56-40% decrease in C/EBPα, PPARγ and aP2 expression compared to -/- fluorescence and compared it with the placebo supplement. Additionally, the T-84 cells wt. Again, cGMP roughly doubled protein expression in sGCβ1 and wt cells compared were incubated with FN, EGCG or placebo and stimulated with TNF-alpha, IF-gamma to control. Under basal conditions, BAT marker gene expression was decreased by -/- and IL-1-beta. After the cytokine stimulation the mRNA expression of IP-10, IL-8 and approximately 80% in sGCβ1 cells compared to wt cells. This decrease was prevented TNF-alpha was measured by quantitative Real-Time PCR (qRT-PCR). by addition of cGMP. Results: Stimulation of T-84 cells increased the expression of IP-10 (gamma-interferon These results show that sGC deletion leads to dysfunctional BAT differentiation and inducible protein 10), TNF-alpha and IL-8. By preincubation with FN at 10 µM the mRNA emphasize the central role of cGMP signaling in BAT differentiation. Further expression of IP-10 was strongly reduced (log2-ratio -14). The TNF-alpha mRNA was investigation of sGC/cGMP signaling in BAT might reveal new drugable targets bringing also but less decreased by FN. EGCG displayed an inhibition pattern similar to FN. BAT-dependent pharmacological therapy to treat obesity and related disease into closer Placebo did not influence the mRNA expression of the chemokines and TNF-alpha. reach. Discussion & Conclusion: Clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. Down-regulation of inflammatory gene References products could be achieved in vivo by botanicals without clinically relevant side effects. Haas, B., P. Mayer, K. Jennissen, D. Scholz, M. B. Diaz, W. Bloch, S. Herzig, R. Fassler and A. Pfeifer (2009). Protein kinase g controls brown fat cell differentiation and References mitochondrial biogenesis. Sci Signal 2(99): ra78. [1] H. Hoensch, B. Groh, L. Edler, W. Kirch (2008). Prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. World J Gastroenterol, 14, 2187-2193. 164
162 Comparative Inhalation toxicity of Carbon-Nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) Hofmann T.1, Lan M. - H.1, Wiench K.2, Wohlleben W.3,1, van Ravenzwaay B.2, The CXCR7 C-terminal domain mediates efficient CXCL12 uptake and degradation Landsiedel R.1 Hoffmann F., Müller W., Schütz D., Schulz S., Stumm R. 1BASF SE Experimental Toxicology and Ecology, 67063 Ludwigshafen am Rhein, Universitätsklinikum Jena Institut für Pharmakologie und Toxikologie, Drackendorfer Str. Germany 1, 07747 Jena, Germany 2BASF SE Product Safety, GV/TB, 67063 Ludwigshafen, Germany 3BASF SE Polymer Physics, 67063 Ludwigshafen am Rhein, Germany CXCL12-signaling mediated by the G protein-coupled CXCR4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. Carbon black is a spherical carbon anomaterial whereas multi-wall Carbon nanotubes The second CXCL12-receptor CXCR7 modulates CXCL12/CXCR4-signaling by acting (MWCNT) are cylindrical and graphene is a laminar allotrope of carbon. Processing and as a CXCL12-scavenger. Given the distinct functions of CXCR4 and CXCR7, we handling as well as abrasion processes can set free inhalable CNT particles. Results of hypothesized that trafficking and receptor stability are differently regulated by the distinct rodent studies collectively show that regardless of the process by which CNTs were C-terminal domains. Here, we examined epitope-tagged wild type and C-terminal mutant synthesized and the types and amounts of metals they contained, CNTs were capable of receptors expressed in human embryonic kidney cells (HEK293) with respect to producing inflammation, epithelioid granulomas, fibrosis, biochemical and or trafficking, stability, 125I-CXCL12 radioligand degradation, and G protein-coupling. We toxicological changes in the lungs (Lam et al. 2004, Muller et al. 2005, Ma-Hock 2009, found that the 24 C-terminal residues of CXCR7 were sufficient for CXCR7 to undergo Pauluhn 2010). Graphene possess similar physical properties as CNT but may different rapid spontaneous internalization. Replacement of the CXCR7 C-terminal domain with toxicological property. We performed short-term inhalation studies in rats to compare the that of CXCR4 (CXCR7-4tail mutant) abolished spontaneous internalization but toxic potency of four different CNT, two graphenes and one carbon black. The materials permitted ligand-induced internalization in conjunction with C-terminal phosphorylation. are characterized thoroughly according to the OECD list. Conversely, replacement of the CXCR4 C-terminal domain by that of CXCR7 caused The four MWCNT caused morphological changes as descriped above. Several ligand-independent internalization of CXCR4. Receptor-mediated 125I-CXCL12-uptake, biochemical and cytological parameters in the broncho-alveolar lavage fluid were release of 125I-CXCL12-degradation products, and degradation of the receptor protein strongly increased consistent with the histological findings. Two MWCNT exhibited a itself were accelerated with receptors bearing the CXCR7 C-terminus. While the CXCR7 higher toxic potency than two other MWCNTS and findings caused by one Graphene typ C-terminus was sufficient to abolish G protein coupling in the CXCR4-7tail mutant, were even less severe. The graphene with lower surface area as well as low surface replacement of the CXCR7 C-terminus, CXCR7 second intracellular loop or both area carbon black did not cause any adverse effects up to 10 mg/m3. domains with the corresponding CXCR4 domain did not generate a G protein-coupled The short-term inhalation studies were able to descriminate different toxic potencies of CXCR7 chimera. Taken together, we provide evidence that the CXCR7 C-terminal carbon-based nanomaterials and is hence used for the selection of less toxic materials domain influences the ligand-uptake/degradation rate, G protein-coupling, and stability for further product development as well as to define and prioritize higher-tier of the receptor. This suggests that heterologous regulatory pathways targeting the toxicological testing of nanomaterials. CXCR7-C-terminal domain may effectively control CXCR7 functions.
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Synthesis and analytical assessment of possible DNA adducts formed after Contribution of components of STW 5 to its anti-inflammatory action activation of the tobacco alkaloid myosmine Hoser S.1, Winkelmann V.1, Abdel-Aziz H.2, Kelber O.2, Weiser D.2, Nieber K.1 Högg C., Zwickenpflug W., Gudermann T. 1Universität Leipzig Institut für Pharmazie; Pharmakologie, Talstr. 33, 04103 Leipzig, Walther-Straub-Institut Abt.: Toxikologie, Nußbaumstraße 26, 80336 München, Germany Germany 2Steigerwald Arzneimittelwerk GmbH Wissenschaftliche Abteilung, Havelstr. 5, 64295 Darmstadt, Germany Myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not STW 5 (Iberogast®), a multi-component herbal drug, is successfully used in the therapy understood in detail. Myosmine can be activated by peroxidation and N-nitrosation of functional dyspepsia and irritable bowel syndrome (IBS). Previous studies revealed yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) which is well known as reactive effects of STW 5 on disturbed motility and inflammatory processes. Although the anti- intermediate during activation of a variety of tobacco specific N-nitrosamines (TSNA). inflammatory properties of STW 5 are well examined, the contribution each of the Therefore, myosmine may be a potential candidate for possible mutagenic or individual herbal constituents to the anti-inflammatory effect remains unclear. carcinogenic risk to human health. Furthermore, myosmine N-nitrosation yields the Therefore, we studied the effects of STW 5 and its components on inflammation-induced tobacco specific nitrosamine N-nitrosonornicotine (NNN), which is classified as cell death and on the release of the pro-inflammatory cytokine TNF-α. The aim of these carcinogenic to humans. Considerable efforts have been undertaken, especially in investigations was to analyse additive or synergistic effects of the components. organic synthesis, to verify and elucidate the significance of the HPB precursor the The experiments were carried out on CaCo-2 cells after stimulation with LPS (10 ng/ml) pyridyloxobutyl (POB) intermediate and its DNA-adducts, which were analysed only in for 2 hours. Cytotoxicity was evaluated using a commercially available LDH (lactate animal experiments till now. The formation of 3-pyridylmethanol was observed under dehydrogenase)-assay. Furthermore, the release of TNF-α after LPS (100ng/ml) myosmine peroxidation and identified as a metabolite in rat urine after application of stimulation of differentiated THP-1 cells was measured using a commercially available myosmine to rats. The formation of the 3-pyridylmethanol intermediate, might provide for ELISA. the reactive electrophilic picolinium ion which could interact with DNA. These possible STW 5 (62.6 - 500.5 µg/ml) reduced LPS (10 ng/ml)-induced cell death in a adducts might be of special interest to elucidate the role of myosmine concerning its concentration-dependent manner with a maximum inhibition of 50.0 %. The herbal uptake by smokers and passive-smokers in contrast to non smokers ingesting the components in equivalent concentrations contributed to the inhibitory effect of STW 5 to substance by consumption of food. DNA adducts may help to obtain more information different extents. The maximum inhibition differed in a wide range between the about possible risk assessment of myosmine and to differentiate between the activation components. from the other nicotinoids and TSNA. The specific DNA adducts, 5-methyl-1,3-bis- STW 5 (500.5 µg/ml) reduced significantly the release of TNF-α by 87 % in LPS (100 pyridin-3-ylmethyl-1H-pyrimidin-2,4-dion and 3-(3'’-picolyl)thymidine have been ng/ml)-stimulated differentiated THP-1 cells while having no effect in untreated cells. In synthesised as reference substances. The former was prepared by addition of concentrations equivalent to STW 5 caraway, milk thistle, lemon balm and greater diisopropylazodicarboxylate (DIAD) to a mixture of 3-pyridylmethanol, thymine and celandine had no effect on the LPS-induced increase in TNF-α release. Bitter candytuft, triphenylphosphine. The reaction product was identified using NMR (1H, 13C, COSY, peppermint, chamomile, liquorice and angelica reduced the TNF-α release, though less HMQC, HMBC). For synthesis of 3-(3'’-picolyl)thymidine the hydroxyl groups of pronounced as compared to STW 5, indicating a possible synergistic effect. thymidine were initially acetylated using acetic acid anhydride and 4- Our results indicate a multi-target effect of STW 5. The anti-inflammatory effect may be dimethylaminopyridine (DMAP). The existence of this thymidine adduct was confirmed due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of using NMR. This adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin the release of the pro-inflammatory cytokine TNF-α from immune cells. The individual (DTAF) to enhance the sensitivity using HPLC-fluorescence chromatography and used herbal components seem to contribute by different mechanisms of action to the overall as reference substances for analysis of DNA samples from biological tissue. effect of STW 5. Supported by DFG grant (TY 81/1-1).
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Immune cell-induced local steroidogenesis in the lung: implications for asthmatic Protectors and traitors: opposing functions of human paraoxonases in disease and therapeutic intervention atherosclerosis and cancer Hostettler N.1, Brunner T.2, Brunner T.2 Horke S., Witte I., Förstermann U. 1Universität Bern Institut für Pathologie, Murtenstraße 31, 3010 Bern, Switzerland Universitätsmedizin Mainz, Institut für Pharmakologie, Obere Zahlbacher Straße 67, 2Universität Konstanz Biologie/Biochemische Pharmakologie, Universitätsstraße 10, 55131 Mainz, Germany 78457 Konstanz, Germany
Cancer and cardiovascular diseases such as atherosclerosis are the most important Glucocorticoids are steroid hormones with potent anti-inflammatory properties. Synthetic causes of death in western societies. Common to both diseases is a deregulation of cell glucocorticoids are frequently used for the therapeutic treatment of inflammatory death, with significant contribution of inflammatory processes. Enhanced oxidative stress disorders, such as asthma. Endogenous glucocorticoids are predominantly produced in plays a dominant role in such events as it forms a vicious cycle with inflammation and the adrenal glands in response to emotional, physical and immunological stress. Recent controls multiple forms of cell demise. Therefore, anti-oxidative enzyme systems gained years, however, revealed several alternative sources of these immunoregulatory steroid considerable interest since control of reactive oxygen species (ROS) has the capacity to hormones. Thus, we have found that the intestinal epithelium is a rich source of regulate cell death in either direction. The human enzyme family of paraoxonases glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local consists of three members, known as PON1, PON2 and PON3. While PON1 is found immune homeostasis (1). As the intestinal and the pulmonary epithelium have much in predominantly in the circulation, PON2 and PON3 are intracellular enzymes with common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered established anti-oxidative functions. It has been shown that both PON2 and PON3 are whether the lung mucosa is also capable of synthesizing immunoregulatory protective against atherosclerosis. Underlying mechanisms of their protective and anti- glucocorticoids in response to immune cell activation. The murine lung was found to oxidative functions however remained elusive. Here we demonstrate that both enzymes expresses all enzymes required for the synthesis of corticosterone from cholesterol. locate to the endoplasmic reticulum (ER) and mitochondria where they fulfill vital While most enzymes where expressed in a constitutive manner, Cyp11a1, encoding functions in the control of ROS generation. In particular, PON2 and PON3 were shown P450ssc, was strongly induced in response to immunological tress. Treatment of mice to interact with coenzyme Q10 which diminishes mitochondrial ROS formation. As a with T cell-activating anti-CD3 antibody of macrophage-activating lipopolysaccharides consequence, these enzymes reduce execution of mitochondrial apoptosis, such as induced strong local glucocorticoid synthesis, which was effectively blocked by the cardiolipin peroxidation, cytochrome C release and caspase activation. Moreover, PON2 corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured and PON3 reduced ER stress-triggered cell death, i.e. by diminishing JNK signaling and were produced bona fide in the lung tissue. Surprisingly, allergen-induced allergic airway CHOP expression. While these results elucidate their protective role in cardiovascular inflammation failed to trigger local glucocorticoid synthesis despite the massive diseases, it also establishes a relevant function in survival of tumor cells. In accordance, infiltration of neutrophils, eosinophils and T cells. In contrast to that in the intestinal we demonstrate that both enzymes are frequently found overexpressed in various epithelium local glucocorticoid synthesis in the lung was found to be dependent on the tumors. In cancer cell culture studies, overexpression of both enzymes granted presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. In considerable resistance against chemotherapeutics. In turn, knock-down of PON2 line with the notion that the lung metabolizes steroid precursors we found that ex vivo caused spontaneous apoptosis of several cancer cell lines. Finally, our analyses also cultured lung tissue metabolized 3H-dehydrocorticosterone to 3H-corticosterone in an revealed that PON2-knockout mice show severe alterations of the hematopoetic stem 11ß-hydroxysteroid-dependent manner (2). While both, lung and intestine are capable of cell compartment, which implies a significant role in leukemias where these enzymes are producing immunoregulatory glucocorticoids in response to immunological stress and frequently found overexpressed. Together, our results propose PON2 and PON3 as new thereby likely contribute to the maintenance of local immune homeostasis, the two putative anti-tumor candidates and demonstrate the efficacy of interventions targeting mucosal tissues use different enzymatic pathways. The identification of this novel cellular redox-balance. immunoregulatory mechanism in the lung offers novel opportunities for therapeutic intervention of asthmatic disease.
References 1. Noti, M, Sidler, D., and Brunner, T. Extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? Sem. Immunopathol. 31(2):237-48. 2009 2. Hostettler N, Bianchi B, Gennari-Moser C, Kassahn D, Schoonjans K, Corazza N, Brunner T. Local glucocorticoid production in the mouse lung is induced by immune cell stimulation. Allergy. 10.1111/j.1398-9995.2011.02749.x.
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MicroRNA expression profiling in placentas of different gestational age Effects of subchronic inhalation exposure to diesel engine exhaust in the 5xFAD Hubeny A.1, Schwenn A.1, Saljè K.2, Herr F.3, Zygmunt M.3, Siegmund W.2, Kroemer H. mouse model of Alzheimer’s Disease. K.1, Grube M.1 Hullmann M.1, Albrecht C.1, van Berlo D.1, Gerlofs-Nijland M. E.2, Hillmann A.3, Boere 2 2 4 3 1 2 1 1Institut für Pharmakologie Allgemeine Pharmakologie, Felix-Hausdorff-Str. 3, 17487 A. , Fokkens P. , Weggen S. , Bayer T. , Krutmann J. , Cassee F. , Schins R. Greifswald, Germany 1Leibniz Research Institute for Environmental Medicine Particle Research, Auf´m 2Institut für Pharmakologie Klinische Pharmakologie, Felix-Hausdorff-Str. 3, 17487 Hennekamp 50, 40225 Düsseldorf, Germany Greifswald, Germany 2National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands 3Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Ferdinand-Sauerbruch- 3Department of Psychiatry, University Medicine Göttingen, Göttingen, Germany Straße, 17475 Greifswald, Germany 4Department of Neuropathology, Heinrich Heine University, Düsseldorf, Germany
During the last decade small RNA molecules has been identified as important regulators In recent years, it has been suggested that nanoparticles generated from combustion for gene expression. These microRNAs (miRNA) are single-stranded transcripts, which processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of are expressed in many cell types, where they modulate RNA stability and translation neurodegenerative diseases such as Alzheimer’s disease (AD). The aim of our current and, therefore, controlling various cellular mechanism and tissue development. Against study was to investigate the effects of subchronic exposure to diesel engine exhaust this background, in the present study the miRNA expression and potential target genes (DEE) in the 5xFAD mouse model, which is characterised by progressive behavioural were studied in the human placenta as a tissue demonstrating various developmental deficits as well as amyloid plaque formation and neuron loss. Ten weeks old female changes in a limited period of time. 5xFAD mice and their nontransgenic littermates were exposed by whole body inhalation TaqMan®Array MicroRNA cards for profiling of 365 miRNAs in placentas of different to diluted DEE (~1 mg particles/m3) or clean air (controls) for 3 or 13 weeks (5 gestation times revealed a significant expression of 277 miRNAs by comparing days/week and 6 hour/day). Subsequently, all animals were subjected to a series of placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). behavioural tests. At ten days post-exposure, mice were sacrificed to investigate lungs When comparing the analyzed groups, 106 of these miRNAs expose a continuous up- and brain tissues for pathological and biochemical and molecular-biological changes. In (including miR-20a, miR-379) or downregulation (including miR-9, miR-200a). While line with the expectations, the 5xFAD mice displayed typically age-dependent comparing placentas of early gestation to term placentas, 30% (e.g. miR-9, miR-429) behavioural deficits and amyloid plaque formation in cortex and hippocampus. A were up- and 70% (e.g. miR-126, miR-373) were downregulated. By focusing on the significant DEE exposure-related effect was observed for the string suspension test, latter group with early preterm placentas the ratio is opposed. Here, 60% of miRNAs representing a measure of motor coordination/grip strength. DEE exposure was also showed higher (e.g. miR-107) and 40% lower expression (e.g. miR-627, miR-191). With associated with mRNA expression changes of markers of inflammation and oxidative emphasize on these miRNAs, a computational prediction algorithm using the miRò stress in specific brain regions, including the olfactory bulb. Histopathology of plaque- database predicted a potential interaction between corticotropin-releasing hormone load in cortex and hippocampus (from a limited number of animals investigated so far) (CRH) and the miRNAs miR-9 and miR-429. Specific expression analyses validate an did not reveal clear evidence for increased plaque formation due to the DEE exposures. inverse expression between miRNAs and target with a reduced expression of miR-9 Further research is needed to evaluate the effects of long term exposure to (93%) and miR-429 (94%) in term placentas, while CRH is upregulated 114fold. This nanoparticles in the central nervous system. interaction was verified on functional level by reporter gene assay. A significantly This work is supported by funds from the Research Committee of the Medical Faculty of suppressed luciferase activity of the reporter plasmid containing the 3´-UTR sequence the University of Düsseldorf (9772-365), the DFG Graduate School GRK1033 and the complementary to CRH was exhibit for the predicted miR-9 binding site. Here, the RIVM - Centre for Environmental Health, Bilthoven, Netherlands. miRNA-mRNA-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted miR-429 binding site is not significant. The results demonstrate a gestation dependent expression of placental miRNAs, which may help to explain gestational changes in gene expression and highlight the potential 172 of miRNAs as biomarker for pregnancy-related pathologies.
Cytoprotective effect of the small GTPase RhoB expressed upon Ras glucosylation by Clostridium sordellii Lethal Toxin 170 May M.1, Hülsenbeck J.1,2, Schulz F.1,3, Fritz G.4, Genth H.1 1Medizinische Hochschule Hannover Toxikologie, Carl-Nezberg-Str. 1, 30625 Hannover, Germany Skin experimental trauma healing under Scilla indica Knuth oil extract local 2Universitätsmedizin Mainz Toxikologie, Obere Zahlbacher Str. 67, 55131 Mainz, treatment Germany 1 2 1 1 1 Hudu M. , Karvouni H. , Kotsiou A. , Magiatis P. , Tesseromatis C. 3Fraunhofer-Institut für Toxikologie und Experimentelle Medizin, Nikolai-Fuchs-Str. 1, 1Universitat Athen Pharmakologie, Mikras Assias 75, 11527 Athen, Greece 30625 Hannover, Germany 2Aretaieio Uni Klinik Pathologie, Vas. Sophias 76, 11528 Athen, Greece 4Heinrich-Heine-Universität Düsseldorf Toxikologie, Universitätsstrasse 1, 40225 Düsseldorf, Germany Since Homo sapiens era wound treatment by early civilizations was based on the use of local flora. Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks Scilla Indica Knuth Liliaceae (S.i) is a perennial herb with a pear shaped, tunicated bulb, critical survival pathways, including the PI3K/Akt, the RalGEF/Ral, or the Raf/ERK, and bearing fibrous roots, white flowers and leaves on the stem resembling U. maritima. It is results in apoptosis. In this study, Ras glucosylation is presented to result in expression used as cardiac tonic, against inflammation, ulcers and sinus diseases. Traditionally the of the cell death-regulating small GTPase RhoB based on transcriptional activitation. powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are RhoB expression depends on K-Ras inhibition, as siRNA-mediated knock-down of applied to corns of the feet soles. The plant contains steroid glycosides (bufadienolides specifically K-Ras (neither H-Ras nor N-Ras) provokes RhoB expression. RhoB 1-3%), glucoscillarene A, proscillaridine A, scillarene A, scillcyanoside ,scilliglaucoside expression further depends on inhibition of PI3K/Akt, as activation of PI3K/Akt using a ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat PI3K activator prevents RhoB expression downstream of inactivated Ras. Newly bran products. synthesized RhoB is GTP-loaded and rapidly degraded in a proteasome- and a The study is focused on the investigation of the restoration quality of a skin dorsal caspase-dependent manner, providing first evidence for caspase-dependent incision wound in Wistar rats degradation of a Rho family protein. Although often characterised as a pro-apoptotic In three groups, control (A1) and experimental (A2,A3 ) of rats weighing 350- 450g local protein, RhoB suppresses caspase-3 activation in TcsL-treated fibroblasts. Conclusions: application of dichloromethane extract of S.i in vehicle olive oil preparations of 50 and 1. Rapid and efficacious Ras inactivation by TcsL turns out to be particularly useful in 100mg were used. the characterisation of Ras inactivation-induced RhoB expression as an immediate-early Animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was gene response. 2. The finding on the cytoprotective activity of RhoB in TcsL-treated cells performed by lancet on depilated dorsal area skin until the muscular aponeurosis and re-enforces the concept that RhoB exhibits cytoprotective rather than pro-apoptotic were treated for 4 days with 60λ of each preparation. activity on cellular background of inactive Ras. Group A3 showed increased remodelling of trauma area (limited width). Granulomatous tissue was more pronounced in length, width and surface in group A2. The macroscopic ulcus dimensions were better in group A3 while the microscopic view were similar in A2 and A3 and better in comparison to control A1. A tendency to trauma remodelling was observed, without statistical significance (t =0.3)
References Panduranga,P.G et al. International Journal of Pharma and Bio Sciences VOL 2(3) 2011 K. Choudhary et al Am-Euras. J. Bot., 1 (2): 38-45, 2008
RhoB expression: The activity of the rhoB promoter is suppressed by RhoA (through a not yet identified pathway or Ras (through the PI3K/Akt pathway. Ras glucosylation by TcsL results in de-suppression of the rhoB promoter and RhoB expression.
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173 whereas T370, T376 and T379 were secondary sites. Moreover, the partial agonist morphine induced only the phosphorylation of S375 but not of T370, T376 of T379. Our results show, that MOR phosphorylation occurs in a hierarchical and agonist-selective The calcium-binding protein Annexin A4 modulates cAMP responsive element manner directly regulating receptor sequestration. (CRE)-dependent transcriptional activity and interferes with CREB-Ser133 phosphorylation Husser X., Nunes F., Heinick A., Schmitz W., Müller F. U. Institut für Pharmakologie und Toxikologie, WWU Münster AG Müller, Domagkstr. 12, 176 48149 Münster, Germany
The calcium-binding protein Annexin A4 (ANXA4) is involved in diverse cellular Assessment of MDA effects from toxicity studies with regard to endocrine processes including e.g. vesicular transport, ion channel regulation and transcriptional modulation 1 2 3 regulation. Upon Ca2+ binding ANXA4 undergoes conformational changes, which lead Jäger R. , West R. , Collins M. to oligomerization of ANXA4 to homotrimers and cause an increased affinity for 1Bayer HealthCare Product Stewardship Industrial Chemicals, Aprather Weg 18a, 42096 membrane phospholipids, which in turn provokes the translocation from the cytosol and Wuppertal, Germany the nucleoplasm to plasma and nuclear membranes. In order to examine the effect of 2Dow Chemical Health & Environment Research Laboratories, 1803 Building, Midland, ANXA4 on CRE- and CREB-(cAMP response element-binding protein) dependent Michigan MI 48674, United States transcription, a series of transient transfections using a luciferase reporter gene driven 3Global Isocyanates Ltd., Bridgewater House, Whitworth Street, Manchester, M1 6LT, by the CREB target promoter of the inducible cAMP early repressor (ICER) was carried Great Britain out. When the luciferase reporter construct was co-transfected with ANXA4, there was significant reduction of basal (DMSO) luciferase activity (control: 1±0.03, ANXA4: Methylene dianiline (MDA; CAS No. 101-77-9) is on the USEPA List 2 for endocrine 0.8±0.1; ANXA4 vs. control P disruption screen testing. In a yeast androgen screening (YAS) assay (in vitro assay on receptor binding or blocking) MDA revealed a significant anti-androgenic activity at a References concentration of 0.01 mM. To assess the biological relevance of this observation under Friedrich MW, Aramuni G, Mank M, Mackinnon JA, Griesbeck O. Imaging in vivo conditions the existing data from animal toxicity studies with MDA were reviewed CREBactivation in living cells. J Biol Chem. 2010 Jul 23;285(30):23285-95. Epub 2010 for indications of possible endocrine modulating (EM) effects (Weight-of-Evidence May 18. PubMed PMID: 20484048; PubMed Central PMCID: PMC2906321. approach). In addition, literature was searched for relevant studies on MDA and structural analogues using the American Chemical Society SciFinder client-server software.
174 Structural analogues indicated several drivers for EM activity, notably the diphenolic ring structure. However, in vitro receptor binding assays showed MDA had no androgenic or anti-estrogenic activitiy. One report described a weak estrogenic binding at 0.2 mM New aspects of pimecrolimus – A possible role in interferon signaling pathway MDA, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mM. Overall it is concluded that MDA does Hußner J.1, Sünwoldt J.1, Bien S.1, Begunk R.1, Gratz M.2, Kroemer H. K.1, Meyer zu 1 not bind to the estrogen receptor. Endocrine related effects of MDA were investigated in Schwabedissen H. several species and dose routes in unvalidated research-type protocols. Guideline 1Institut für Pharmakologie Ernst-Moritz-Arndt-Universität, Greifswald, Germany 2 subchronic and chronic toxicity studies with rodents revealed neither adverse organ Biotronik SE & Co. KG, Erlangen, Germany weight changes nor histopathological alterations of sex organs. The main systemic effects from the oral 13-week studies were body weight reduction and histopathological The implantation of Drug Eluting Stents (DES) after coronary artery intervention was an changes of the thyroid and bile duct (lowest LOAEL: 7.5 mg/kg bw). MDA was important step treating coronary artery disease. Indeed, cytotoxic compounds like carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. sirolimus used on DES are responsible for reduced in-stent restenosis in comparison Non-neoplastic effects in the thyroid (rats and mice) were observed (lowest LOAEL for with bare metal stents. Pimecrolimus, a potent anti-inflammatory drug has also been systemic toxicity: 9 mg/kg bw). MDA inhibits iodide oxidation which with concomitant investigated for its efficacy on DES. Preclinical studies in pigs revealed promising anti- decreased thyroid hormone formation is known to induce thyroid tumors. proliferative effects of pimecrolimus on neointima formation. However, in humans, pimecrolimus coated stents exerted adverse effects. In summary, in vitro screening tests revealed no consistent endocrine related effects of We hypothesize that compared to the highly active sirolimus, pimecrolimus may MDA. In vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. influence additional cellular processes leading to the worse outcome. In order to identify There were no histopathological changes of gonads and accessory sex organs and no those processes we conducted in vitro studies in human coronary artery endothelial cells evidence of sex hormone related EM. The evidence from the full dataset on MDA does (HCAEC) and smooth muscle cells (HCASMC). not indicate androgenic or estrogenic effects. Overall, based on a weight of evidence BrdU in vitro assays of HCAEC treated with pimecrolimus examined an IC50 value of assessment there are insufficient alerts for EM activity to suggest further testing should 6.787 µM [5.745 to 8.017] which is much higher than IC50 values of sirolimus described be done. in literature [0.1nM to 1nM]. GeneChip array analysis comparing gene expression in pimecrolimus and sirolimus treated HCAEC and HCASMC showed significant induction of several genes involved in interferon signaling. In detail, the expression of IFNβ activated genes like IRF9 and IFITM1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. Gene 177 regulatory effects were validated by real time PCR. Incubation with IFNβ itself showed similar effects in up regulation of genes involved in interferon signaling. Furthermore, we were able to demonstrate a significant increase of IFNβ secretion in HCASMC and The GTPase ARFRP1 controls the assembly of ApoA-I to and the lipidation of HCAEC treated with pimecrolimus. However, comparison of IFNβ and pimecrolimus on chylomicrons in the Golgi of intestinal epithelium 1 1 1 2 3 4 proliferation of HCAEC and HCASMC revealed different cellular responses. While IFNβ Jaschke A. , Chung B. , Hesse D. , Kluge R. , Heeren J. , Joost H. - G. , Schürmann 1 significantly decreased HCASMC and increased HCAEC proliferation, treatment with A. pimecrolimus lead to anti-proliferative effects on both cell types. 1Deutsches Institut für Ernährungsforschung Potsdam Rehbrücke Experimentelle In conclusion, pimecrolimus activates pathways involved in interferon signaling but Diabetologie, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany exerts different pharmacological effects, compared to the endogenous compound 2Deutsches Institut für Ernährungsforschung Potsdam Rehbrücke Max-Rubner- suggesting that INFβ secretion is not the major factor contributing to the difference in Laboratorium, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany pimecrolimus function. 3Universitätsklinikum Hamburg-Eppendorf Institut für Biochemie und Molekulare Zellbiologie, Martinistraße 52, 20246 Hamburg, Germany 4Deutsches Institut für Ernährungsforschung Potsdam Rehbrücke Pharmakologie, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany 175 Background: The uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the Identification of novel phospho acceptor sites of the mu opioid receptor mucosa, and subsequent synthesis and export of chylomicrons. The GTPase ADP- regulating receptor internalization ribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family and controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins Illing S., Just S., Doll C., Schulz S. which in turn bind several Rab-GTPases. The aim of the study was to define the role of Uniklinikum der FSU Jena Pharmakologie und Toxikologie, Drackendorfer Straße 1, ARFRP1 in intestinal nutrient absorption. 07747 Jena, Germany Methods: For the generation of intestine-specific null mutants Arfrp1flox/flox mice were crossed with mice expressing the Cre recombinase under the control of the intestine- The opioid alkaloid morphine is among the most potent clinically used analgesics. specific villin promoter (vil-Cre) and Arfrp1 expression was suppressed by siRNA in However, the clinical utility of morphine to treat chronic pain is limited by its rapid Caco2-cells. The phenotype in respect to lipid absorption and chylomicron production in development of tolerance and dependence. The stimulation of the mu opioid receptor the intestinal epithelium and in Caco2-cells, respectively, was analyzed. (MOR) with DAMGO or morphine results in different patterns of receptor phosphorylation Results: Arfrpvil-/- mice were viable but showed an early postnatal growth retardation and trafficking. So far, the three major phosphorylation sites of the mu opioid receptor (mean body weight of Arfrp1vil-/- was 43.3±5% lower than that of control mice at the age namely serine 363 (S363) threonine 370 (T370) and serine 375 (S375) have been of 28 days) Arfrp1vil-/- mice displayed reduced triglycerides, free fatty acids and glucose identified using phosphosite-specific antibodies. However, mutations of these three plasma levels indicating that the growth retardation is the result of a malabsorption. residues to alanine (S363A/T370A/S375A) did not prevent agonist-dependent Uptake of glucose and amino acids were unaffected by the deletion of Arfrp1. In internalization. In the present study, we have constructed a series of phosphorylation- contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in Arfrp1vil-/- deficient mutants showing that mutation of at least six residues mice. Arfrp1vil-/- enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed (S363A/T370A/S375A/T376A/T379A/T383A) was required to completely block agonist- fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride driven endocytosis. Consequently, we generated phosphosite-specific antibodies to content. In addition, while the release of apolipoprotein A-I (ApoA-I) was dramatically T376 and T379 which enabled us to provide direct evidence for agonist-dependent decreased ApoA-I accumulated in the Arfrp1vil-/- epithelium and was predominantly co- phosphorylation of these sites. Our analysis of time- and dose-dependent localized with Rab2. phosphorylation of MOR revealed that S375 was the primary phosphorylation site, S42
Conclusion: Our results demonstrate that the growth retardation of Arfrpvil-/- mice is a identification of Protease Inhibitor 16 (PI16). Here we report the generation of a mouse consequence of impaired intestinal fatty acid absorption. We suggest that ARFRP1 is line where PI16 can be deleted globally or conditionally using the Cre/LoxP system. required for the assembly of AopA-I to the chylomicrons and for the further lipidation of After electroporation of the Pi16floxneo targeting vector in embryonic stem cells and chylomicrons in the Golgi of intestinal epithelial cells. This finally leads to an secretion of injection into murine blastocysts we gained a mouse line that carried the targeted chylomicrons with a markedly reduced triglyceride content. modification of the Pi16 allele. Global PI16 deficiency (Pi16lox/lox) per se did not lead to a cardiac phenoytpe (HW/BW (mg/g): Pi16+/+ = 5.4 ± 0.2 (n = 6), Pi16lox/lox = 5.9 ± 0.7 (n = 4), P > 0.05; Fibrosis (%): Pi16+/+ = 3.3 ± 0.2 (n = 7), Pi16lox/lox = 3.9 ± 0.8 (n = 5), P > 0.05; Fractional Shortening (%): Pi16+/+ = 29.8 ± 0.7 (n = 7), Pi16lox/lox = 26.4 ± 2.5 (n=5), 178 P > 0.05). In addition we carried out an immunohistochemical analysis of PI16 expression using PI16-deficient mice as negative controls. PI16 localized to cardiac fibroblasts, to the epididymis and the trachea. In the failing heart we detected RhoA influences adhesion and spreading of cardiac fibroblasts via complex accumulation of PI16 preferentially in fibrotic areas. We are currently applying cardiac regulation of cytoskeletal proteins stress models to gain a broader understanding of the function of PI16 and its potential 1 2 1 1 1 1 as a therapeutic target molecule. Jatho A. , Zieseniss A. , Schenk K. , Ramba B. , Würtz C. , Lutz S. 1University of Göttingen Department of Pharmacology, Robert Koch Straße 40, 37077 Göttingen, Germany 2University of Göttingen Department of Cardiovascular Physiology, Humboldtallee 23, 37073 Göttingen, Germany 181
The monomeric GTPase RhoA is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. Therefore we intended to Enantioselective determination of R- and S-hyoscyamine in mammalian plasma analyze the effect of RhoA in cardiac fibroblasts by using a lentivirus-based knockdown and urine samples 1 2 2 1 approach. By doing so, we could show that the knockdown of this GTPase by about John H. , Eyer F. , Zilker T. , Thiermann H. 50% resulted in a massive change in cell morphology, but displayed no effect on cell 1Bundeswehr Institute of Pharmacology and Toxicology Analytical Chemistry, viability. The appearance of the cells in the 2D-culture changed from a fibroblast-typical Neuherbergstrasse 11, 80937 Munich, Germany stretched morphology with intracellular stress fibers to a more epithelial-like cell 2Second Medical Clinic, Technical University Munich Toxicological Department, morphology. By morphometrical analyses we demonstrate that fibroblasts with reduced Ismaninger Str. 22, 81675 Munich, Germany RhoA expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5- fold. Moreover, the depletion of RhoA significantly influences the adhesion velocity, as Atropine (Atr) is the racemic mixture of the tropane alkaloids S- and R-hyoscyamine within the first hour after cell detachment about 20% of the RhoA knockdown cells (hyo). S-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors reattach, but only 5% of the respective control cells. (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, Based on these findings, we investigated the distribution and composition of different whereas R-hyo does not (distomer). Atr is used for clinical intervention of poisoning with cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. We organophosphorus (OP) pesticides or nerve agents. Despite well known differences in found, that the amount of b-actin is not reduced in RhoA knockdown cells, however, the pharmacological behavior, individual pharmacokinetics of both Atr enantiomers have distribution is markedly changed. In these cells internal star-shape bundles of actin could rarely been addressed in the literature [1,2,3]. Therefore, we initially developed a non- be found instead of the commonly appearing stress fibers in control cells. In contrast, the chiral liquid-chromatography-electrospray tandem mass spectrometric method (LC-ESI cortical actin fibers, mainly consisting of g-actin, were not affected. In addition, smooth MS/MS) that allows quantification of Atr and additional natural and synthetic tropane muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in RhoA alkaloids from plasma after simple deproteinization [4]. To discriminate both Atr knockdown cells compared to control cells by 33%. This reduction might be responsible enantiomers the sample preparation step was expanded by an enzymatic pretreatment. for the more relaxed cell shape. Samples were incubated either with diluted human serum (not containing In summary, the knockdown of RhoA influences cell adhesion and the morphological atropinesterase, AtrE, procedure A) or with diluted rabbit serum (procedure B). Rabbit characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and serum contains AtrE (EC 3.1.1.10) which is suitable for stereospecific hydrolysis of S- viability. hyo into tropine and tropic acid while R-hyo remains unaffected. After sample precipitation, hyoscyamines were quantified by the LC-ESI MS/MS method. Following procedure A the concentration of total hyo and following procedure B remaining R-hyo were determined. S-hyo was calculated by the difference between these concentrations 179 [3]. The impact of potential matrix ingredients that may appear in samples from OP- poisoned patients under Atr therapy were evaluated (oximes, OP agents, carbamates) [3]. The assay was applied to diverse toxicological and pharmacological samples. Using site-specific fluorescence labeling to study uptake of Pasteurella multocida i) Measurement of natural S-hyo in an extract of atropa belladonna leaves as well as in toxin into eucaryotic cells plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to R-hyo occurred. ii) Analysis of plasma obtained Jehle D., Aktories K., Orth J. from an OP-poisoned female patient under Atr therapy revealed faster elimination of S- Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Abteilung 1, hyo when compared to R-hyo [3]. iii) In contrast, no enantioselective differences were Albertstraße 25, 79104 Freiburg, Germany obvious in healthy anaesthetized swine after intravenous injection of Atr [5]. Data indicate that the enzymatic enantioselective procedure represents a useful tool to Pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal characterize in vivo behavior of R- and S-hyo allowing to reveal individual kinetics. pharyngeal space of animals. P. multocida is of particular importance in the livestock management of pigs. Under special conditions infection of pigs with P. multocida leads References to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones [1] Aaltonen et al. Eur. J. Clin. Pharmacol. 26 (1984) 613-617 accompanied by a shortening and twisting of the snout. The causative agent of the [2] Siluk et al. J. Chromatogr. B 859 (2007) 213-221 atrophic rhinitis was found to be the bacterial protein toxin PMT (Pasteurella multocida [3] John et al. Anal. Chim. Acta 680 (2010) 32-40 toxin). After entering the cell the 146-kDa toxin activates various signal transduction [4] John et al. Anal. Bioanal. Chem. 396 (2010) 751-663 pathways by stimulating heterotrimeric G proteins of the Gαq, Gα13 and Gαi family. The [5] John et al. Drug Test. Anal. (in press) underlying mechanism of the activation of heterotrimeric G proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the G protein. The uptake of PMT into cells is not comprehensively understood. Therefore, we utilized a recently described technique, called “sortagging” (Popp MW et al. Nat Chem Biol. 2007;3: 707), to specifically couple fluorescence tags to the N- or C-terminus of PMT. 182 The enzyme sortase A (SrtA) from Staphylococcus aureus attaches proteins to the bacterial cell wall. The substrates are recognized by an LPXTG motif. SrtA cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. We RKIP protects from pressure overload induced heart failure 1 2 1 1 introduced these motifs into PMT to express SrtA-recognized toxin and coupled the toxin Kahlert K. , Hein L. , Lohse M. J. , Lorenz K. with fluorescence tags, respectively. Fluorescently labeled PMT was used to study the 1Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 uptake of the toxin into eucaryotic cells by laser scanning microscopy. Würzburg, Germany 2Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstraße 25, 79104 Freiburg i. Brsg., Germany
180 Failing hearts are unable to adequately supply the body with blood and oxygen. Common therapeutic strategies interfere with cardiac remodelling, reduce cardiac pre- and afterload or aim at direct improvement of cardiac contractility. Cardiac contractility is Characterization of PI16-deficient mice mainly controlled by β1-adrenergic receptors. Resensitization of b-adrenergic receptors by inhibition of G-protein coupled receptor kinase 2 (GRK2) is discussed as a potential Jentzsch C.1, Regn M.1, Braun A.2, Calzada-Wack J.3, Esposito I.3, Nieswandt B.2, 1,4 strategy to treat heart failure. We characterized Raf kinase inhibitor protein (RKIP) as a Engelhardt S. physiological inhibitor of GRK2 and found RKIP to increase contractility of neonatal 1 TUM Institut für Pharmakologie und Toxikologie, Biedersteiner Strasse 29, 80802 cardiomyocytes. The present study evaluated the role of RKIP in heart failure. We München, Germany assessed its effects on cardiac function in pressure overload induced heart failure and 2 Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine Vascular determined the expression patterns of RKIP in failing hearts of humans and mice. Medicine, Josef-Schneider-Straße 2, Building D15, 97080 Würzburg, Germany Transverse aortic contriction (TAC) was performed on 7-week-old C57/BL6 mice to 3 Helmholtz Zentrum München, German Research Center for Environmental Health induce heart failure by pressure overload of left ventricles. After three weeks of TAC, Institute for Pathology, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany echocardiography showed distinct signs of decreased cardiac function in wild-type mice. 4 Munich Heart Alliance, München, Germany Fractional shortening was reduced and left ventricular diameters were increased. Histological analyses revealed increased interstitial fibrosis, caspase- and TUNEL- Cells within the myocardium communicate by secreted factors and this has been assays indicated myocyte apoptosis. Western blot analysis showed significant suggested to contribute to cardiac remodeling. To identify novel factors secreted by the upregulation of RKIP expression in failing hearts compared to non-banded control myocardium, we have previously reported a genetic yeast screen which led to the hearts. Interestingly, this upregulation of RKIP expression was also detected in failing S43
human hearts and in samples of patients with aortic valve stenosis but not in healthy References control samples. [1] Kaiser, E., Kroll, C., Ernst, K., Schwan, C., Popoff, M. R., Fischer, G., Buchner, J., To assess the effects of RKIP overexpression on heart failure, we analysed heart Aktories, K. and Barth, H. (2011). Membrane translocation of binary actin-ADP- function and structure of RKIP transgenic mice and wild-type mice 3 weeks after TAC. ribosylating toxins from Clostridium difficile and Clostridium perfringens is facilitated by While left ventricular hypertrophy was increased to similar extents in wild-type and RKIP Cyclophilin A and Hsp90. Infect. Immun. 79, 3913-3921. trangenic mice, RKIP mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. In contrast to wild-type mice, the expression of the heart failure markers BNP and ANP was not upregulated in banded RKIP transgenic mice after 3 weeks of TAC. 185 Taken together, cardiac overexpression of RKIP prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. Therefore, increased RKIP expression may be an interesting target to prevent NADPH Oxidase 4 essentially contributes to nociceptive processing in detrimental effects from increased left ventricular pressure. neuropathic pain states
Kallenborn-Gerhardt W.1, Schröder K.2, Del Turco D.3, Lu R.1, Kynast K.1, Kosowski J.2, Niederberger E.1, Shah A. M.4, Brandes R. P.2, Geisslinger G.1, Schmidtko A.1 1pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology Goethe University, 183 Frankfurt am Main, Germany 2Institute of Cardiovascular Physiology/ZAFES Goethe University, Frankfurt am Main, Germany Simultaneous determination of antibiotic drugs in different matrix 3Institute of Clinical Neuroanatomy/Neuroscience Center Goethe University, Frankfurt Kahnt M., Oertel R., Kirch W. am Main, Germany TU Dresden Institut für klinische Pharmakologie, Fiedlerstraße 27, 01305 Dresden, 4Cardiovascular Division King’s College London British Heart Foundation Centre, Germany London SE5 9PJ, Great Britain
Background: This study describes the development of an analytical method for a Recent data indicate that reactive oxygen species (ROS) such as superoxide or simultaneous determination of antibiotic drugs in biological samples like plasma, urine hydrogen peroxide contribute to nociceptive processing during persistent pain, and that and tissue. Besides the LC/MS/MS method should be used for examinations of sewage. administration of ROS scavengers can effectively reduce the nociceptive behavior in The main focus of this study was the chromatographic separation. The most frequently different animal models of pain. Yet, little is known about the primary source of ROS prescribed antibiotic drugs Clarithromycin, Erythromycin, Clindamycin, Cefuroxim, production and how ROS contribute to nociceptive processing. NADPH oxidase (Nox) Doxycyclin, Amoxicillin, Levofloxacin, and Ciprofloxacin were selected for the screening enzymes produce ROS in different tissues including the central nervous system. Here, method. we investigated whether or not the Nox isoform Nox4 is involved in nociceptive Method: A relatively short operation time and a sufficient separation were reached by processing. Nox4 is expressed in a subset of nonpeptidergic nociceptors and myelinated column, eluent, and gradient optimization with POPLC (Phase Optimized Liquid dorsal root ganglia neurons. Neuropathic pain behavior induced by peripheral nerve Chromatography). In the first step columns with the five stationary phases C18 EPS 2, injury was attenuated in Nox4 deficient mice. Furthermore, persisting neuropathic pain C18 SH 2, Phenyl 2, CN 2, and C30 were used to determine the retention times of the behavior was also reduced after tamoxifen-induced deletion of Nox4 in adult transgenic drugs in an isocratic mode. The stationary phases, the column length and the retention mice, indicating that Nox4 essentially contributes to neuropathic pain signaling. Hence, times were fed in the POPLC software and the optimal column was calculated. This Nox4 might be a novel target for future pain therapy. column contained different stationary phases and was compared with customary columns. Supported by the Deutsche Forschungsgemeinschaft (DFG-SFB 815/A14). Results: Using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. That was not possible with the customary columns. With the optimal column the time of measurement was too long. Using a mobile phase gradient the measuring time could be reduced. 186 Discussion: With LC-MS/MS a complete chromatographic separation of all analytes is not necessary. But when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per Interactions of nanoparticles with an in vitro model of the alveolar-capillary barrier peak. Especially when using positive and negative ionization in the MS a good and the role of the alveolar macrophage separation is mostly necessary. To determine only the eight antibiotic drugs an Kasper J.1, Hermanns M. I.2, Bantz C.3, Utech S.3, Koshkina O.4, Maskos M.4,5, Unger optimized column is not necessary, but for a screening method with more than twenty 1 1 drugs the POPLC system is very helpful. R. E. , Kirkpatrick C. J. 1 University Medical Centre Institute of Pathology, Langenbeckstrasse 1, 55101 Mainz, Germany 2ikfe GmbH Institut für klinische Forschung und Endwicklung, Parcusstraße 8, 55116 Mainz, Germany 184 3Johannes Gutenberg University Mainz Institute of Physical Chemistry, Saarstr. 21, 55122 Mainz, Germany 4BAM Bundesanstalt für Materialforschung und –prüfung, Unter den Eichen 87, 12205 Cellular uptake of Clostridium difficile toxin CDT is facilitated by host cell factors Berlin, Germany 1 1 1 2 2 1 Ernst K. , Kaiser E. , Kroll C. , Schwan C. , Aktories K. , Barth H. 5IMM Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Str. 18-20, 55129 Mainz, 1Universitätsklinikum Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein- Germany Allee 11, 89081 Ulm, Germany 2Albert-Ludwigs-Universität Freiburg Institut für experimentelle und klinische Complex multicellular in vitro coculture models represent a promising tool regarding e. g. Pharmakologie und Toxikologie, Albertstr. 25, Otto-Krayer-Haus, 79104 Freiburg, cellular interactions with nanoparticles, since they more closely mimic the cellular Germany composition of the body. Therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (NCI H441) on top and Clostridium (C.) difficile infection can result in pseudomembranous colitis. The bacteria microvascular endothelial cells (ISO-HAS-1) on the bottom side of a filter-membrane to produce several exotoxins including toxins A and B, the causative agents of this study nanoparticle cytotoxicity and cellular uptake. With a coculture period of about 10 disease. Some hypervirulent strains of C. difficile additionally produce the binary ADP- days the cells achieve a more differentiated and polarized state and develop a tight ribosyltransferase CDT which is suggested to increase the severity of the disease. CDT barrier, which can be measured via TER (transepithelial electrical resistance). consists of the binding/translocation component CDTb which mediates the transport of Regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles the separate enzyme component CDTa into the cytosol of target cells, where CDTa (aSNPs, 30 nm) the coculture took up much less aSNPs than conventional ADP-ribosylates actin. We have investigated the uptake mechanism of CDT and in monocultures. Besides, we could not verify a specific uptake mechanism (e. g. clathrin-, particular the intracellular membrane translocation of CDTa. Our data indicate that CDT caveolae-mediated endocytosis) via immunofluorescence staining of the cells. However, requires acidification of the endosomal lumen for translocation of CDTa across we detected aSNPs incorporated in flotillin-1 and -2 labelled vesicles. Former studies endosomal membranes into the cytosol. Bafilomycin A1, an inhibitor of endosomal concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles acidification protects Vero (African Green Monkey kidney) cells from intoxication with (aSNPs, 30 nm) revealed that our coculture behaved much more robustly compared to CDT. Consistently, translocation of CDTa was observed when the acidic conditions of conventional monocultures. However, regarding inflammatory responses (e. g. sICAM, the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. Next, IL-8 increase) the coculture responded more sensitively than conventional we tested whether host cell factors are involved in membrane translocation of the toxin. monocultures. In a further development we added a third cell type, the alveolar Radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein macrophage (AM), to our coculture. Since AMs embody the front-line of alveolar defense Hsp90 as well as cyclosporine A, an inhibitor of cyclophilins delayed the intoxication of against inhaled pathogens and particles, they play a central role in regulating lung cells with CDT but not with toxins A and B [1]. This result was confirmed by analyzing immunity. As model we applied the human acute monocytic leukemia cell line, THP-1 the ADP-ribosylation status of actin from such cells in the presence or absence of the (prestimulated with 8 nM PMA for 4d) apically to the epithelial monolayer of the inhibitors. In addition, we excluded that the inhibitors of Hsp90 and cyclophilins have any coculture. Our preliminary studies concerning inflammatory responses of the triple- effect on receptor binding, endocytosis or enzyme activity of CDT. The data strongly culture (H441/ISO-HAS-1 with THP-1) revealed a higher sensitivity of the triple-culture suggest that the participation of Hsp90 and cyclophilin is crucial for translocation of compared to the double coculture. The triple-culture responded with an increased IL-8 CDTa into the cytosol. Comparable results were obtained for the related binary iota toxin release upon LPS or TNF-a stimulation. In conclusion, this triple-culture model offers a of C. perfringens. In vitro purified immobilized Hsp90 and cyclophilin A specifically bound promising prospect to mimic more closely realistic cell interactions with nanoparticles in to the enzyme components of CDT and iota toxins. In conclusion, the results imply a the distal lung. common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins. Our current investigations focus on the participation of FK506-binding proteins (FKBPs), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins.
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Ethanol in herbal medicinal products for children: No toxicological concerns Effect of an herbal preparation, STW 5, in a stress-induced model of functional Kelber O.1,2, Steinhoff B.3,2, Nauert C.4, Biller A.5, Adler M.6, Abdel-Aziz H.1, Okpanyi S. dyspepsia. N.1, Kraft K.7,2 Khayyal M. T.1, Abdel-Aziz H.2,3, Wadie W.1, Zaki H.1, Kelber O.3, Weiser D.3 1Steigerwald Arzneimittelwerk GmbH Scientific Department, Havelstr. 5, 64295 1Faculty of Pharmacy, Cairo University Pharmacology Department, Kasr-El-Aini Street, Darmstadt, Germany Kairo 11562, Egypt 2Kooperation Phytopharmaka Working Group “Efficacy and Safety”, Plittersdorfer Str. 2Institut für Pharmazeutische Chemie Pharmakologie, Hittorfstr.58, 48149 Münster, 218, 53173 Bonn, Germany Germany 3Bundesverband der Arzneimittel-Hersteller, Ubierstr. 71-73, 53173 Bonn, Germany 3Steigerwald Arzneimittelwerk GmbH Wissenschaftliche Abteilung, Havelstr. 5, 64295 4Cassella-med GmbH & Co. KG, Gereonsmühlengasse 1, 50670 Köln, Germany Darmstadt, Germany 5Dr. Loges + Co. GmbH, Schützenstr. 5, 21423 Winsen, Germany 6Institute for Integrative Medicine Siegen, Sohlbacher Str. 20, 57078 Siegen, Germany The involvement of psychological factors, especially stress, are known to play an important 7University of Rostock Chair of Naturopathy, Center of Internal Medicine, Ernst- role in functional gastrointestinal diseases (1,2) probably by affecting the brain-gut axis. Heydemann-Str. 6, 18057 Rostock, Germany Based on the good correlation between stress & functional dyspepsia (FD), many animal models for FD have been developed where animals are subjected to various kinds of Ethanol is a component of many herbal fluid preparations [1], since it is an excellent psychological stress either during the neonatal period or in adulthood. This stress was found extraction solvent for the phytochemical components of herbal drugs and contributes to to induce gastric motor dysfunction resembling symptoms of FD. Two models for stress- the stability of these medicines. Toxicological and pharmacokinetic evaluations [2] have induced FD were performed in order to choose the more adequate one for studying shown that the small amounts of ethanol applied with therapeutic doses are safe even in sensitivity changes of the fundus to various mediators as well as changes in some relevant children. Despite that these medicines have been used safely since many decades, they hormone levels in the blood for subsequently testing the efficacy of treatment with STW 5 (a have occasionally been subject of discussion in the public, triggered by the increasing 9 component herbal preparation of proven clinical efficacy in FD and in irritable bowel problem of recreational abuse of alcoholic beverages by children and young persons [3, syndrome (3,4) ). In one model, maternal separation (5) was performed on weanling rats 4]. Therefore, there is a growing need of a systematic evaluation of pharmacovigilance starting from postnatal day 2 for 3 h each day for 3 weeks. Rats were then allowed to data on these medicines. mature to an adult age. The other model was that of restraint stress (RS) (6,7). Adult For evaluating the experience gained from the therapeutic use of these medicines, 16 animals were restrained for 90 min/ day for 1 week. The animals of both models were pro- and retrospective studies with 10 herbal medicinal products containing ethanol at eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to doses of 40 to 240 mg per single dose, depending on the age group, have been carbachol, potassium chloride, serotonin and adrenaline was tested. Blood samples were analyzed, covering 49 816 patients of 0-12 years of age. In these studies, altogether 15 taken to assess levels of ghrelin, corticosterone releasing factor (CRF) and corticosterone. adverse drug effects have been described, none of which was attributable to the ethanol The sensitivity of fundus strips from restrained rats towards the agents tested, partly content of the medicines. representing autonomic responsiveness, was more depressed than those from maternally In a survey of the worldwide use of these and other 6 herbal medicinal products it was separated ones. Levels of ghrelin, CRF and corticosterone were also more elevated in the shown that during the past few years, more than 764 Mio daily doses have been sold, RS model. That model was therefore chosen to test the efficacy of STW 5 in restoring the corresponding to more than 33 Mio of patients (data obtained from manufacturers; deranged parameters. A group of animals received STW 5 orally once daily starting figures available partly from 1993 onwards, partly from 2003/4 onwards). From the treatment 1 week before exposing them to RS and continuing treatment for a further week packages sold in Germany in the years between 2005 and 2009, 48.1 Mio were during subjection to RS. STW 5 was effective in normalizing the depressed stomach fundus attributable to self-medication, 10.8 Mio to prescriptions reimbursed by health insurance responses exhibited by animals subjected to RS and to normalize to a large extent the (IMS, Frankfurt). As non prescription medicines are reimbursed in Germany only in deranged blood levels of ghrelin, CRF and corticosterone. The findings lend further children, at least the latter part of the prescriptions can be attributed mainly to children. evidence to the role of the brain-gut axis in FD and gives supportive evidence for the first All of these medicines are registered or licensed by regulatory authorities. Adverse time for the clinical usefulness of STW 5 in this condition. effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. References This set of data supports the conclusion drawn from the experience of a safe use over 1. Holtmann G et al. (2004) Dig. Dis. Sci. 49, 672–679. decades, i.e. that the ethanol content of herbal medicinal products does not give any 2. Holtmann G et al. (2004) Wien Med Wochenschr 154:528-534. causes for concern regarding their safety even in children. 3. Wagner H (2006) Phytomedicine 13:122-129. Dedication: This contribution is dedicated to Prof. Dr. Hilke Winterhoff, Institute for 4. Feinle-Bisset C and Andrews JM (2003) Curr. Treat. Options Gastroenterol. 6, 289– Pharmacology and Toxicology, University of Münster, who died on 9 May 2010. She has 297 initiated this work. 5. Cheung, CK et al (2010) Gastroenterology,138 : S-766 6. Zhang H et al. (2008) Phytomedicine, 15: 602-611. References 7. Zheng J et al. (2009) Am J Physiol Regul Integr Comp Physiol., 296: R1358- R1365. 1. Gundermann, K.-J. et al., 2010. Therapy of gastrointestinal diseases in the pediatric population: Review of clinical data for the phytomedicine STW 5. Neurogastroenterol Motil 22, S1, 69 2. Kelber, O. et al., 2008. Ethanol in herbal medicinal products for children. Pharm Ind; 190 70;1124-1128. 3. Gemeinsamer Bundesausschuss (G-BA), 2011. Beschluss über die Änderung der Arzneimittel-Richtlinie in § 8 und in Anlage III: Alkoholhaltige Arzneimittel zur oralen Effects of the sequence context on the repair excision of 8-oxo-7,8- Anwendung. Bundesanzeiger 21 vom 8. Februar 2011. dihydroguanine and the related inhibition of gene transcription 4. HMPC, 2010. Reflection paper on ethanol content in herbal medicinal products and traditional herbal medicinal products used in children. EMA/HMPC/85114/2008. Kitsera N., Allgayer J., Epe B., Khobta A. Johannes Gutenberg University of Mainz Institute of Pharmacy and Biochemistry, Staudingerweg 5, 55128 Mainz, Germany
There is good evidence that oxidative damage to DNA leads to down-regulation of 188 transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine DNA glycosylase (OGG1), which generates harmful repair intermediates [1,2]. We have recently shown that the magnitude of inhibition of Effects of prucalopride in 5-HT4a receptor overexpressing mice transcription of an EGFP reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8- 1 1 2 1 Keller N. , Gergs U. , Dhein S. , Neumann J. oxodG) varies between the opposing DNA strands of the gene [3]. 1Institute for Pharmacology and Toxicology Medical Faculty, Martin-Luther-University We now have addressed the question, to which extent the transcription inhibitory potential Halle-Wittenberg, Magdeburger Str. 4, 06112 Halle (Saale), Germany of 8-oxodG depends on its position in the gene and on the DNA microsequence 2Clinic for Cardiac Surgery Heart Centre, University of Leipzig, Strümpellstr. 39, 04289 surrounding the modified nucleobase. To investigate the effect of position, we produced Leipzig, Germany plasmid vectors containing single 8-oxodG or dG (underlined) in the second position of an AGC trinucleotide. Measurements of EGFP expression in transfected mammalian host cells Prucalopride was introduced as a new selective 5-HT4 receptor agonist that is approved showed that a single 8-oxodG caused a strong inhibition of gene transcription. The for treating obstipation. Whereas one could expect - due to the fact that 5-HT4 receptors magnitude of this effect for 8-oxodG situated in the transcribed DNA strand of the gene was are functionally expressed in the human heart - in clinical trials prucalopride did not the same as in the non-transcribed DNA strand. Similarly, there was no quantitative show cardiac effects. This is quite surprising because other 5-HT4 receptor agonists difference between the effects of 8-oxodG present in the 5′- versus 3′-UTR of the gene. The have been withdrawn from the market just for that reason. In this study we used results thus indicate that inhibition of transcription by this base modification does not depend prucalopride for in vitro studies with atrial preparations from transgenic (TG) mice with on the position in the gene. Further comparisons were done between the effects of 8-oxodG cardiac myocyte-specific overexpression of the human 5-HT4a receptor. Isolated nucleobases localised in the same DNA strand but within different sequence contexts. Gene electrically driven (1 Hz) left and spontaneous beating right atria of TG mice were expression analyses in the repair proficient host cells showed that the degree of inhibition of compared with those of wild type (WT) littermates. Moreover, we used isolated transcription caused by single 8-oxodG was dependent on the neighbouring nucleotides. electrically driven (1 Hz) human right atrial preparations from patients undergoing Among three tested sequence motifs, the minimal effect on the gene transcription was cardiac surgery. Finally GR113808, a 5-HT4 receptor antagonist, was added. found to correlate with a significantly less efficient base excision by the purified human Prucalopride exhibited a dose dependent positive inotropic effect in left atria and a OGG1 protein. The results thus support the initiatory role of OGG1 in the mechanism of positive chronotropic effect in right atria of TG mice with a logEC50 of -6.8 and -7.1, transcriptional repression. In addition, the finding of the effect of DNA sequence on the base respectively (p<0.05 vs. WT, n=3). In human atrial tissue prucalopride also acts as an excision activity of OGG1 suggests that repair rates of single base modifications in genome agonist, leading to an inotropic effect. All effects could be antagonized by 10 µM could also be heterogeneous. GR113808. We could demonstrate that prucalopride acts as an agonist at the 5-HT4 receptor in our transgenic mice model and also in human right atrial preparations. These References findings suggest that tachycardia and arrhythmias are possible side effects, which [1] A. Khobta, et al., DNA Repair (Amst) 8 (2009) 309-317. should be carefully looked for. [2] A. Khobta, et al., Nucleic Acids Res 38 (2010) 4285-4295. [3] N. Kitsera, et al., Nucleic Acids Res 39 (2011) 5926-5934.
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191 supplementation of the TRPC6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). In contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), Construction of plasmids for the investigation of position-specific effects of 8- and the TRPC6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion oxoG on transcription 21.3 ± 1.22 g). We conclude that a TRPC6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and Allgayer J., Kitsera N., Epe B., Khobta A. the resulting post transplant weight gain. The results indicate a role for TRPC6 in the Johannes Gutenberg University of Mainz Institute of Pharmacy and Biochemistry, regulation of pulmonary vessel permeability and support the concept that perfusion of Staudingerweg 5, 55128 Mainz, Germany donor lungs with TRPC6 blockers may prevent edema formation caused by ischemia- reperfusion injury shortly after lung transplantation. Interference of DNA base modifications with gene transcription is an important biological consequence of genotoxic damage [1]. An efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxoG) at a defined position in the EGFP gene in a plasmid vector was recently developed in our lab. The method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of 194 a synthetic oligonucleotide containing 8-oxoG in a chosen position. We further showed that this single lesion inhibits transcription after excision by OGG1 [2]. In order to determine to which extend the observed effect depends on the position of the Regulation of human inducible nitric oxide synthase (iNOS) expression by non- modified base in the gene, we constructed several new plasmid vectors which allow coding RNAs (ncRNAs) incorporation of the same DNA oligonucleotide containing 8-oxoG or G in different Kleinert H., Schmitz K., Koch K., Hahn S., Pautz A. positions in different DNA-strands. DNA sequence cassettes were designed to contain a Universitätsmedizin der Johannes Gutenberg-Universität Mainz Institut für 5′-CATTGCTTCGCTAGCACG nucleotide sequence in different orientations, which was Pharmakologie, Obere Zahlbacher Str. 67, 55101 Mainz, Germany flanked by two unidirectional BsrDI recognition sites (5′-GCAATGNN). Adapters containing the restriction sites for directional cloning into the 5′- or the 3′-UTR of the The transcriptome analyses of human cells showed that additionally to the 30.000 plasmid-borne EGFP gene were added. The produced plasmid vectors thus allow the protein coding sequences the human DNA codes for much more (450.000 ?) non-coding insertion of a single 8-oxoG in the same sequence context into opposing DNA strands in RNAs 1. Beside the ribosomal, and transfer RNAs (rRNA and tRNA) involved in the the 5’UTR and in the 3’UTR. Incorporation of 8-oxoG was accomplished with equal mechanism of translation there are also short (snRNA, soRNA, miRNA) and long non- efficiency for all plasmid vectors employed. The presence of the 8-oxoG was verified by coding RNAs (ncRNAs) implicated in regulation of gene expression (splicing, translation, conversion of plasmid DNA into the open circular form by E. coli Fpg DNA glycosylase. chromatin packaging etc.). The effect on transcription was quantitatively measured by flow cytometry after Matsui et al. described regulation of IL-1β-induced iNOS expression by an antisense transfection into HeLa cells. An inhibitory effect of single 8-oxoG on gene transcription RNA (as-3-UTR-RNA) in rat hepatocytes 2. A promoter located on the antisense strand was observed in all positions assessed in both DNA strands. 3’ to the last exon (exon 27) of the rat iNOS gene drives the expression of an as-RNA complementary to the 3’-UTR of the rat iNOS mRNA. Using different sense primers with References homology to the 3'-UTR sequences of the human iNOS gene for specific RT-PCR [1] A. Khobta, B. Epe, Mutat Res. (2011) doi:10.1016/j.mrfmmm.2011.07.014 reactions (detecting only an as-RNA) we were not able to detect such an as-3-UTR-RNA [2] N. Kitsera, et al., Nucleic Acids Res 39 (2011) 5926-5934. in human cells. In addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3’-flanking genomic sequences (as used by Matsui et al. in the rat system) of the human iNOS gene in front of a luciferase reportergene into DLD-1 cells revealed no promoter activity of these sequences. 192 Korneev et al. described the expression of a 2 kb anti-iNOS-ncRNA in different brain tumors and showed reciprocal expression to the iNOS mRNA in human embryonic stem cells 3. Analyzing the expression of this anti-iNOS-ncRNA in cytokine-treated DLD-1 Modulation of TRPV3 channel activity by cellular cholesterol content cells also showed a basal expression of this as-RNA and an enhancement of the expression by cytokine-treatment. Downregulation of the anti-iNOS-ncRNA by siRNAs Klein A., Tannert A., Schaefer M. reduced whereas overexpression enhanced CM-induced iNOS expression in human Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, DLD-1 cells. This indicates that this anti-iNOS-ncRNA regulates cytokine-induced iNOS Härtelstraße 16-18, 04107 Leipzig, Germany expression in a positive manner.
Keratinocytes are the major cell type of epidermis and are responsible for the formation References of an outer barrier, the statum corneum, to protect an organism against harmful 1 Huttenhofer, A. et al. Non-coding RNAs: hope or hype? Trends in genetics : TIG 21, environmental influences. For generation of this barrier, keratinocytes pass through a 289-297 (2005). complex differentiation program that is accompanied by synthesis of lipids, like 2 Matsui, K. et al. Natural antisense transcript stabilizes inducible nitric oxide synthase cholesterol and ceramides. Finally, the differentiation of keratinocytes leads to messenger RNA in rat hepatocytes. Hepatology 47, 686-697 (2008). apoptosis. Another function of keratinocytes is to sense environmental factors, some of 3 Korneev, S. A. et al. Novel noncoding antisense RNA transcribed from human anti- which are decoded by members of the transient receptor potential (TRP) ion channel NOS2A locus is differentially regulated during neuronal differentiation of embryonic stem family. TRPV3, for example, is predominantly expressed in keratinocytes, and decodes cells. RNA 14, 2030-2037 (2008). different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°C. Less is known about the influence of cholesterol on TRPV3 signalling. We modified the cholesterol content of HEK293 cells stably transfected with TRPV3 and performed FLIPR-based calcium measurements. These experiments revealed that cholesterol enrichment robustly potentiates TRPV3 by 195 sensitizing it to lower agonist concentrations. We verified these results with whole-cell patch-clamp measurements. In contrast, TRPV2, another heat-sensing channel, was not affected by cholesterol modification. Since former studies showed a defective formation Using directed evolution to improve functional expression of class B G-protein of epidermal barrier in TRPV3-/- mice, our results imply that a cholesterol-regulated coupled receptors TRPV3 signalling may contribute to the progression of differentiation or initiation of Klenk C., Scott D. J., Plückthun A. apoptosis of keratinocytes. Universität Zürich Institut für Biochemie, Winterthurerstrasse 190, 8057 Zürich, Switzerland
The class B of G-protein coupled receptors (GPCRs) comprises 15 peptide hormone- 193 binding receptors which regulate important endocrine and neuroendocrine functions of the human body. Several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, Inhibition of transient receptor potential channel 6 decreases post transplant which makes them attractive targets for drug therapy. To develop new compounds edema formation in an ex vivo transplantation model targeting these receptors a detailed understanding of the molecular structure is required 1 2 1 2 1 1 1 which has not been succeeded to date. Structural studies of proteins by X-ray Klein S. , Urban N. , Dhein S. , Schaefer M. , Bauer L. , Bittner H. , Mohr F. - W. crystallography or NMR spectroscopy generally require large and homogenous 1Herzzentrum Leipzig Klinik für Herzchirurgie, Strümpellstr. 39, 04289 Leipzig, Germany 2 quantities of protein. For GPCRs this prerequisite is difficult to achieve as the vast Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr. majority of GPCRs exhibits low endogenous expression and is very unstable in solution. 16-18, 04107 Leipzig, Germany Therefore, improved expression conditions are necessary for the efficient characterization of new GPCR structures. Here we present a method to optimize class B Ischemia-reperfusion injury causes severe problems in the early period after lung GPCRs for improved heterologous expression and increased thermostability by means transplantation. Since transient receptor potential (TRP) channels are important of directed evolution. Libraries of class B GPCRs were obtained by random mutagenesis regulators of vascular permeability and tone, we investigated the influence of a TRPC6 and were expressed in a heterologous expression system in which functional GPCR is blocker on pulmonary function after simulated transplantation, using an ex vivo model of targeted to the inner membrane of E. coli. Mutants that display increased receptor isolated perfused and ventilated rabbit lungs. To this end, heart-lung blocks were expression levels and ligand binding were selected by flow cytometry using fluorescently excised and mounted in an artificial thorax chamber. Negative pleural pressure labeled ligands. Repetitive cycles of randomization and selection allow to gradually ventilation was initiated, and lungs were perfused with albumin-containing Tyrode- increasing the level of protein expression and stability. With this evolutionary approach Solution (100 ml min-1). After equilibration in a stable perfusion and ventilation mode for ® key residues within the receptor sequence can be identified rapidly that are responsible 30 min, lungs were flush-perfused with Perfadex solution, followed by an ischemic for improved biophysical properties without affecting the pharmacological features of the storage for 4 h on ice. Subsequently, ventilation and perfusion were re-initiated to receptor. Such GPCR mutants will become a valuable tool on the way to express high simulate a transplantation situation. In the oxygenated group, pulmonary artery pO2 was quantities of stable receptor protein for subsequent structural studies. adjusted to 120 mmHg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery pO2 of 50 mmHg. Both transplantation conditions were conducted in the absence or in the presence of the TRPC6 blocker larixol acetate (5 µM). Hemodynamic and ventilatory parameters were continuously monitored. The weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. This weight gain was inhibited by S46
196 autophagy was observed several days (96 h) after TMZ treatment and MGMT proficient cells did not display significant autophagy. Thus, the data show that MGMT protects against TMZ induced autophagy, pointing to O6MeG as the critical lesion responsible for Use of a somatostatin receptor 2 phosphorylation motif to probe agonist-selective induction of autophagy. Using colon cancer cell lines proficient and deficient in MMR, we activation of other GPCRs show that MMR is required for TMZ-induced autophagy. Because DSBs, which emerge during the processing of O6MeG, are repaired preferably by homologous recombination Kliewer A. , Pöll F., Schulz S. (HR) LN-229 cells stably down-regulated for HR were tested for autophagy induction. Friedrich-Schiler Universität Jena, Universitätsklinikum Jena Institut für Pharmakologie & The data indicate that DSBs are involved in TMZ-induced autophagy. Because Toxikologie, Drackendorfer Str. 1, 07747 Jena, Germany autophagy following TMZ treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. Using an early stage autophagy Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma octreotide for the treatment of acromegaly, Cushing’s disease and carcinoid tumors. cells to TMZ-induced apoptosis. Taken together our data point out that TMZ induces Whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was autophagy in glioma cells and that autophagy protects glioma cells against TMZ-induced designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other apoptosis. O6MeG is the lesion responsible for autophagy induction. Furthermore, the somatostatin receptor subtypes. Somatostatin and octreotide stimulate the complete data also shows that MMR and DSBs are involved in the induction of autophagy after phosphorylation of at least six carboxyl-terminal serine and threonine residues namely TMZ treatment. Work was supported by BMBF (02NUK016). S341, S343, T353, T354, T356 and T359, which in turn leads to a robust endocytosis of the sst2 receptor. Surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of S341 and S343. Somatoprim and KE108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. Here, we performed an in vitro study comparing the 199 effects of pasireotide, somatoprim and KE108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. rAAV-mediated Gene Replacement Therapy Restores Vision and Delays Further somatostatin, octreotide, pasireotide, somatoprim and KE108 were tested for -/- functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2- Degeneration in the CNGB1 Mouse Model of Retinitis Pigmentosa 1 2 2 2 1 1 tail. This approach allows detection of receptor activation by phospho-specific sst2 Koch S. , Sothilingam V. , Garcia Garrido M. , Tanimoto N. , Becirovic E. , Fred K. , 2 1 2 1 antibodies. Compared to octreotide, somatoprim activates sst2 but has a higher activity Seeliger M. , Biel M. , Mühlfriedel R. , Michalakis S. on sst5. KE108 and pasireotide are partial agonists at the sst2 receptor. Pasireotide, 1Ludwig-Maximilians-Universität München Department of Pharmacy, Center for KE108 and somatoprim show comparable effects on sst5 receptor. However, none of Integrated Protein Science Munich (CIPSM), Butenandtstr. 5-13, 81377 München, these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 Germany receptor. 2Eberhard Karls Universität Tübingen Institute for Ophthalmic Research, Centre for Ophthalmology, Schleichstr. 4/3, 72076 Tübingen, Germany
Retinitis pigmentosa (RP) is a severe human retinal disease characterized by a 197 progressive degeneration of rod photoreceptors and a secondary loss of cone function. In most cases, RP finally leads to legal blindness. Mutations in the regulatory subunit of the rod cyclic nucleotide-gated (CNG) channel (CNGB1a) have been found in patients -/- Cadmium modulates AhR-associated gene expression in the rat intestine after suffering from RP. We used CNGB1-deficient (CNGB1 ) mice to establish a gene oral exposure replacement therapy as a potential treatment for RP by means of recombinant adeno- associated viral (rAAV) vectors. The packaging limitations of rAAV vectors required a Kluxen F. M.1,2, Höfer N.1,2, Becker E.2, Degen G. H.1, Diel P.2 1 capacity-optimized vector of the large CNGB1a cDNA (approx. 4 kb). Therefore, we IfADo – Leibniz Research Centre for Working Environment and Human Factors at the replaced regulatory elements within the expression cassette and used a short mouse TU Dortmund, Ardeystrasse 67, 44139 Dortmund, Germany 2 rhodopsin promoter element for rod-specific expression. After injection of therapeutic Sports University Cologne Institute of Cardiovascular Research and Sports Medicine, rAAVs (serotype 8) into the subretinal space of 2-week-old CNGB1-/- mice, we assessed Department of Molecular and Cellular Sports Medicine, Am Sportpark Müngersdorf 3, the restoration of vision by analyzing i) protein expression and localization, ii) retinal 50927 Köln, Germany function and morphology and iii) vision guided behavior. We found that treated CNGB1-/- mice expressed full-length CNGB1a and CNGA1, which were previously downregulated. Cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. We Both proteins co-localized in rod outer segments and formed regular CNG channel have recently identified cross-talk of estrogen receptor (ER) and aryl hydrocarbon complexes in the treated area of the CNGB1-/- retina. Using electroretinography (ERG) receptor (AhR) in the rat uterus where 17beta-estradiol (E2) and CdCl2 modulate AhR- we observed a distinct rescue of rod-driven responses. Moreover, CNGB1 replacement associated genes via ER after i.p. injection in a similar fashion (Kluxen et al., Arch significantly preserved outer segment morphology and delayed retinal degeneration. Toxicol DOI 10.1007/s00204-011-0787-x). However, the predominant route of exposure Finally, treated CNGB1-/- mice performed significantly better than untreated mice in a to cadmium in non-smokers is via diet. Moreover, uterus expresses mainly the receptor modified water maze task designed to test for rod-mediated, vision-guided behavior. In subtype ERalpha, whilst small intestine and colon express mainly ERbeta. Thus, we summary, this work provides a proof-of-concept for the treatment of rod channelopathy- now investigated by real-time RT-PCR the effects of cadmium (2mg/kg b.wt CdCl2 associated retinitis pigmentosa by rAAV-mediated gene replacement. (Cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (EE2)) on AhR- associated gene expression (i.e., Ahr, Cyp1a1, Gsta2, Nqo1) in the small intestine of rats after oral exposure (3 days gavage). The animals were also co-treated with Cd2 and pure anti-estrogen (2.5 mg/kg b.wt ZK191703 (ZK)) or EE2, to asseess whether ER- mediated processes are involved. We also measured Cyp1a1 mRNA expression in two 200 estrogen receptor negative colon cancer cell lines (HT-29 and CaCo2) treated for 5 days with CdCl2 (1µM) and E2 (0.01µM). The dose-dependency of cadmium induced AhR target gene modulation was studied in a second animal experiment, with administration A Testing Strategy for the Identification of Endocrine Disruptors of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt CdCl2 equivalent to 5, 50, Kolle S., Ramirez T., Kamp H., Buesen R., Flick B., Strauss V., van Ravenzwaay B. and 150 ppm) and EE2 (0.08 mg/kg b.wt) as steroid reference. BASF SE Experimental Toxicology and Ecology, Carl-Bosch-Str. 38, 67056 In summary we present two major results: The metalloestrogen cadmium modulates Ludwigshafen, Germany dose-dependently the AhR-associated gene expression in the intestine after oral exposure. Yet, since the cadmium induced modulation of AhR target genes was not Most endocrine disruptors interact with hormone receptors or steroid biosynthesis and antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in metabolism, thereby modifying the physiological function of endogenous hormones. ER-negative colon cells in vitro, we propose that ER-independent mechanisms might Here, we present an alternative testing paradigm for detection of endocrine modes of play a role in this effect. actions that replace and reduce animal testing through refinement. Receptor mediated This project has been supported by funds for the DFG graduate collegue 1427 endocrine effects were assessed using the yeast based receptor mediated transcriptional activation YES/YAS assays and effects on steroid hormone biosynthesis were assessed using the human cell line H295R in the steroidogenesis assay. In our testing paradigm we propose to complement the in vitro assays with a single in vivo 198 repeated dose study in which plasma samples are analyzed for their metabolome profile. The combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources Temozolomide induces autophagy in glioma cells that protects against apoptosis and allows for a sound assessment of the endocrine disruption potential of compounds. Thus, this proposal constitutes a potentially attractive alternative to EPA’s Endocrine Knizhnik A., Roos W. P., Kaina B. Disruptor Screening Program. Data on 14 reference substances for which the in vitro Medical Center of the Johannes Gutenberg University Mainz Institute for Toxicology, YES/YAS and steroidogenesis assays and the in vivo metabolome analysis were Obere Zahlbacher Strasse 67, 55131 Mainz, Germany performed to assess their putative endocrine mode of action is presented here.
Autophagy is a process induced by nutrient limitation and cellular stress, which governs degradation of long-lived proteins and whole organelles, thereby maintaining a balance between synthesis, degradation and recycling of cellular components. Alkylating agents, in particular temozolomide (TMZ), are used as first line chemotherapy of gliomas. TMZ induces several DNA adducts, among which O6-methylguanine (O6MeG) is the most cytotoxic lesion. If not repaired by O6-methylguanine-DNA methyltransferase (MGMT), O6MeG/T mismatch is recognized by the mismatch repair system (MMR) that performs futile repair cycles. During this process secondary lesions (i.e. DNA single-strand brakes) are formed, which block DNA replication in the next replication cycle, leading to DNA double-strand breaks (DSBs). These DSBs eventually signal to apoptosis and other genotoxic endpoints. Here, we wished to address the question whether autophagy is part of the cellular response triggered by O6MeG. We also assessed whether autophagy influences apoptosis induced by TMZ in glioma cells. We show that TMZ induces autophagy in U-87 MG and LN-229 glioma cell lines. The maximum amount of S47
201 se. Modern toxicology is based on the insight into toxic pathways. For nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) In vitro skin irritation and corrosion testing serving different regulatory and their uptake, distribution and clearance. The use of dermal penetration studies in classification schemes vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3]. In vitro methods for specific effects (such as inflammation, pulmonary toxicity, Kolle S.1, Mehling A.2, van Ravenzwaay B.1, Landsiedel R.1 1 sensitization) are currently awaiting validation (for both chemicals/molecules as well as BASF SE Experimental Toxicology and Ecology, Carl-Bosch-Str. 38, 67056 nanomaterials). In the meantime alternative short-term in vivo studies with optimized Ludwigshafen, Germany 2 biological readouts can deliver information on the toxic pathways as well as the BASF Personal Care and Nutrition GmbH Product Safety, Henkel Str. 67, 40589 biokinetics and dose-response relation of nanomaterials in the body. A short-term Düsseldorf, Germany inhalation test for nanomaterials has already been used successfully [4]. Testing strategies based on those methods engage less animals and provides more significant Skin corrosion or irritation refers to irreversible or reversible tissue damage to the skin data than classical testing. Moreover data from these methods will serve as a following the application of a test substance. Traditionally, this was tested using the in benchmark and a validation for the in vitro models under evaluation. vivo Draize skin irritation test. Due to animal welfare considerations and regulatory provisions such as the European Cosmetics Regulation, in vitro tests using different References reconstructed human skin models were developed and have now gained regulatory [1] Schulze Ch, Kroll A, Lehr CM, Schäfer UF, Becker K, Schnekenburger J, Schulze acceptance (OECD TG 431 and OECD 439). Depending on the up-front prediction of the Isfort Ch, Landsiedel R, Wohlleben W (2008): Not ready to use - overcoming pitfalls severity of the effects (skin corrosive, skin irritant, or nonirritant) by expert judgments or when dispersing nanoparticles in physiological media Nanotoxicology 2: 51-61 structure activity relationship, different testing schemes can be applied, e.g. the top- [2] Landsiedel R, Schulz M, Kapp MD, Oesch F: "Genotoxicity Investigations on down or bottom-up strategies proposed for skin irritation testing by COLIPA (MacFarlane Nanomaterials: Methods, Preparation and Characterization of Test Material, Potential et al., 2009). We have used the EpiDerm™ (Mattek, USA) and compared it to the results Artifacts and Limitations - Many Questions, Some Answers", Mutation Research 681: of Draize skin irritation test in vivo which had been performed due to regulatory 241-258 requirements. Over 100 substances have been tested including a wide range of different [3] Gamer AO, Leibold E, van Ravenzwaay B (2006). The in vitro absoprtion of microfine chemical classes. The test results were assessed according to the UN-GHS, EU-CLP, zinc oxide and titanium dioxide through porcine skin, Toxicol In Vitro 20:301 Brazilian and US EPA guidelines. We could confirm a good correlation of the in vitro [4] Ma-Hock L, Treumann S, Strauss V, Brill S, Luizi F, Mertler M, Wiench K, Gamer AO, results with the in vivo data for corrosive and non-irritating substances. Irritating van Ravenzwaay B, Landsiedel R (2009). Inhalation toxicity of multi-wall carbon substances were occasionally incorrectly classified by not more than one class off the in nanotubes in rats exposed for 3 months. Toxicol. Sci. 112:468 vivo-based classifcation. Moreover, the in vitro data could serve all four different regulatory classification schemes.
204 202 Contribution of nanoparticles to neutrophil-induced DNA damage in human intestinal Caco-2 cells. Predicting Eye Irritation of Agrochemical Formulations according to different Classification Schemes by in vitro methods (Bovine Corneal Opacity and Einfluss von Nanopartikeln auf Neutrophil-induzierte DNA Schäden in humanen Permeability and EpiOcular Eye Irritation Test) intestinalen Caco-2 Zellen. Kolle S.1, Knieriem T.2, Mayer W.2, Buechse A.3, Rey Moreno M. - C.1, van Cott A.4, van 1 1 2 1 1 1 1 Kolling J. , Gerloff K. , Förster I. , Albrecht C. , Schins R. Ravenzwaay B. , Landsiedel R. 1 1 Leibniz Research Institute for Environmental Medicine Particle Research, Auf'm BASF SE Experimental Toxicology and Ecology, Carl-Bosch-Str. 38, 67056 Hennekamp 50, 40225 Düsseldorf, Germany Ludwigshafen, Germany 2 2 Leibniz Research Institute for Environmental Medicine Molecular Immunology, Auf'm BASF SE Agricultural Products - Formulation Development, Carl-Bosch-Str. 38, 67056 Hennekamp 50, 40225 Düsseldorf, Germany Ludwigshafen, Germany 3 BASF SE Global Research Crop Protection - Data Management and Biometrics, Carl- Nanoparticles (NP) are increasingly used in various field of industry which necessitates Bosch-Str. 38, 67056 Ludwigshafen, Germany 4 evaluation of their safety. Also in the food industry, NPs have gained strong interest, for BASF Corporation Agricultural Products - Global Product Safety & Registration, 26 example as food additives or to improve food packing. However, the potential risks of Davis Drive, Raleigh, NC 27709 United States ingested NP have been rarely investigated. Inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of The bovine corneal opacity and permeability (BCOP) test has been adopted by OECD adverse health effects of toxic NP. In the present study, a co-incubation model of for the identification of corrosive and severe ocular irritants (GHS category 1) for single human polymorphonuclear neutrohils (PMNs) and Caco-2 human intestinal epithelial component substances and multi-component formulations. Eye irritation tests (EIT) cells was used as a model to address potential genotoxic effect of NP during intestinal using human reconstructed tissue models (such as EpiOcular) have been described to inflammation. Oxidative DNA damage induction (measured by the Fpg-modified comet predict ocular non-irritants (GHS no category). Thus the ultimate repaltement of the assay) was induced in the Caco-2 cells by activated PMN and this effect increased with Draize rabbit eye irritation test (OECD TG 405) by a combined or tiered testing strategy increasing PMN to Caco-2 cell ratio. The crucial involvement of the phagocyte NADPH could be possible. oxidase complex could be demonstrated using treatment of Caco-2 cells with bone The purpose of this study was to evaluate whether the BCOP with additional corneal marrow-derived PMN from NADPH oxidase deficient mice. DNA damage by PMN as histology together with the EIT could be used to predict eye irritancy of agrochemical well as H2O2 was increased in buthionine sulphoximine (BSO) pre-treated Caco-2 cells, formulations according to different classification schemes including UN GHS and EPA illustrating the importance of the cellular glutathione (GSH) status in these target cells. systems. We have performed the BCOP (plus histology) and the EIT of 50 agrochemical GSH depletion in Caco-2 cells could also be shown upon treatment with various types of formulations for which in vivo eye irritation data were already available (for registration NP. Our data suggests that ingested NP may increase the susceptibility of the colon purposes). Using the OECD TG guideline evaluation scheme for opacity and mucosa to genetic damage during the occurrence of intestinal inflammation. permeability in the BCOP was not predictive for the agrochemical formulations assessed Supported by the DFG-Graduate College GRK1427 here, while corneal histology grades and the EpiOcular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. 205
203 Impact of xenobiotic metabolism of okadaic acid on viability and oxidative stress status in HepG2 cells Kolrep F., Hessel S., Ehlers A., Lampen A. The Use of Alternative Methods for Toxicity Testing of Nanomaterials 1 1 1 2,1 1 Bundesinstitut für Risikobewertung Lebensmittelsicherheit, Max-Dohrn-Str. 8-10, 10589 Kolle S. , Ramirez-Hernandez T. , Vogel S. , Wohlleben W. , Lan M. - H. , van Berlin, Germany Ravenzwaay B.3, Fabian E.3, Oesch F.4, Schulz M.1, Landsiedel R.1 1 BASF SE Experimental Toxicology and Ecology, 67063 Ludwigshafen am Rhein, The ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic Germany 2 acid (OA) is responsible for diarrhetic shellfish poisoning. It is recently known that both BASF SE Polymer Physics, 67063 Ludwigshafen am Rhein, Germany the rat and the human hepatic cytochrome P450 monooxygenases (CYP) are able to 3BASF SE Product Safety, GV/TB, 67063 Ludwigshafen, Germany 4 metabolize this toxin. Currently, there is a lack of data about the toxicity and mode of JG Universität Mainz Toxikologie, Mainz, Germany action of OA after xenobiotic metabolism. The aim of our study was the measurement of the toxicity and oxidative stress status in HepG2 cells incubated with OA in the absence Nanomaterials offers extraordinary opportunities. The nano-structure can change the and presence of S9 mix. physical and chemical properties, and often also alters the biological effects. Hence the Pure OA, as well as OA pre-activated with liver homogenisates (S9 mix) were used to toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no treat human HepG2 cells that have nearly undetectable levels of functional CYP but new quality of a general nano-specific toxic effect has been observed. Therefore the express phase II enzymes. The experiments were performed with both human and rat established testing methods are generally suitable. It is, however, difficult to apply the S9 fraction plus cofactors of phase I enzymes. The cell viability was measured after 4 h nanomaterials to in vitro test systems, since it is the nature of these materials to change using MTT-test and xCelligence real time cell monitoring system. Furthermore, levels of their surface properties and agglomeration stateand the uptake and distribution in the intracellular reactive oxygen species (ROS) were detected by 2´,7´-dichlorofluorescein body may differ from their larger-scale materials. diacetate and additionally by measuring the intracellular glutathione content. While the methods for topical effects may be used for nanomaterials without further In the presence of both human and rat S9 mix OA showed a higher toxicity than the modification, the in vitro methods for genotoxicity testing require the dispersion in culture parental substance. OA pre-incubated in rat S9 mix was toxic at 75 nM OA. Strong media. The use of reproducible and well-documented dispersion protocols andthe effects could be observed when OA was pre-activated with human S9-mix at a characterization of its particle size distribution is de rigueur .[1]. For many concentration of 50 nM OA. Pure OA was non-toxic in that concentration range. We nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per S48
could also detect an increase of oxidative stress in HepG2 cells treated with OA in the 208 presence of all investigated S9-Mix. These results suggest that OA is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of Modulation of cGMP signals by phosphodiesterases in smooth muscle cells intracellular ROS in HepG2 cells. Krawutschke C., Koesling D., Russwurm M. Ruhr-Universität Bochum Pharmakologie und Toxikologie, Universitätsstrasse 150, 44780 Bochum, Germany
206 Within the cardiovascular system, cGMP mediates smooth muscle relaxation and inhibition of platelet aggregation. cGMP is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric Ballast Water Treatment – Emerging Health Risks oxide (NO), respectively. Besides the cGMP-forming enzymes, the cGMP-degrading Werschkun B., Banerji S., Krätke R. phosphodiesterases strongly determine amplitude and shape of cGMP signals. Bundesinstitut für Risikobewertung, Max-Dohrn-Strasse 10, 10589 Berlin, Germany In vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cGMP degradation: PDE5, the cGMP-specific phosphodiesterase is The introduction of invasive marine species into new environments by ships’ ballast activated directly by cGMP binding to its GAF domains; this activation if further stabilized water, via ships’ hulls and other vectors has severe impacts on the oceans. In 2004 the by cGMP-dependent protein kinase-mediated phosphorylation. PDE1, the International Maritime Organisation (IMO) launched the International Convention for the Ca2+/Calmodulin-stimulated PDE constitutes the majority of cGMP-hydrolyzing activity Control and Management of Ships Ballast Water and Sediments which requires ballast in smooth muscle cells at least in the presence of high intracellular Ca2+ concentrations. water to be treated in order to eliminate alien aquatic species. Ballast water treatment And lastly PDE3, the cGMP-inhibited PDE displays some cGMP-degrading activity, may include mechanical, physical or chemical measures. Any ballast water management although cGMP binding to its catalytic domain is primarily thought to inhibit the cAMP- system using active substances needs IMO approval. Therefore identification of active degrading activity of PDE3. substances, relevant chemicals and submission of specified datasets on their physical, cGMP signals measurable in radioimmunoassays require stimulation with cGMP- chemical and toxicological properties is required in order to assess the safety for the increasing vasodilator concentrations that are orders of magnitudes higher than those aquatic environment and for human health. The BfR is the German federal agency required for relaxation. Thus, we developed fluorescent sensors for real-time responsible for health risk assessment and has been involved in more than twenty measurement of cGMP signals in single cells. By using these indicators, we analyzed assessment and approval processes so far. the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cGMP signals elicited by physiologically relevant vasodilator The majority of IMO approved systems are based on oxidative principles such as concentrations. chlorination and ozonation. These methods can generate disinfection by-products (DBPs), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. The formation of DBPs is well known from the disinfection of drinking water. Some DBPs are regulated under drinking 209 water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. The formation of DBPs may vary significantly depending on the treatment system as well as on environmental parameters like Functional interaction of SCAI with the tumor suppressing SWI/SNF chromatin temperature, pH and composition of the organic matter within the aquatic environment. remodeling complex In sea water, sources for DBP formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. Kreßner C., Grosse R., Brandt D. Institut für Pharmakologie, Karl-von-Frisch-Straße 1, 35032 Marburg, Germany In order to address possible health and environmental risks from DBPs formed during ballast water treatment a conference on emerging risks from ballast water treatment was Invasive behavior of cancer cells is a prerequisite for metastasis, which is the leading held at BfR in October 2011. Here we summarise the main conference findings and cause of death from cancer. The gain of invasive capacities is attributed to the identify areas for future research. Two presentations corroborated that significant accumulation of genetic alterations and subsequent changes in gene expression. amounts of DBPs can be formed in sea water and a presentation on the toxicological Recently, we characterized SCAI (Suppressor of Cancer Cell Invasion) as a novel properties of DBPs pointed out that many have genotoxic properties. Accordingly, the transcriptional modulator which inhibits SRF gene transcription by interfering with the determination of DBP species and generated concentrations under different ballast myocardin-related transcription factor MAL. Depletion of SCAI causes an increase in ß1- water treatment conditions was seen as a mayor task. integrin gene transcription which leads to an increased invasive migration of tumor cells. The general accessibility of DNA to transcription factors relies on dynamic changes in Different approaches for health and environmental risk assessments were also chromatin architecture. During this, the SWI/SNF chromatin remodeling complex uses discussed. Appropriate human exposure scenarios and methods for exposure the ATPase activity of its key component Brahma (BRM) to facilitate nucleosome sliding. assessment, taking into account common approaches used in risk assessment were Notably, loss of function of BRM has been linked to cancer development and presented during the conference. A suitable approach based on derived PEC-values for progression, suggesting that this complex functions as a tumor suppressor. Here we exposure quantification was proposed in order to improve the procedure available for show a physical and functional interaction between SCAI and the chromatin remodeler risk assessment of chemical agents used for ballast water treatment. BRM in human cancer cells. Reporter gene analysis show that SCAI requires the SWI/SNF complex to supress the activity of SRF. Consistent with this, expression of an ATPase-deficient BRM mutant is able to relief the inhibitory effect of SCAI on MAL/SRF. Gene expression data points towards an overlapping pattern of SCAI and BRM controlled target genes. Moreover, loss of BRM phenocopies the effects of SCAI on 207 invasive cellular migration into 3-dimensional Matrigel. Based on our results, we propose cooperative functions of BRM and SCAI in the suppression of an invasive phenotype.
Agonist-Selective Signaling of µ-Opioid Receptors in T Lymphocytes Kraus J., Börner C., Lanciotti S., Koch T., Höllt V. Inst. für Pharmakologie und Toxikologie, Leipzigerstr. 44, 39120 Magdeburg, Germany 210
Opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. In addition to their central effects, opioids modulate a great variety of immune Regulation and subcellular localization of a truncated hyaluronic acid synthase effector cell functions, which may result in unwanted side effects during opioid treatment. isoenzyme The effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of G protein coupled receptors. Kretschmer I., Dai G., Grandoch M., Fischer J. W. Recent data support the concept that G protein coupled receptors function as dynamic Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der entities, which may occupy multiple conformations and activate multiple signaling Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany pathways in a ligand-dependent manner. Consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonist- Hyaluronan (HA) is a major component of extracellular matrices and is thought to control selective signaling". cellular phenotypes such as proliferation and migration. Therefore, HA synthesis may Little is known about agonist-selective signaling of µ-opioid receptors in immune effector play an important role in the pathophysiology of atherosclerosis. There are three major cells. In a first attempt to understand if and why such different profiles among different HA-synthase isoenzymes (HAS1-3). The HAS3 gene is alternatively spliced. HAS3 opioids occur we investigated effects of different opioids in human Jurkat T cells. We transcript variant 2 (HAS3v2) encodes the smallest HAS isoenzyme which has a report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and beta- different C-terminus and contains only two transmembrane domains compared to HAS3 endorphin induce internalization of a µ-opioid receptor-green fluorescent reporter transcript variant 1. The aim of the present study was to investigate whether HAS3v2 is construct, whereas morphine and buprenorphine did not induce internalization. The expressed by vascular cells, how it is regulated and where it is localized in cells and internalization was dependent on p38 MAPK and phospholipase D2. In line with this, we whether it indeed synthesizes HA. observed marked phosphorylation of p38 MAPK and activation of phospholipase D2 HAS3v2 mRNA expression was monitored by quantitative real-time RT-PCR. Protein induced by the internalizing opioids, but no or little such activity by morphine and expression was determined by Western blotting using a polyclonal antibody that was buprenorphine. As a physiological result, fentanyl, methadone, loperamide and beta- raised in rabbit. An N-terminal EYFP-HAS3v2 fusion protein and a DDK-tagged HAS3v2 endorphin treatment of primary human T cells and Jurkat T cells resulted in a strong, up construct were expressed for subcellular localization studies and co-immunoprecipitation to 100 fold induction of IL-4, which was dependent on p38. In contrast, morphine and (Co-IP). Endogenous HAS3v2 mRNA was expressed in both vascular smooth muscle buprenorphine only showed a weak, approximately one order of magnitude lower cells (VSMC) and endothelial cells. Furthermore, Western blotting revealed HAS3v2 induction of IL-4. protein expression in VSMC and platelets. In VSMC HAS3v2 mRNA expression was By inducing IL-4 opioids significantly modulate the T helper cell balance into the type 2 strongly up-regulated in response to interleukin-1β (IL-1β, 10 ng/ml) whereas stimulation direction, which influences various immune responses, e. g. the antiviral, T helper cell with interleukin-10 (10 ng/ml), platelet-derived growth factor-BB (10 ng/ml), transforming type 1-mediated response. Considering the vital necessity of opioid use in humans, it is growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) an intriguing goal to identify analgetically feasible opioids that have little or no had no effect. Transfection of HEK cells with EYFP-HAS3v2 fusion protein revealed immunosuppressive or -modulatory effects. localization to the endoplasmic reticulum but not to the plasma membrane. Furthermore, Co-IP experiments showed that tagged HAS3v2 proteins were precipitated together S49
suggesting formation of multimeric HAS3v2 complexes. Transfection of HAS3v2 did not 213 cause increased secretion of HA into the cell culture supernatant in HEK cells. In conclusion, HAS3v2 is present in vascular cells and responds to inflammatory cytokines such as IL-1ß. Because of the intracellular localization and the lack of HA Engineering of modified elastin-like proteins to enhance reendothelialization of secretion in HAS3v2 transfected cells, HAS3v2 may serve intracellular functions apart coronary stents from HA synthesis. 1 2,2 3 3 4 4 Kurtbay G. , Boeck M. , Beyer S. , Schweder T. , Hammer E. , Schmidt F. , Sternberg K.2, Bornscheuer U.5, Kroemer H. K.1, Petersen S.2, Meyer zu Schwabedissen H.1 1Ernst Moritz Arndt University of Greifswald Department of Pharmacology, Felix- Hausdorff-Strasse 3, 17489 Greifswald, Germany 211 2University of Rostock Institute for Biomedical Technology, Friedrich-Barnewitz-Straße 4, 18119 Rostock, Germany 3Ernst Moritz Arndt University of Greifswald Institute of Pharmacy, Pharmaceutical The tubulin antagonist pretubulysin shows strong vascular-disrupting properties Biotechnology, Felix-Hausdorff-Strasse 3, 17489 Greifswald, Germany in vitro 4Ernst Moritz Arndt University of Greifswald Institute of Functional Genomics, Friedrich- 1 2 1 1 2 1 Kretzschmann V. , Ullrich A. , Zahler S. , Vollmar A. , Kazmaier U. , Fürst R. Ludwig-Jahnstr. 15a, 17489 Greifswald, Germany 1LMU München Department Pharmazie, Pharmazeutische Biologie, Butenandtstr. 5-13, 5Ernst Moritz Arndt University of Greifswald 5Institute of Biochemistry, Dept. of 81377 München, Germany Biotechnology & Enzyme Catalysis, Felix-Hausdorff-Strasse 3, 17489 Greifswald, 2Universität des Saarlandes Institut für Organische Chemie, Postfach 151150, 66041 Germany Saarbrücken, Germany Introduction: Vascular-disrupting agents (VDAs) have emerged as a novel promising class of anti- Drug-eluting stents (DES) are commonly used in the treatment of acute artery occlusion. cancer therapeutics. We aimed to elucidate the vascular-disrupting potential of the new However, even if released cytotoxic drugs reduced neointimal proliferation significantly tubulin-depolymerizing agent pretubulysin (PT). PT is a synthetically accessible there is still the risk of in-stent thrombosis. It is presumed that this is due to reduced re- precursor of tubulysin, a myxobacterial compound that has recently been found to exert endothelialization. It has been suggested that coating the stent with biomolecules may potent tumor cell death-inducing properties. In this study, we focused on the action of PT provide a new approach to circumvent the lack of healing of the endothelial layer. One on endothelial cells in vitro in comparison to the lead VDA combretastatin A-4 phosphate approach would be the use of biomolecular signals, such as (poly)peptides and growth (CA4P). factors. RGD and REDV are peptide motifs, known to enhance cell attachment and We investigated the effects of PT on key features of vascular disruption using human spreading. dermal microvascular (HMEC-1) and human umbilical vein endothelial cells (HUVECs): Aim: (i) PT induced a concentration-dependent disassembly of established endothelial tubes The aim of our study was to evaluate the efficacy of proteins, derived from elastin-like on Matrigel in vitro as well as in an ex vivo aortic ring model. (ii) PT rapidly increased proteins (ELP) and artificial modified by incorporating with the amino acid motifs endothelial permeability within 1 h, as measured by impedance sensing (xCELLigence, RGD,REDV and P15, in terms of endothelial healing on stents and other cardiovascular Roche) and Transwell® assays. (iii) Moreover, using immunocytochemistry and confocal devices. microscopy, we found that PT leads to a disruption of microtubules and cell junctions Results: (VE-cadherin, claudin-5), and to a strong induction of F-actin stress fibers. Interestingly, In this work, we generated vectors encoding for different biopolymers consisting of PT showed in all assays a potency comparable to CA4P. Regarding cytotoxicity, PT- various bioactive signal molecule sequences. The peptides, e.g. based on the elastin- treated cells did neither undergo apoptosis (analysis of subdiploid DNA content) or like matrix (VPGIG)2-VPGKG-(VPGIG)2, were synthesized using heterologous necrosis (PI -staining) within 24 h, nor reduce their metabolic activity (CellTiter Blue® expression. After optimizing culture conditions and extraction procedures their biological assay) after 1 or 24 h. HUVECs were even able to reassume their normal morphology activity was assessed using human umbilical vein endothelial cells (HUVEC). Elastin like after washing out of PT/CA4P. proteins with differently incorporated bioactive signals (REDV, RGD and a small P15 In summary, PT exhibits the typical features of a microtubule-targeting VDA: it disrupts peptide) were linked covalently via carbodiimide coupling to poly(L-lactide) (PLLA) films. endothelial tubes, cause barrier breakdown and induces cytoskeletal rearrangements HUVEC growth was determined on these modified surfaces using the BrdU assay (cell without inducing cell death. Since PT shows similar effects than the lead VDA CA4P, proliferation) and resazurin assay (cell viability). The chemically modified PLLA surfaces this natural compound might represent an interesting novel VDA. These findings warrant conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation investigations into the in vivo potential of PT. (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified PLLA. These results indicate that the synthesized ELP incorporated with amino acid motifs promote This work was supported by the German Research Foundation (DFG, FOR 1406, FU an accelerated endothelialization of the biodegradable stent material PLLA. 691/9-1). Discussion: In summary, we were able to generate elastin-like proteins modified by bioactive sequences. Those sequences enhanced endothelial cell proliferation and adhesion. Further studies are warranted to determine the activity on smooth muscle cells of these 212 peptides.
References Lung inflammation plays a key role in carbon nanoparticle-induced adjuvant [1] Patterson J, Martino MM, Hubbell JA. Biomimetic materials in tissue activity engineering. Mater Today 2010;13:14–22. 1 1 2 1 [2] Hutmacher DW. Biomaterials offer cancer research the third dimension. Nat Kroker M. , Sydlik U. , Weighardt H. , Unfried K. Mater 2010;9:90–3. 1 Leibniz-Institut für umweltmedizinische Forschung Partikel-Zell-Interaktion, Auf´m [3] Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular Hennekamp 50, 40225 Düsseldorf, Germany microenvironments for morphogenesis in tissue engineering. Nat Biotechnol 2 Leibniz-Institut für umweltmedizinische Forschung Immunologie, Auf´m Hennekamp 50, 2005;23:47–55. 40225 Düsseldorf, Germany [4] Davis ME, Motion JPM, Narmoneva DA, Takahashi T, Hakuno D, Kamm RD, et al. Injectable self-assembling peptide nanofibers create intramyocardial Several epidemiological studies indicate a correlation of human exposure to ultrafine microenvironments for endothelial cells. Circulation 2005;111:442–50. particulate air pollution caused by incomplete combustion processes and an increase in [5] Jamal M, Bassik N, Cho JH, Randall CL, Gracias DH. Directed growth of the incidence of pulmonary immune diseases like asthma. As a possible mechanism fibroblasts into three dimensional micropatterned geometries via selfassembling behind this pathological phenomenon, the adjuvant effect of lung inflammation induced scaffolds. Biomaterials 2010;31:1683–90. by poorly soluble environmental particles has been hypothesised. The aim of our study [6] Rai B, Teoh SH, Hutmacher DW, Cao T, Ho KH. Novel PCL-based honeycomb was to investigate the causal link between carbon nanoparticle-induced lung scaffolds as drug delivery systems for rhBMP-2. Biomaterials inflammation and modulations of immune cell populations during processes leading to 2005;26:3739–48. sensitization, and allergic immune responses of the airways. [7] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–6. Therefore mice were treated with ovalbumin (OVA) alone or in combination with carbon [8] Chen RR, Silva EA, Yuen WW, Mooney DJ. Spatio-temporal VEGF and PDGF nanoparticles (CNP) by pharyngeal aspiration. The induction of inflammation and the delivery patterns blood vessel formation and maturation. Pharm Res immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph 2007;24:258–64. nodes (PBLN) at the level of sensitization, and at the level of the immune response. [9] Guler MO, Hsu L, Soukasene S, Harrington DA, Hulvat JF, Stupp SI. Presentation OVA-specific IgE antibodies were measured in blood serum, and the development of of RGDS epitopes on self-assembled nanofibers of branched peptide allergic airway inflammation was studied after OVA challenge. amphiphiles. Biomacromolecules 2006;7:1855–63. Results at the level of sensitization showed that CNP-induced immediate airway [10M. C. Berg, S. Y. Yang, P. T. Hammond, M. F. Rubner, inflammation had immune adjuvant activity resulting in an increase of specific cell Controlling Mammalian Cell Interactions on Patterned Polyelectrolyte Multilayer populations in PBLN and in a stimulation of asthma-specific Th2 cytokines. A specific Surfaces reduction of the neutrophilic lung inflammation by application of the compatible solute Langmuir 2004, 20,1362. ectoine significantly reduced the adjuvant effects of CNP. In OVA-sensitized mice, [11] E. Ruoslahti, avIntegrins as receptors for tumor targeting by circulating ligands, application of CNP 12 hours prior to allergen challenge, led to a significant increase in Annu. Rev. Cell Dev. Biol. 1996, 12, 697. inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. Co- [12] Rajangam K, Behanna HA, Hui MJ, Han XQ, Hulvat JF, Lomasney JW, et al. application of 1 mM ectoine together with CNP reduced the particle-induced effects. Heparin binding nanostructures to promote growth of blood vessels. Nano Lett Our data show a link between neutrophilic lung inflammation and adjuvant effects of 2006;6:2086–90. CNP. A specific reduction of neutrophils by the application of ectoine attenuated this NP [13] Sarah C. Heilshorn, Kathleen A. DiZio, Eric R. Welsh, David A. Tirrell induced adjuvant effect, indicating that particle-induced lung inflammation rather than Endothelial cell adhesion to the fibronectin CS5 domain in artificial the direct interaction of nanoparticles with immune cells is the critical step in extracellular matrix proteins. Biomaterials 24, 2003, 4245–4252 environmentally modulated pulmonary immune diseases like asthma. [14] Girotti A, Reguera J, Rodriguez-Cabello JC, Arias FJ, Alonso M, Testera AM. Design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes. J Mater Sci Mater Med 2004;15:479–84. [15] Rui R. Costa, J. F. Mano, Stimuli-Responsive Thin Coatings Using Elastin-Like Polymers for Biomedical Applications Adv. Funct. Mater. 2009, 19, 3210–3218
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214 reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. Likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the NP. Pathophysiological distribution and relevance of Kv7.2/3/5 potassium channels in the CNS of 6-OHDA lesioned rats These data demonstrate that inhalation of finer NP is not necessarily associated with higher toxicity compared to the coarse material. The results were obtained with two Lambrecht C.1, Creed M.2, Sander S. E.1, Nobrega J. N.2, Richter A.1 1 organic particles of rather different size and composition but are in contrast to the more Freie Universität Berlin, Faculty of Veterinary Medicine Institute of Pharmacology and severe effects seen with several anorganic NP when compared to the corresponding Toxicology, Koserstr. 20, 14195 Berlin, Germany 2 coarse particles. Centre for Addiction and Mental Health Neuroimaging Research Section, 250 College St, Ontario Toronto M5T 1R8, Canada
L-DOPA-induced dyskinesias (LID), i. e. the development of abnormal involuntary movements, are a frequently occurring side effect of long-term therapy of Parkinson's 217 disease. Once established, LID is difficult to treat. Recently published research demonstrates antidyskinetic effects of Kv7.2-7.5 channel openers (retigabine, flupirtine) in a rat model of LID. The present study sought to clarify if altered expression of Comparing Inhalation Toxicity of Nanomaterials: Inhalation Studies with K 7.2/3/5 channels in the CNS is involved in the pathophysiology of this animal model of 16 Materials v 1 1 1 2 3,1 4 LID. To investigate this question brains from (1) naive, (2) SHAM lesioned, (3) 6-OHDA Landsiedel R. , Strauss V. , Treumann S. , Wiench K. , Wohlleben W. , Wiemann M. , 2 lesioned and vehicle-treated, and (4) 6-OHDA lesioned, L-DOPA-treated dyskinetic rats van Ravenzwaay B. were subjected to in situ-hybridization. The mRNA values [left (intact) – right (lesioned) 1BASF SE Experimental Toxicology and Ecology, 67063 Ludwigshafen am Rhein, side] of individual regions of the brain were compared between the four groups. Most Germany 2 alterations were found in the subregions of caudate-putamen (Kv7.2/3/5 subunits), cortex BASF SE Product Safety, GV/TB, 67063 Ludwigshafen, Germany 3 piriformis (Kv7.2 subunit), substantia nigra pars reticulata and pars compacta (Kv7.3 BASF SE Polymer Physics, 67063 Ludwigshafen am Rhein, Germany 4 subunit) and nucleus accumbens core (Kv7.5 subunit). The results of the in situ- ibe R&D gGmbH, Münster, Germany hybridization substantiate the importance of Kv7 potassium channels in the pathophysiology of LID. In view of the observed antidyskinetic effects of Kv7 potassium Within the NanoCare Project a standard short-term inhalation test to examine the toxicity channel openers in previous studies, they confirm the status of retigabine and flupirtine of inhaled aerosols from nanomaterials has been developed. The inhalation toxicity of as interesting candidates for the treatment of LID. nano- andpigmentary materials was studied: BaSO4, ZnO, CeO2, Al-doped CeO2, ZrO2, The study was funded by the Micheal J. Fox Foundation. amorphous silica, surface-coated amorphous silica, Titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the OECD sponsorship program. Quartz dust TiO2 and ZnO were tested as pigmentary materials. Rats were exposed nose-only to three concentrations of one of 215 these materials, 6 h a day for five consecutive days. Positive controls were exposed to quartz dust or pigmentary ZnO, negative controls to clean air. A wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 Phosphodiesterase 2A is upregulated in failing hearts and activates cardiac days and 3 weeks after the last exposure. Among these parameters, polymorphnuclear myofibroblast formation and CTGF synthesis via cGMP hydrolysis granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type Lämmle S., Emons J., Wittköpper K., Würtz C., Zimmermann W. - H., Lutz S., El- and localization of inflammation. Among these substances, we identified BaSO4 as Armouche A. having the lowest toxicity. All MWCNTs were most potent in producing progressive UMG Göttingen Abteilung Pharmakologie, Robert-Koch-Str. 40, 37075 Göttingen, inflammation in the lung; granulomas in lung and lung associated lymph nodes were Germany observed without indication for fibrosis. The NOAECs of the 16 substances ranged between < 0.1 and >50 mg/m3. Generally the material was only found in the lung The failing heart is characterized by excessive extracellular matrix production by (surface and macrophages) and in the draining lymph nodes. Surface modified myofibroblasts (MyoCFs) causing fibrosis and myocardial stiffening. MyoCFs represent amorphous silica was also found in the spleen. The data demonstrate that the method is phenotypically transformed cardiac fibroblasts (CFs) and are characterized by the able to differentiate the toxic potential of different nanomaterials and to expression of contractile proteins like a-smooth muscle actin (α-SMA) and enhanced indicate regression or progression of the effects effects. Moreover the lung burden and secretion of growth factors (e. g. CTGF). Identification of intracellular enzymes that potential translocation to other tissues was detecable. modulate this transformation process is desired to therapeutically modulate pro-fibrotic Comparing the material properties and effects of the 16 materials, no general relationship progression in heart failure. We show that PDE2A, a phosphodiesterase isoform, that is between the toxicity and either particle size, specific surface area or aerosol particle number able to hydrolyse cGMP and cAMP, is markedly upregulated in failing hearts from concentration was found. Hence we must not expect to find a gerneral "nanotoxicology" or a patients with end-stage heart failure (2-3-fold, p<0.05, n=8). Notably, PDE2A protein unifying dosimetry for all nanomaterials. We must rather be prepared to test individual abundance is 4-fold higher in MyoCFs compared to cardiomyocytes from neonatal rat nanomaterials for their effects. And to develop grouping concepts not only based on hearts (p<0.05, n=7). To this end we tested whether PDE2A modulates the material properties but also on biopersistence, biokinetics and biological effects. transformation of CFs isolated from neonatal rat hearts to MyoCFs. Indeed, as assessed by immunoblotting and fluorescent microscopy (α-SMA, phalloidin, DAPI), adenoviral References PDE2A overexpression induced α-SMA expression (3.2-fold p<0.05, n≥8) and to a lower Part of this studes has been sponsored by BMBF (nanoCare). extent CTGF synthesis (1.5-fold, p<0.05, n≥5). Mechanistically, PDE2A showed preferential subsarcolemmal localisation with diminished total cGMP levels (-56%, p<0.05, n≥5). Consistently, parallel stimulation with atrial natriuretic peptide (ANP), a selective activator of membrane-bound guanylyl cyclase, normalized CTGF synthesis indicating that PDE2A controls cGMP in a discrete subdomain near the plasma 218 membrane. Moreover PDE2A overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). Endpoint-centric search for toxicological information and data to support the These data implicate PDE2A-dependent subsarcolemmal cGMP regulation in information retrieval for regulatory programs 1 2,3 4 1 5 1 myofibroblast formation and potentially cardiac fibrosis. Therefore, targeting PDE2A may Landsiedel R. , Wächter T. , Sauer U. G. , Wareing B. , Huhse B. , Langsch A. , 1 3 3 3 5 5 3 lead to regression of the fibrotic remodeling associated with heart failure. Hareng L. , Zschunke M. , Bodor C. , Mönnich J. , Grune B. , Luch A. , Alvers M. R. , 6 2 Spielmann H. , Schroeder M. 1BASF Product Safety Experimental Toxicology and Ecology, Ludwigshafen, Germany 2Biotechniologisches Zentrum (BIOTEC), TU Dresden Lehrstuhl für Bioinformatik, 216 Dresden, Germany 3Transinsight GmbH Semantic Technologies, Dresden, Germany 4Scientific Consultancy - Animal Welfare, Neubiberg, Germany 5Bundesinstitut für Risikobewertung (BfR), Berlin, Germany Comparative Inhalation Toxicity of Organic Nanoparticles and Larger Particles of 6 the Same Chemical Identity FU Berlin Institut für Pharmazie, Berlin, Germany
Lan M. - H.1 1 2 2 1,3 , Rey-Moreno M. , Wiench K. , van Ravenzwaay B. , Wohlleben W. , The EU REACH regulation No 1907/2006 requires industry to ensure the safety of Landsiedel R.1 1 chemical use and manufacturing. All substances manufactured or imported in quantities BASF SE Experimental Toxicology and Ecology, 67063 Ludwigshafen am Rhein, above one tonne per year must be registered. Information requirements for the dossiers Germany increase with increasing tonnage or once hazards are suspected. Searching for 2BASF SE Product Safety, GV/TB, 67063 Ludwigshafen, Germany 3 substance specific literature and the compilation of hazard data for safety assessments BASF SE Polymer Physics, 67063 Ludwigshafen am Rhein, Germany are highly challenging procedures. The novel web-based search engine Go3R, accessible free of charge at www.Go3R.org, Several anorganic nanoparticles (NP) causedhigher inhalation toxicity than the has been created to allow quickly finding relevant hazard information and data. Go3R corresponding coarse particles (Oberdoerster et al. 2005). We examined an organic provides an endpoint-centered literature search to all scientists and regulatory pigment and a polymer dispersion each as nanomaterial and as the chemical identical authorities seeking for toxicological information. Furthermore, Go3R specifically coarse material in short-term inhalation studies in malerats. highlights information on animal testing alternatives. Search results are presented The polymer was an anionic acrylic ester copolymer containing free carboxylic groups. automatically linked to an “intelligent table of contents” which enables the user to sort Three different particle sizes were synthesized by varying polymerization conditions: 12, the literature listed in PubMed or the Toxicology Data Network (TOXNET) in a fast and 80 or 250 nm. Although polymeric acrylic ester was reported to be irritating to the comprehensive manner. Retrieved documents are automatically organized in categories respiratory tract at 3 mg/m3, all three tested polymers - including the NP (12 and 80 nm) relating to the IUCLID 5 chapters. Hereby, the user can browse directly through the - did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. entire 21 million documents without even having to start the search with an initial query. The organic pigment was a poorly soluble pyrrol with an intense orange color. The NP The semantically enriched platform supports the user during query formulation, allows (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm for bibliographic analysis, and specifically highlights information related to the width and 0.3 to 3 µm length) are both needle-like. They were tested at 1, 3, 10 and 30 replacement, reduction, and refinement of animal experiments. mg/m3 for the NP and 3, 10 and 30 mg/m3 for the coarse materials. Mild and partly S51
Introduction. Activation of Gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic CB1 receptors. The aim of the present experiments was to study the mechanism of the endocannabinoid production. Specifically, we wanted to clarify the role of Ca2+ release from intracellular stores in triggering endocannabinoid production. Methods. Patch-clamp- and Ca2+ imaging experiments were performed on Purkinje cells in mouse cerebellar brain slices. Glutamatergic excitatory postsynaptic currents (eEPSCs) were elicited by electrical stimulation of parallel fibers. The Gαq/11 protein- coupled metabotropic glutamate receptor 1 (mGluR1) was activated by superfusion of (RS)-3,5-dihydroxyphenylglycine (DHPG) or – more physiologically – by burst stimulation of the parallel fibers. Results. Both DHPG superfusion and burst stimulation of parallel fibers elicited an increase in intracellular Ca2+ concentration in the postsynaptic Purkinje cells. DHPG superfusion and burst stimulation suppressed eEPSCs, and this suppression was abolished in the presence of the mGluR1 antagonist CPCCOEt. The suppression of the eEPSCs was also sensitive to the CB1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and CB1 receptors. The suppression of the eEPSCs was attenuated after depletion of the endoplasmic reticulum Ca2+ stores by thapsigargin, cyclopiazonic acid and IP3. Conclusion. The results indicate that after activation of the Gαq/11 protein-coupled metabotropic glutamate receptor 1 (mGluR1) of the postsynaptic neuron Ca2+ is released from the endoplasmic reticulum. This Ca2+ release significantly contributes to the production of endocannabinoids. The endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic CB1 receptors. Go3R search interface: Search results in Go3R are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. 221
The guanine nucleotide exchange factor Dock9 controls Reelin dependent Cdc42- 219 effects on radial migration Pichler M.1,2, Chai X.3, Fan L.4, Bouche E.2, Schlosser A.5, Zhao S.3,6, Hein L.1, Schumacher S.7, Frotscher M.6, Bock H.2,8, Leemhuis J.1,2 1 Towards a differentiation therapy of acute myelogenous leukemia with histamine Albert-Ludwigs University Freiburg Pharmacology, Albertstrasse 25, 79104 Freiburg, Germany H2-receptor agonists 2 Laue S., Burhenne H., Seifert R. Albert-Ludwigs University Freiburg Zentrum für Neurowissenschaften, Albertstrasse 23, 79104 Freiburg, Germany Hannover Medical School Institute of Pharmacology, Carl-Neuberg-Str. 1, 30625 3Albert-Ludwigs University Freiburg Department of Anatomy, 79104 Freiburg, Germany Hannover, Germany 4Lanzhou University, China School of Life Science, Lanzhou China 5Albert-Ludwigs University Freiburg Center for Biological Systems Analysis, 79104 Acute myelogenous leukemia (AML) is a devastating malignancy characterized by a Freiburg, Germany differentiation block of myeloid progenitor cells. Recently, histamine dihydrochloride in 6University Hamburg Center for Molecular Neurobiology, Hamburg, Germany combination with interleukin 2 has been approved as orphan drug for the consolidation 7University Ulm Institute of Molecular and Cellular Anatomy, Ulm, Germany treatment of AML (1). It is assumed that histamine exerts its effects by activating the 8Albert-Ludwigs University Department of Medicine II, 79104 Freiburg, Germany histamine H2-receptor (H2R) in human neutrophils, resulting in improved anti-tumor function of T killer cells by inhibiting NADPH oxidase-catalyzed superoxide formation. During mouse brain development projection neurons migrate from the ventricular zone Previous studies had shown that histamine also induces myeloid differentiation (2). to the neocortical plate. The glycoprotein Reelin controls cortical layering by signaling Considering the fact that all-trans-retinoic acids constitutes a powerful differentiation through the lipoprotein receptors very low density lipoprotein receptor (Vldlr) and the therapy of acute promyelocytic leukaemia, a specific subtype of AML (3), we initiated a apolipoprotein-E-receptor-2 (Apoer2). Reelin-deficient reeler mice have architectonic study to explore the possibility that H2R-mediated myeloid differentiation provides an anomalies especially in the cortex, hippocampus and the spinal cord. Reelin activates alternative or complementary strategy to treat leukemias associated with differentiation Cdc42 via the lipoprotein receptor Apolipoprotein E Receptor 2 (Apoer2), the blocks. As model system, we used HL-60 cells. cytoplasmic adapter protein Disabled-1 (Dab1) and PI3-kinase to induce filopodia In HL-60 cells, histamine and various H2-receptor agonists induced concentration- formation as well as to increase vesicle traffic. Moreover, Reelin activates Cdc42 and dependent increases in cAMP levels. Interestingly, ligands differentially increased Rac1 to increase growth cone motility. During radial migration Cdc42 plays an integral cytosolic calcium concentration and extracellular receptor kinase (ERK) pathways, role in polarizing the centrosome and defining the direction of movement. It remains indicative for ligand-specific H2R conformations. H2R activation resulted in myeloid elusive whether Reelin-dependent Cdc42 activation controls radial migration. differentiation as assessed by enhanced formyl peptide receptor-mediated increases in We identified the CZH-protein family member and guanine-nucleotide-exchange-factor cytosolic calcium concentration. H2R agonists showed no signs of cytotoxicity. Dock9 as being involved in Reelin-dependent activation of Cdc42. Dock9 mediates Intriguingly, following H2R activation, the majority of the formed cAMP was exported into Reelin- and Cdc42-induced filopodia and spine formation but not neuronal vesicle traffic. the extracellular space via multi-drug resistance protein (MRP) 4, indicating that export shRNA-induced knockdown of Dock9 but not of Cdc42 mimics the migrational defects is a more important pathway for signal termination than cleavage of cAMP by during brain development in the reeler mouse lacking Reelin protein. phosphodiesterases. Despite effective cAMP export, even a short-term exposure (30 Alterations in the Reelin signalling pathway can be detected in many different psychiatric minutes) of cells was sufficient to induce expression of functionally active formyl peptide disorders, like schizophrenia and bipolar disorders and DNA variations in Reelin receptors. These data indicate that in contrast to previously held dogma, induction of signalling molecules are known. Also altered cortical Cdc42 signalling pathways are myeloid differentiation does not require continuous presence of a cAMP signal. From a linked to schizophrenia; this includes sequence variation in Dock9 and heterogeneity in therapeutic point of view this is very important since “spike” therapy with cAMP- bipolar disorder. increasing substances may be sufficient to induce a therapeutic effect in AML, thereby also reducing toxic side effects. Currently, we are systematically exploring the effects of H2R agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. 222 References 1. Martner A, Thoren FB, Aurelius J, Söderholm J, Brune M, Hellstrand K. Immunotherapy with histamine dihydrochloride for the prevention of relapse in acute Gαi2 proteins are negative regulators of glucose homeostasis myeloid leukemia. Expert Rev Hematol 3:381-391 (2010) Leiss V.1, Flockerzie K.1, Schürmann A.2, Nürnberg B.1 2+ 2. Seifert R, Höer A, Schwaner I, Buschauer A. Histamine icreases cytosolic Ca in HL- 1Institut für Pharmakologie und Toxikologie Abteilung für Pharmakologie und 60 promyelocytes predominantly via H2 receptors with an unique agonist/antagonist Experimentelle Therapie, Wilhelmstr. 56, 72074 Tübingen, Germany profile and induces functional differentiation. Mol Pharmacol 42:235-241 (1992) 2Deutsches Institut für Ernährungsforschung Abteilung für Experimentelle Diabetologie, 3. Ablain J, de The H. Revisiting the differentiation paradigm in acute promyelocytic Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany leukemia. Blood 117:5795-5802 (2011) The regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. Many of these cellular signalling pathways are initiated by binding of the ligand to a G-protein coupled receptor (GPCR), 220 e.g. noradrenaline inhibits insulin secretion upon binding to a Gi-coupled receptor. Upon GPCR activation the heterotrimeric G-protein is activated and both the α-subunit and βγ- dimers are released and interact with their specific target proteins. By the usage of Role of calcium from intracellular stores in the production of endocannabinoids Bordetella pertussis toxin (PTX) as a common Gαi inhibitor Gαi-dependent signalling involved in retrograde synaptic signaling pathways are interrupted which leads to increased insulin secretion, and significantly Lederer M., Szabo B. improves glucose tolerance. Since the Gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in Gαi2-deficient Albert-Ludwigs-Universität Freiburg, Institut für Pharmakologie und Toxikologie AG mice. Surprisingly and in contrast to the PTX data, glucose tolerance was unchanged in Szabo, Albertstrasse 25, 79104 Freiburg i. Br., Germany the Gαi2-deficient mice compared to wild type controls. However, the plasma insulin levels were significantly reduced upon glucose challenge. These findings point to S52
disturbed islets function and improved peripheral insulin sensitivity. Analysing Gαi2- New potassium channel openers (KCO) of the thieno-thiadiazine(TTD)-type initially deficient islets we show that islet size and number of nuclei are reduced. Nevertheless, developed as agonists for the SUR1-type KATP channels (Nielsen et al., J Med Chem 45: in vitro insulin secretion is improved at low (3 mM) and high (16 mM) glucose 4171, 2002) were characterized in SUR2B-type KATP channels as agonists and ant- concentrations and can be further stimulated upon PTX-treatment. These data indicate agonists, if R contains a quaternary (methyl-cycloalkyl) and a tertiary (R = cycloalkyl) that Gαi2 proteins influence islet development and inhibit insulin secretion. In addition, carbon, respectively (Lemoine et al., this journal 375, R45, 2007). To investigate the these findings support our hypothesis that Gαi2-deletion influences peripheral insulin selectivity of TTD-derivatives for myocardial KATP channels the membrane potential sensitivity. Therefore, we investigated glucose homeostasis and pAKT-levels after two actions of compounds were tested in HEK 293(Kir6.2/SUR2A)-cells and compared to hours feeding ad libitum in Gαi2-deficient mice. Under feeding conditions no differences HEK 293(Kir6.1/SUR2B)-cells as a model for smooth muscle-type KATP channels. in plasma insulin levels were visible although blood glucose levels were significantly Membrane potential was measured by fluorescence (excitation 505 nm, emission 530 reduced in Gαi2-targeted mice. pAKT-levels of liver and skeletal muscle were unaltered, nm) using 0.125 mg/ml of the dye R7260 (Molecular Probes). Standard-KCO induced whereas AKT phosphorylation in white adipose tissue was significantly increased, hyperpolarisation with ~10-fold smaller potency (pEC50) in SUR2A. TTD-compounds indicating improved glucose uptake of adipocytes. with CH3-cycloalkyl residues not only lost potency but also intrinsic activity for channel In conclusion, Gαi2 is a negative regulator of both insulin secretion and peripheral insulin activation (Emax) in SUR2A. Possibly, this loss of Emax would be much greater in native sensitivity and important for the maintenance of glucose homeostasis. heart cells with a normal channel density. In contrast, TTD-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in SUR2A and SUR2B-type KATP channels. Concluding, selectivity of KCO for KATP channel-subtypes cannot only be achieved by a different affinity but also by a 223 selective stimulation of the channel of interest.
Stereoselectivity of the enantiomers of BMS-191095 in activating and blocking SUR2-type KATP channels Lemoine H.1, Wrobel T.1, Rauhaus K.1, Schönlau J.2, Weber H.2 1Heinrich-Heine Universität, Inst. für Lasermedizin, Mol. Wirkstoff-Forschung, Universitätsstr. 1, 40225 Düsseldorf, Germany 2Heinrich-Heine Universität, Pharmazeutische Chemie, Universitätsstr. 1, 40225 Düsseldorf, Germany
BMS-191095 (3R,4S)- (3S,4R)-
radioligand binding, pKD (-log M) SUR2A 5.63 ± 0.04 4.99 ± 0.04 SUR2B 5.90 ± 0.03 5.20 ± 0.03
membrane potential test, pEC50 (-log M) SUR2A 5.06±0.03(A) 5.32±0.03(B)
SUR2B 6.15±0.03(A) 5.59±0.02(B) Fig 1: Formula (3R,4S)-BMS-191095 was introduced as cardioselective antiischemic ATP-sensitive potassium channel (KATP) opener (KCO) in 1997 (Rover et al., J Med Chem:40, 24). We were interested if the cardioselectivity was based on a true selectivity for the myocardial KATP channel-subtype (SUR2A/ Kir6.2) and extended our investigation on both enantiomers of BMS-191095. HEK 293 cells stably expressing SUR2A/Kir6.2 and 225 SUR2B/ Kir6.1-channels were used to measure the affinity of the enantiomers as competitors of [3H]-P 1075 (Bray & Quast, J Biol Chem 267: 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane Activation of SK3/K 2.3 channels modulate the response of LPS-stimulated potential test using the dye R7260 (Molecular Probes, 0.125 mg/ml, excitation 505 nm, Ca microglial cells emission 530 nm). The affinity of compounds in radioligand binding was slightly higher in Letsche T.1,2, Dolga A.1, Gold M.2, Doti N.1,3, Bacher M.2, Chiamvimonvat N.4, Dodel SUR2B than in SUR2A-type channels, but the enantiomeric ratio in SUR2A channels 2 1 matched that one determined for the SUR2B-type indicating some conformity of the R. , Culmsee C. 1 binding pockets of SUR2A and SUR2B-proteins. Surprisingly, however, the membrane Philipps Universität Marburg Institut für Pharmakologie und Klinische Pharmazie, Karl- potential tests revealed that the (3R,4S)-enantiomer acted as agonist (A) whereas the von-Frischstraße 1, 35032 Marburg, Germany 2 (3S,4R)-enantiomer acted as antagonist (B): (3R,4S)-BMS-191095 induced membrane Philipps Universität Marburg Klinik für Neurologie, Baldinger Straße, 35032 Marburg, hyperpolarisation whereas (3S,4R)-BMS-191095 repolarised cells prestimulated with Germany 3 submaximally effective concentrations of diazoxide. Concluding, BMS-191095 is not CNR Institute for Biostructure and Bioimaging, Naples, Italy 4 selective for SUR2A as compared to SUR2B-type KATP channels. Its enantiomers University of California Department of Medicine, Davis, California United States activate and block SUR2-type KATP channels in a stereospecific manner. Small-conductance calcium activated potassium (KCNN/SK/KCa2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. So far, little is known about the function of KCa2 channels in non- 224 neuronal cells. The aim of this study was to investigate the expression of KCa2 channels in microglial cells and their potential function in microglial activation and maintenance. Expression of KCa2 channel subtypes in microglial cells was assessed by mRNA analysis, Western blots and immunocytochemistry. Lipopolysaccharide (LPS)-induced Thieno-thiadiazine derivatives with full agonistic activity at SUR2B-type K ATP microglial proliferation was evaluated by the xCELLigence impedance-based system channels act as partial agonists at cardiac SUR2A-subtypes and MTT assays, and immunogenic activation of microglia was determined by Oldenhage C., Grittner D., Schmidt C., Lemoine H. measuring cytokines and nitric oxide (NO) release into the cell culture medium. The Heinrich-Heine Universität, Inst. für Lasermedizin, Mol. Wirkstoff-Forschung, KCa2.2 and KCa2.3 channel activator CyPPA (25 µM) and specific inhibitory peptides (50 Universitätsstr. 1, 40225 Düsseldorf, Germany µM) were applied to distinguish effects mediated by the KCa2 channel subtypes. All KCa2 channel subtypes were detected on mRNA and protein levels in resting and in SUR2A SUR2B LPS-activated microglial cells. xCELLigence real-time measurements and MTT assays demonstrated that LPS (200 ng/ml) induced microglial proliferation. The KCa2.2/KCa2.3 EC50 Emax EC50 Emax channel activator CyPPA reduced LPS-induced microglial proliferation in a Standard-KCO -log M % -log M % concentration-dependent manner. Specific peptide inhibitors of KCa2.3 channels, but not of K 2.2 channels, reversed the CyPPA-effects on LPS-induced microglial proliferation. P 1075 7.37 97.4 8.10 99.6 Ca CyPPA alone did not alter the production of TNF-alpha or IL-6, but strongly reduced the Bimakalim 7.04 97.8 7.90 99.6 LPS-dependent cytokine production. Interestingly, chelation of extracellular calcium by Diazoxide 4.19 96.7 5.11 98.1 EDTA induced differential cytokine kinetics by decreasing LPS-dependent IL-6 production while TNF-a production was not affected. Moreover, using inhibitory TTD-compounds with agonistic activity SK3/KCa2.3 channel peptides, we demonstrated that SK3/KCa2.3 channels modulate LPS-induced cytokine IL-6 production in a calcium-dependent manner, while the TNF-a CH3-Cyclobutyl 7.17 42.4 7.59 97.0 release was independent of extracellular calcium. CH -Cyclopentyl 7.11 54.1 7.99 98.9 3 In summary, the present study revealed that KCa2.3 channel stimulation reversed
CH3-Cyclohexyl 6.60 66.4 7.29 98.0 microglial activation. Thus, KCa2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in CNS diseases. TTD-compounds with antagonistic activity Cyclobutyl 6.64 89.8 6.60 104.3 Cyclopentyl 7.07 90.2 6.93 102.8
Cyclohexyl 6.65 76.2 6.30 69.8
S53
226 Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. Up to now, no curative systemic therapy Mitochondria-derived ROS lead to enhanced amyloid beta formation exists emphasizing the high clinical importance of new therapies for HCC. Therefore, the identification and characterization of novel drugable targets is a relevant goal. Leuner K.1, Kurz C.2, Eckert S. H.2, Schiller C.2, Reichert A.3, Müller W. E.2 1 Cyclin-dependent kinase 5 (Cdk5) is well characterized for its function in CNS FAU Erlangen Molekulare und Klinische Pharmazie, Cauerstraße, 91058 Erlangen, development and disease. Recently, few reports indicate functions of Cdk5 in cancer. Germany 2 Cdk5 was shown to regulate tumor growth, and our group discovered that Cdk5 Goethe Universität Pharmakologie für Naturwissenschaftler, Max-von-Laue-Str.9, regulates angiogenesis. Since HCC is a highly vascularized tumor and anti-angiogenic 60438 Frankfurt, Germany 3 treatment (Sorafenib) has shown some therapeutic benefit, we hypothesize that Cdk5 is Goethe Universität Molecular Bioenergetics Group, Theodor-Stern Kai 7, 60590 an interesting target for HCC therapy. The aim of this study was to characterize the Frankfurt, Germany function of Cdk5 in HCC. Histology of tissue micro arrays indicates an increased expression of Cdk5 in human Intracellular amyloid beta (Aß) oligomers and extracellular Aß plaques are key players in HCC tissue in comparison to healthy liver tissue of the same patient. To investigate the the progression of sporadic Alzheimer disease (AD). Still, the molecular signals function of Cdk5 in HCC, we analyzed the impact of both pharmacological inhibition of triggering Aß production are largely unclear. We asked whether mitochondria-derived Cdk5 and specific downregulation of Cdk5 with RNA interference on HCC cells. reactive oxygen species (ROS) are sufficient to increase Aß generation and thereby Pharmacological inhibition of Cdk5 with the small molecule roscovitine (R-Roscovitine, initiate a vicious cycle further impairing mitochondrial function. Seliciclib) decreased proliferation and clonogenic survival, induced G2/M cell cycle Results: arrest and cell death, and reduced motility of HUH7 and HepG2 cells. Transient Complex I and III dysfunction were induced in a cell model using the respiratory downregulation (siRNA) and stable knockdown (shRNA) of Cdk5 also reduced inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced proliferation, clonogenic survival, migration and invasion of HUH7 cells. In a ROS levels. Both treatments lead to elevated levels of Aß. Presence of an antioxidant subcutaneous HCC xenograft model, treatment with roscovitine reduced tumor growth rescued mitochondrial function and reduced formation of Aß demonstrating that the and angiogenesis, indicated by decreased tumor weight and volume, and reduced observed effects depended on ROS. Conversely, cells overproducing Aß showed vessel density. Moreover, cotreatment of HCC cells with roscovitine and tumor necrosis impairment of mitochondrial function such as comprised mitochondrial respiration, factor related apoptosis inducing ligand (TRAIL) resulted in an over-additive additive strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, effect on the induction of apoptosis. This coincided with reduced phosphorylation and the capability of these cells to generate Aß was partly reduced by an antioxidant activity of the anti-apoptotic transcription factor Stat3 at Ser727 that is directly indicating that Aß formation was also ROS-dependent. Moreover, mice with a genetic phosphorylated by Cdk5, and Tyr705. In line with this, the expression of the anti- defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aß apoptotic protein Mcl-1 is reduced by inhibition of Cdk5. levels in vivo. Our results point to an important function of Cdk5 in HCC and suggest Cdk5 as an Conclusion: interesting pharmacologically druggable target for HCC therapy. Several lines of evidence show that mitochondria-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aß itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD. 229
Delivery of mono-biotinylated RNaseA into macrophages with streptavidin- conjugated Clostridium botulinum C3 toxin 227 1 2 2 1 1 Lillich M. , Chen X. , Weil T. , Barth H. , Fahrer J. 1Universitätsklinikum Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein- Allee 11, 89081 Ulm, Germany Comparison of methods to derive health-based guidance or limit values for 2 chemicals Universität Ulm Institut für Organische Chemie III, Albert-Einstein-Allee 11, 89081 Ulm, Germany Licht O., Voss J. - U., Mangelsdorf I. Fraunhofer ITEM Chemikalienbewertung, Nikolai-Fuchs-Str. 1, 30625 Hannover, Clostridium botulinum produces the ADP-ribosyltransferase C3, which modifies and thereby Germany inactivates exclusively the small GTP binding proteins Rho-A,-B and –C. Recently, we discovered a specific endocytotic internalization of C3 toxin in macrophages and myeloid Health-based guidance or limit values are derived for chemicals to compare measured leukaemia cells, but not in epithelial cells [1]. Thus, C3 toxin provides a tool to target cells of or estimated exposure concentrations with these values. If the exposure is below the the monocyte/macrophage lineage, which are involved in various diseases and are of great limit value, adverse effect for human health can be regarded as negligible, e.g. the clinical interest. We used a biochemical crosslinking approach to design a delivery system exposure is expected to be tolerable. In Germany such values have been derived since based on an enzymatic inactive C3bot mutant (C3Mut) and streptavidin. The C3 portion years for chemicals that can be found in soil, water and air as well as human blood and mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for urine (biomonitoring). In a research project sponsored by the German binding of biotinylated cargo molecules to the transporter. Umweltbundesamt several methods used by the agency are compared to the method In vitro, the generated C3Mut-streptavidin bioconjugate showed specific and concentration laid in REACH guidance document R.8 to derive a Derived No Effect Level (DNEL). dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic The aim was to identify possibilities for standardization as well as to figure out specific mobility shift assay. Cell fractionation experiments indicated an uptake of the bioconjugate elements in individual methods. In addition to extrapolation factors the public availability into the cytosol of J774A.1 macrophages. In the next step, mono-biotinylated bovine of guidance and specific derivations as well as procedures for consensus on the limit pancreatic ribonuclease A (RNaseA) was used as a model cargo for the delivery of value were evaluated. macromolecules by the bioconjugate. RNaseA is a highly stable, well studied protein which The comparison of extrapolation factors revealed that, although they are named catalyzes the degradation of RNA. Mono-biotinylated RNaseA interacts in a specific and differently such as extrapolation, safety or assessment factors, they are used in a concentration dependent manner with the C3Mut-streptavidin bioconjugate in vitro as comparable manner. Factors for interspecies and intraspecies extrapolation are analysed with dot blot technique. The C3Mut-streptavidin bioconjugate efficiently mediates presented in more detail. The standard factor for such extrapolation is 10 in most cases. the internalization of biotinylated RNaseA into J774A.1 macrophages as analyzed with laser In the REACH guidance this factor consists of a part for allometric scaling and remaining scanning microscopy in fixed cells. This finding was also confirmed by live cell imaging. differences. Other factors are only defined in some methods, like a factor for Furthermore, cell fractionation showed a cytosolic delivery of biotinylated RNaseA in the extrapolation from LOAEL to NOAEL, data quality or data gaps. A factor for data quality presence of C3Mut-streptavidin. As expected we could not observe a cytotoxic effect of is not laid down in the basic scheme for setting of indoor air guidance values, but is used biotinylated wild-type RNaseA on J774A.1 macrophages, which is attributable to the in some of the recent derivations of limit values. Also the WHO guidelines for drinking- presence of ribonuclease inhibitor protein in mammalian cells. water quality use comparable factors to account for adequacy of studies or database In summary, the C3Mut-streptavidin bioconjugate mediates the efficient internalization of and nature and severity of effect. biotinylated (macro)molecules into macrophage like cells, and therefore represents a A transparent and documented derivation is necessary for acceptance of the value. The useful tool for the transduction of exogenous molecules into macrophages. In addition, derivation methods as well as the evaluation document on a specific substance are cytotoxic RNaseA mutants are available and will be used in further studies. available through publications or the internet in nearly all cases. For the DNEL only the numeric value is available at the ECHA website, but not any information on starting point References and extrapolation factors. [1] J. Fahrer, J. Kuban, K. Heine, G. Rupps, E. Kaiser, E. Felder, R. Benz, H. Barth Although all guide or limit values are derived in a comparable way, differences, however, (2010): Selective and specific internalization of clostridial C3 ADP-ribosyltransferases exist in some details. In most cases detailed explanation is lacking when deviating from into macrophages and monocytes. Cell Microbiol, 12: 233-47. standard or default assumptions. Often such deviation is based on expert judgement.
230 228
Cytotoxic effects of gold complexes in different tumour cell lines Cyclin dependent kinase 5 (Cdk5) and its function in Hepatocellular carcinoma 1 1 1 1 2 2 1 1 1 2 3 3 4 1 Lisowsky S. , Josch S. , Havermann S. , Fritz G. , Kunz P. C. , Wetzel C. , Wätjen W. Liebl J. 1 , Stamm S. , Günther M. , Mayr D. , Kirchner T. , De Toni E. N. , Vollmar A. , Heinrich-Heine-Universität Düsseldorf Institut für Toxikologie, Universitätsstraße 1, Zahler S.1 1 40225 Düsseldorf, Germany Ludwig-Maximilians-Universität München Pharmazeutische Biologie, Butenandtstr 5-13, 2Heinrich-Heine-Universität Düsseldorf Institut für anorganische Chemie, 81377 München, Germany 2 Universitätsstraße 1, 40225 Düsseldorf, Germany Ludwig-Maximilians-Universität München Pharmazeutische Biotechnologie, Butenandtstr 5-13, 81377 München, Germany 3 Organometal compounds such as cisplatin or the second generation complexes Ludwig-Maximilians-Universität München Pathologie, Thalkirchner Str. 36, 80337 carboplatin and oxaliplatin have become more and more important as antitumor agents. München, Germany 4 Nevertheless there is still an increasing demand for novel metal-based compounds. This Ludwig-Maximilians-Universität München Medizinische Klinik und Poliklinik 2, is necessary due to severe side effects and the occurence of resistent tumour cells. Marchioninistraße 15, 81377 München, Germany S54
In this context we investigated the cytotoxic effects of imidazole-based phosphane induced by endothelial NO production but that cGKI and TrpC6 channels are not gold(I) complexes as potential agents for cancer treatment. Initially we have used the functionally coupled in vascular smooth muscle. MTT-Assay to examine the toxic potential of the gold complexes in H4IIE rat hepatoma -/- cells. In this context CW60 (a diphosphane ligand with azoyl substituents R2P(CH2)2PR2, We thank Profs Birnbaumer (NIH) and Freichel (Homburg) for providing us with TrpC6 , R= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with and TrpC3-/- mice and Prof. Flockerzi (Homburg) for the antibodies against the TrpC an IC50 value of 6,5mM (24h incubation). Further investigations revealed that CW60 channels. induced an apoptotic cell death in H4IIE demonstrated by the activation of caspase 3/7 (48h incubation with 10mM CW60). In addition the induction of apoptosis was confirmed by the DNA ladder formation (24h incubation with 5mM CW60). In connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the 233 generation of DNA strand breaks as well as the DCF-Assay to detect the formation of reactive oxygen species. However neither DNA strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. Whole genome microarray analysis of the effects of TCDD and PCB 153 in human Furthermore we analysed if the compound influences intracellular signalling pathways hepatic cell models such as the JNK pathway and the PI3K/AKT but after 24h of incubation neither pAKT 1 1 2 3 1 nor JNK were influenced. The imidazole based phosphane gold (I) complex CW60 Lohr C. , Neser S. , Andresen K. , Andersson P. L. , Schrenk D. 1 shows strong toxic effects in H4IIE cells and turned out to be a promising compound as University of Kaiserslautern Food Science and Toxicology, Erwin-Schroedinger-Str. 52, a potential agent for cancer treatment. 67663 Kaiserslautern, Germany 2 Institute of Biotechnology and Drug Research, Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern,, Germany 3Umeå University Department of Chemistry, SE-901 87 Umeå, Sweden
231 Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants which can exert chronic toxicity in humans and animals (vertebrates). Most effects are mediated by the arylhydrocarbon receptor (AhR) Usage of loop diuretics in hypertensive elderly a ligand-activated nuclear transcription factor. 2,3,7,8-tetrachlorodibenzo-p-dioxin Lochner S., Kirch W., Schindler C. (TCDD), the most potent congener, was attributed by WHO (2005) with a Toxicity Institut für Klinische Pharmakologie, Fiedlerstraße 27, 01307 Dresden, Germany Equivalency Factor (TEF) of 1.0 and classified as a human carcinogen by IARC (1997). TCDD induces damage of immune function, reproduction, organogenesis, growth and Introduction: differentiation of epithelial tissues etc. in rodents. In addition, a number of PCDDs and According to current European guidelines loop diuretics are only recommended in PCBs have been determined as potent tumor promoters in rat liver. PCB 153 is a 'non- treatment of complicated arterial hypertension, e.g. in coexistence of renal or heart dioxin-like' PCB (NDL-PCB) present in food and the feed chain. The human hepatoma failure. Nevertheless, former studies showed a high usage of loop diuretics in cell line HepG2 and primary human hepatocytes (hHeps) were treated with TCDD (10 uncomplicated arterial hypertension. This pharmacoepidemiological research project nM) or PCB 153 (1 µM) for 24 h. Subsequently, RNA was isolated and microarray investigated the current use of loop diuretics in hypertensive elderly. analysis was performed using Agilent Whole Human Genome Oligo Microarray 4x44k. Methods: PCB 153 caused no up or down regulation of gene expression in both cell models. After This retrospective study used data from a community-dwelling population (CD) and from the treatment with TCDD, however, a total of 281 genes were more than 2-fold up 4 nursing homes in Dresden/Germany (NH). Elderly with the diagnosis of arterial regulated in HepG2 e.g. cytochrome P450 1A1 (CYP1A1) (32-fold) a sensitive marker hypertension aged ≥65 were included. Demographical, medical and drug prescription for AhR activation. Additional up regulated genes in HepG2 were; arylhydrocarbon data as well as blood pressure values were analysed and compared descriptively after receptor repressor (AHRR) 15-fold, aldehyde dehydrogenase 3 A1 (ALDH3A1) 11-fold, matching the populations by age and gender. and cytochrome P450 1B1 (CYP1B1) 10-fold. 44 genes were more than 2-fold down Results: regulated in HepG2 cells e.g. -proprotein convertase subtilisin/kexin type 9. Markedly Each population contained 209 hypertensive elderly (mean age 80.4 years [±6], 70.3% different findings were obtained in hHeps, i.e., 117 genes were up regulated, the highest women). Diuretics were prescribed to more than half of patients in both groups (p>0.05). up regulated gene was CYP1B1 with a 95-fold increase in gene expression, followed by Loop diuretics were taken by 63.7% (72/113) of the NH subgroup treated with diuretics. CYP1A1 (41-fold) and ALDH3A1 (21-fold). Only a small group of genes were A diagnosis of renal or heart failure was found only in 38.9% of the patients using loop significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose diuretics in nursing homes (28/72). Although thiazides were preferred as diuretic agents transporter). Comparing both human cell types, there was an unexpected small overlap in the CD population, the rate of patients suffering from renal or heart failure while of genes being up or down regulated. Interestingly, in both cell types, only 30 in common consuming loop diuretics was even lower (22.6%; 12/52; p=n.s.). Torasemide was genes were up regulated, including CYP1A1, CYP1A2, CYP1B1 and ALDH1A3. Only favored in both settings as loop diuretic (NH: 70.8%; CD:77.4%; p= n.s.). platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both Discussion: HepG2 and hHeps. In conclusion, our data indicate pronounced differences in the The high and inappropriate intake of loop diuretics in hypertensive elderly reported in patterns of TCDD-regulated genes between HepG2 cells and hHeps. former studies has again been confirmed. Remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. Furthermore, our results lead us to assume a high estimated number of 234 unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. This loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in Detection of redox modified proteins in nociceptive processing subdiuretic doses. Lorenz J. E.1, Kallenborn-Gerhardt W.1, Heide H.2, Steger M.2, Wittig I.2, Brandt U.2, Conclusion: 1 1 It must be pointed out that loop diuretics are still frequently inadequately prescribed Geisslinger G. , Schmidtko A. 1 because current guidelines recommend loop diuretics only in complicated arterial pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology Goethe University, hypertension. Frankfurt am Main, Germany 2 Molecular Bioenergetic Goethe University, Frankfurt am Main, Germany
Recent data indicate that redox modifications of proteins induced by reactive oxygen species (ROS) contribute to sensitization of pain pathways during persistent pain. 232 However, little is known about the targets of ROS in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. Here, we used the quantitative The role of cGMP/cGKI signaling and TrpC channels in regulation of vascular tone thiol trapping technique termed OxICAT to identify proteins which are redox modified Loga F., Domes K., Hofmann F., Wegener J. during nociceptive processing. We investigated spinal cords of untreated mice, after Pharmakologie und Toxikologie For 923, Biedersteiner Str 27, 80802 München, zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve Germany injury (neuropathic pain model). We identified several proteins with marked changes in their redox states after nociceptive stimulation. Our results show that the OxICAT Signaling by intracellular cGMP and cGMP-dependent protein kinase I (cGKI) is the method is an efficient method to detect redox modifications in proteins and that redox major pathway in vascular smooth muscle, by which endothelial NO regulates vascular modifications seem to play a role in pain processing. tone. Recent evidence suggests that TrpC channels are targets of cGKI in smooth muscle and mediate, at least partially, the relaxant effects of cGMP. We tested this Supported by the Deutsche Forschungsgemeinschaft (SFB 815/A14). concept by investigating the role of cGMP/cGKI signaling on vascular tone and peripheral resistance using cGKI-, TrpC6-, and TrpC3-, and TrpC3/C6-double knock-out mice. We found larger contractile responses to α-adrenergic stimulation in intact aorta from cGKI-, TrpC6-, and TrpC3/C6-double knock-out mice as compared to aorta from 235 Ctr and TrpC3-knock-out mice indicating a functional link between cGKI and TrpC6 channels. No differences were found if the vasodilator tone, provided by the NO generation in the vascular endothelium, was inhibited by L-NAME. Likewise, no Additive antinociceptive effects of a combination of vitamin C and vitamin E after differences were observed in the increase in peripheral resistance by α-adrenergic peripheral nerve injury stimulation using the hind limb perfusion system. Activation of cGKI by 8-Br-cGMP diminished aortic tone and peripheral resistance to a similar extent in control, TrpC6-, Lu R., Kallenborn-Gerhardt W., Geisslinger G., Schmidtko A. TrpC3-, and TrpC3/C6-double knock-out mice. No effect of 8-Br-cGMP was observed in pharmazentrum frankfurt/ZAFES Institute of Clinical Pharmacology, Goethe University, preparations from cGKI-/- mice. To test the co-localization of cGKI and TrpC channels, Frankfurt am Main, Germany we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of Ctr, TrpC3-, and TrpC6-knockout mice. TrpC3 could be detected in both Accumulating evidence indicates that increased generation of reactive oxygen species smooth muscle and endothelial cells whereas TrpC6 was only detected in endothelial (ROS) contributes to the development of exaggerated pain hypersensitivity during cells. The results suggest that absence of cGKI or TrpC6 impairs the vasodilator tone persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic S55
pain. We show that systemic administration of a combination of vitamins C and E 238 inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freund’s Adjuvant. In contrast, vitamin C or vitamin E given alone failed to Evaluation of three different dietary animal models for non-alcoholic fatty liver affect the nociceptive behavior in all tested models. The attenuated neuropathic pain disease (NAFLD) in Lewis rats behavior induced by the vitamin C and E combination was paralleled by a reduced p38 Lupp A.1, Lenhardt I.1, Sun J.2, Schulze Selting A.1, Vranjkovic K.1, Dahmen U.2 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed 1 after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination Institut für Pharmakologie und Toxikologie Universitätsklinikum Jena, Drackendorfer Str. 1, 07747 Jena, Germany ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results 2 suggest that administration of vitamins C and E in combination may exert synergistic Klinik für Allgemein-, Viszeral- und Gefäßchirurgie Universitätsklinikum Jena, antinociceptive effects, and further indicate that ROS essentially contribute to Drackendorfer Str. 1, 07747 Jena, Germany nociceptive processing in special pain states. Supported by the Deutsche Forschungsgemeinschaft (SFB 815/A14). With a prevalence of about 20-30% non-alcoholic fatty liver disease (NAFLD) represents the most common liver disorder in Europe. NAFLD manifestation ranges from steatosis through steatohepatitis (NASH) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. Fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory 236 cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of NAFLD into NASH is still unknown. Thus, besides life style modifications and weight reduction interventions, no established Molecular analysis of the interaction of adenylyl cyclase 1 with calmodulin and pharmacological therapy exists so far. calmodulin mutants To gain further insights into the pathogenesis of NAFLD and NASH and to develop new 1 2 1 Lübker C. , Richter M. , Seifert R. therapeutic strategies, appropriate animal models are essential. Thus, in the present 1Medizinische Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Str.1, study three different dietary animal models for NAFLD were evaluated and compared to 30625 Hannover, Germany the biochemical and metabolic alterations seen with NAFLD and NASH in man. 2University of Kansas Molecular Biosciences, 1200 Sunnyside Avenue, Lawrence, Male adult Lewis rats were given standard food or one of three different diets: fatty liver Kansas 66045, United States diet [FLD], methionine/choline deficient diet [MCD] or methionine/choline deficient plus high fat diet [MCD+HF]. After 1, 2, 4, 6 or 12 weeks of treatment, animals were Adenylyl cyclases (AC) catalyze the conversion of ATP to 3’,5’-cyclic monophosphate sacrificed and body and liver weights, laboratory parameters (ASAT, ALAT) as well as (cAMP), which regulates, as a second messenger, specific cell functions. AC 1 is mainly histopathological changes in the livers and different parameters indicating oxidative expressed in the brain, especially in the hippocampus, neocortex, entorhinal cortex, stress or representing the biotransformation capacity of the livers were analyzed. cerebellar cortex, olfactory bulb and pineal gland and plays an important role in learning, With FLD and MCD+HF a normal body weight gain was observed, whereas with MCD memory, neurodegeneration and pain responses. body weight gain was strongly impaired. Liver weights were mainly increased after AC 1 is stimulated by the eukaryotic Ca2+-sensor calmodulin (CaM), a 148 amino acid MCD+HF. Elevation of ASAT and ALAT values and hepatic steatosis were more protein. CaM possesses nine methionine residues in positions 36, 51, 71, 72, 76, 109, pronounced after MCD and MCD+HF than after FLD. All three diets caused an increase 124, 144 and 145, which are important for interacting with target structures. In CaM- in the oxidative stress in liver tissue, but especially with MCD a tremendous elevation in mutants 206, 213, 214 and 215, the methionine residues are completely (CaM-215) or the hepatic levels of lipid peroxidation products was seen. With regard to liver partially (CaM-206, -213 and -214) replaced by leucine residues. Both amino acids are biotransformation capacity, with all three diets mainly an induction of the cytochrome comparable in terms of hydrophobicity, volume and the preference for forming α-helices, P450 2E1 and 4A1 isoforms expression and activity was observed, which was most but only methionine is oxidizable to a sulfoxide, in contrast to leucine. pronounced after MCD and MCD+HF. In the present study we examined the protein-protein interaction (PPI) of recombinant In summary, the changes induced by MCD or MCD+HF most closely resemble the AC 1, expressed in Sf9 insect cell membranes, with CaM, CaM-206, -213, -214 and -215 alterations described in literature for NAFLD in man and thus should be preferred over by measuring the catalytic activity of AC 1. CaM-mutants show a 3-4-fold lower potency FLD in future investigations on NAFLD and NASH. than CaM, but they are more efficacious than CaM. Most prominently, CaM-215 was 133 % more efficacious than CaM. Such striking differences between CaM and CaM-mutants have not yet been observed for other mammalian effector proteins. As a result of the exchange of all methionine against leucine residues in CaM-215, it is more hydrophobic 239 than CaM and this leads to a better PPI with AC 1. In future studies we will examine the effects of CaM inhibitors, antidepressants and antipsychotics on CaM/AC 1 interaction. Furthermore we will analyze the effects of EP3 receptor-mediated contraction of human pulmonary arteries and inhibition of oxidized CaM and CaM-mutants on the catalytic activity of AC 1. Because oxidative neurogenic tachycardia in pithed rats stress is of great importance in aging, it is important to know more about the above- 1 1 1 1 named interaction in view to the demographic change. Malinowska B. , Kozlowska H. , Baranowska-Kuczko M. , Zakrzeska A. , Kozlowski M.2, Grzeda E.1, Schlicker E.3 Taken together, our data point to a unique CaM/AC 1 interaction that may be selectively 1 targeted by small molecules. In particular, enhancers of these interaction could be useful Medical University of Bialystok Department of Experimental Physiology and Pathophysiology, Mickiewicz 2A, 15-089 Bialystok, Poland to improve memory and learning. 2 Medical University of Bialystok Department of Thoracic Surgery, M. Skłodowskiej-Curie 24A, 15-276 Bialystok, Poland 3University of Bonn Department of Pharmacology and Toxicology, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany 237 EP3 receptors for prostaglandin E2 convey stimulatory and inhibitory effects. E.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their Gender differences in fat distribution and diabetes prevalence in NZO mouse inhibitory activity to reduction of neurotransmitter release from neuron endings. The aim Lubura M., Scherneck S., Zucker A., Schürmann A. of our study was (1) the pharmacological characterization of EP3 receptors in human Deutsches Institut für Ernährungsforschung Experimentelle Diabetologie, Arthur- pulmonary arteries and (2) the examination of the involvement of these receptors in the Scheunert-Allee 114-116, 14558 Potsdam, Germany regulation of the neurogenic tachycardia in pithed rats. L-826266 served as the EP3 antagonist. Background: Excessive fat accumulation in visceral but not subcutaneous fat depots as Experiments were performed in human pulmonary arterial rings isolated from patients well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an undergoing lobectomy during resection of lung carcinoma and in pithed and increased risk for the development of type 2 diabetes in humans. In this study we aimed vagotomised rats. The EP1/EP3 agonist sulprostone (1 nM – 100 mM) concentration- to examine the influence of early fat distribution on onset of type 2 diabetes in mice. dependently contracted human pulmonary artery rings (pEC50 and Emax; 6.89±0.12 and Methods: NZO mice are regarded as insulin resistant model in which only males 106.5±5.2%, relative to the contraction induced by KCl 60 mM). The concentration- become diabetic. We used male and female mice fed with high-fat and standard diet. response curve of sulprostone was not affected by the EP1 antagonist SC 19920 (10 We determined fat distribution by computed tomography for three times and conducted µM) but shifted to the right by L-826266 (10 µM) (apparent pA2 6.22). Extending the oral glucose tolerance tests on two different time points. Besides we assessed body exposure time to L-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 weight and blood glucose levels on weekly basis. (Schild plot-based pA2; concentrations 0.1, 1 and 10 µM). In pithed rats electrical Results: Contrary to previous findings, we observed that not only male NZO mice on stimulation (0.66 Hz, 1 ms, 50 V for 5 s) of the preganglionic sympathetic nerve fibers or high-fat diet develop diabetes. Blood glucose levels at the 16th week of age and total intravenous isoprenaline (0.15 nmol/kg) increased heart rate (HR) by 55 beats/min. pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in Sulprostone (10 – 1000 nmol/kg) did not affect the isoprenaline-induced increase in HR females These results lead to the conclusion that high-fat diet counteracts protective but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. L- action of estrogens against diabetes. Inversely to the findings in humans, female mice 826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the tend to store more fat in abdominal region than males. There was no relationship neurogenic tachycardia by 20%. between early accumulation of fat in abdominal region and onset of type 2 diabetes. In conclusion, EP3 receptors (1) located postsynaptically strongly contract human However, visceral fat was associated with liver fat in males as well as in females. pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying Furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose the heart of rats strongly inhibit the neurogenic tachycardia. levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. However, there was no correlation between hepatic insulin sensitivity L-826266 - 5-bromo-N-[3-(5-chloro-2-naphthalen-2-ylmethyl-phenyl)-acryloyl]-2- (indicated by quantitative insulin sensitivity index-QUICKI) and amounts of hepatic fat we methoxy-benzenesulphonamide conclude that early hepatosteatosis does not predict for glucose intolerance in NZO mice. Conclusion: In the NZO mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. Further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain.
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The deletion of the Cav2.1 a1-gene in the murine forebrain reveals multiple Characterization of a membrane protein expressed in mouse heart and brain behavioral alterations in learning and memory tasks Mannebach S.1, Frohnweiler K.1, Tselnicker I.2, Keren-Raifman T.2, Wissenbach U.1, Mallmann R.1, Castonguay J.1, v.d. Maagdenberg A. M. J.2, Klugbauer N.1 Dascal N.2, Flockerzi V.1 1Albert-Ludwigs-Universität Freiburg Experimentelle und Klinische Pharmakologie und 1Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg, Germany Toxikologie, Abteilung I, Albertstraße 25, 79104 Freiburg, Germany 2Department of Physiology and Pharmacology Sackler School of Medicine, Tel Aviv 2Leiden University Medical Centre Center for Human and Clinical Genetics, University, Tel Aviv, Israel Einthovenweg 20, 2333 CZ Leiden, Netherlands Recently, a novel membrane protein in Drosophila was shown to be localized in Voltage-gated Ca2+ channels of the central nervous system control a multitude of Ca2+ presynaptic vesicles. It appears to mediate a Ca influx after vesicle fusion with the dependent processes such as neurotransmitter release, neuronal excitability, neurite plasma membrane. Disruption of the corresponding gene leads to endocytic defects in 2+ outgrowth, synaptogenesis, plasticity and neuronal survival. The Cav2.1 Ca channel – drosophila [1]. Apparently, this protein plays a role in exo- and endocytosis and could also known as P/Q-type channel – belongs to the subfamily of high voltage activated serve as a Ca channel supplying Ca required for endocytosis. 2+ 2+ 2+ Ca -channels. Ca influx via Cav2.1 Ca channels located at presynaptic nerve We have identified a protein in mouse, c90rf7, which shares 26,3% amino acid terminals triggers vesicle fusion and transmitter release at brain synapses and at the sequence identity with the Drosophila protein. It covers 171 amino acid residues. Using 2+ neuromuscular junction. Thus, Cav2.1 Ca channels play a crucial role in synaptic RT-PCR the full length transcripts could be identified in brain, kidney, pancreas, heart, transmission. spleen, thymus and mast cells. Coexpression of c90rf7 and the CaV2.2 channel in 2+ The global Cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and Xenopus oocytes reduced the amount of the α1B and CaVβ3 subunits of the Ca lethality during the first postnatal weeks and is therefore an unsuitable model to analyze channel in the plasma membrane but did not affect the gating properties of the CaV2.2 2+ the importance of Cav2.1 Ca channels for learning and memory. Therefore, we channel. Expression of c90rf7 alone did not yield any channel activity. We therefore crossed a floxed Cav2.1 mouse line with Nex-Cre transgenic mice to establish a viable, started to produce recombinant protein using the His-SUMO-prokaryotic expression forebrain specific knock-out mouse line (fbKO-mice). Results from Western blot analysis vector. The protein was efficiently expressed as His-SUMO-c9orf7-fusion in E.coli (yield confirmed an efficient knock out of Cav2.1 in hippocampal and cortical preparations, 2mg at 1mg/ml). We are currently preparing the c9orf7 part of the His-SUMO-c9orf7- whereas the expression level in the cerebellum was not altered. fusion protein by Ulp1-protease digestion followed by various chromatographic steps. To investigate the specific role of Cav2.1 channels in hippocampus and neocortex The purified recombinant protein will be used to immunize rabbits to get antibodies. In dependent behavior, we performed tests for motor functions and sensory abilities and in parallel we generated antisera by immunizing rabbits with peptide fragments derived particular learning and memory tasks. Mice with a forebrain specific Cav2.1 knock-out from the c9orf7 sequence. show significant deficits in spatial learning & reference memory and a significant We could not identify any homologues of C9orf7 in the mouse genome and to analyze reduced recognition memory as revealed by the Morris Water Maze and an object its function we are currently generating C9orf7 deficient mouse lines by gene targeting. recognition task. The fbKO-mice exhibit no obvious locomotor deficits during behavioral We have chosen a strategy for conditionally inactivation of the gene with the option to tasks in the Open Field test and Elevated Plus Maze. Some fbKO-mice demonstrate study the cellular localization of C9orf7 by expression of the bgalactosidase gene under episodes of seizures in the Morris Water Maze and during different Rotarod tasks. To the control of the endogenous C9orf7 promoter. By southern blot analysis we´ve already assess motor-function of fbKO-mice in a stress reduced environment, we performed identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed home cage based running-wheel motor-learning tasks. ones and we will proceed with blastocyst injection to get chimeric mice and finally mice In summary, the diverse phenotypes of the forebrain specific knock-out mouse line carrying the introduced mutations in the c90rf gene. emphasize the critical importance of Cav2.1 for learning and memory. References References [1] Yao et al. (2009) Cell 138, 947–960 S. Goebbels et al., Genetic Targeting of Principal Neurons in Neocortex and Hippocampus of NEX-Cre Mice; Genesis 44:611–621 (2006) F. Hofmann, Voltage-dependent calcium channels: from structure to function; Rev Physiol Biochem Pharmacol 139:33-87 (1999) 243 D. Pietrobon, Function and Dysfunction of synaptic Calcium Channels: Insights from Mouse Models; Curr Opin Neurobiol 15:257-265 (2005) B. Todorov et a., Conditional Inactivation of the Cacna1a Gene in Transgenic Mice; Quantification of poly(ADP-ribose) levels in living cells by electrospray tandem Genesis 44:589-594 (2006) mass spectrometry
Martello R., Mangerich A., Sass S., Beneke S., Bürkle A. Universität Konstanz Molekulare Toxikologie, Jacob-Burckhardt-Str 31, 78467 Konstanz, Germany 241 Poly(ADP-ribose) (PAR) is a biopolymer of various chain lengths with a linear or branched structure which is synthesized by poly(ADP-ribose) polymerases (PARPs). Comprehensive characterization of a mouse model of colitis-associated PARPs are involved in various biological processes such as regulation of DNA repair, carcinogenesis reveals predominance of myeloperoxidase-mediated chlorination cell cycle progression, and cell death. Consequently, several PARP inhibitors are chemistry currently in clinical development as chemo- and radiosensitizers as well as 1,2 1 3 1 1 1 Mangerich A. , Zeng Y. , Parry N. M. A. , Ye W. , Prestwich E. , Cui L. , McFaline J. monotherapeutic agents following the concept of synthetic lethality. Pharmacological 1 1 1 4 1,4 1,3 L. , Knutson C. , Mobley M. W. , Taghizadeh K. , Wishnok J. S. , Fox J. G. , and toxicological studies call for an accurate analysis of PARP activity in terms of a 1,4 1,4 Tannenbaum S. R. , Dedon P. C. detailed knowledge of the structure of PAR and a reliable method for its quantification. 1Massachusetts Institute of Technology Department of Biological Engineering, 77 We have developed a sensitive, precise, and accurate bioanalytical method based on Massachusetts Avenue, Cambridge, MA 02139, United States liquid chromatography coupled to electrospray tandem mass spectrometry (LC/MS-MS) 2Universität Konstanz Molekulare Toxikologie, Jacob-Burckhardt-Str 31, 78467 to characterize and quantify PAR with femtmol sensitivity: PAR is extracted from cells Konstanz, Germany and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is 3Massachusetts Institute of Technology Division of Comparative Medicine, 77 characteristic for linear PAR, diribosyladenosine, which is characteristic for branching Massachusetts Avenue, Cambridge, MA 02139, United States points, and adenosine, which represents the terminal part of the polymer. Using this 4Massachusetts Institute of Technology Center for Environmental Health Science, 77 method, we are currently analyzing PAR levels in different cell lines and in primary Massachusetts Avenue, Cambridge, MA 02139, United States human peripheral blood mononuclear cells (PBMCs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent PARP inhibitors. We Helicobacter hepaticus-infected Rag2-/- mice emulate many aspects of human expect that after completing method validation this assay will be useful for a wide range inflammatory bowel disease (IBD), including the development of colitis and colon cancer of applications in pharmacology and toxicology. [Erdman et al., 2009, PNAS 106: 1027-1032]. Toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in H. hepaticus-infected Rag2-/- mice. 244 Infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and F4/80-positive macrophages, by 10 wks post- infection (pi), progressing into colon carcinoma by 20 wks pi. qPCR array-based gene Gene mutagenic potential and metabolite profile of 17β-estradiol in cultured V79 expression profiling revealed that pathophysiological changes were associated with cells expressing human cytochrome P450 1A1 characteristic alterations in the expression of genes related to inflammation, DNA repair, and oxidative stress response. To study inflammation-related macromolecular damage, Martínez Jaramillo D., Lehmann L. colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass University of Wuerzburg, Institute of Pharmacy and Food Chemistry Section of Food spectrometry to quantify a battery of 16 different DNA, RNA and protein damage Chemistry, Am Hubland, 97074 Wuerzburg, Germany products thought to represent the full spectrum of inflammation-related chemistries. Our data revealed a significant predominance of chlorinated DNA-, RNA-, and protein Oxidative metabolism of the female sex hormone 17β-estradiol (E2) is considered to damage products by 20 weeks pi. In contrast, levels of damage products arising from play a major role in the initiation of hormone-induced carcinogenesis. In extrahepatic oxidation, nitration and nitrosation changed only modestly or remained unchanged. Our tissues, E2 undergoes metabolic activation by cytochrome P450-dependent analyses also revealed higher levels of damage products in RNA than in DNA and monooxygenase (CYP) isozyme 1A1 to 2-hydroxy- (2-HO) and to a lesser extent to 4- demonstrated organ-specific differences of oxidative damage products, such as 8-oxo- HO-E2. If not conjugated, these catecholestrogens (CE) can further oxidize to dG and its oxidation products spiroiminodihydantoin and guanidinohydantoin. electrophilic quinones (Q), which may react with DNA and induce thereby mutations. Collectively, these results suggest that neutrophil and myeloperoxidase-induced Conjugation of these CE in extrahepatic tissues is mainly catalyzed by catechol-O- chlorination chemistry may serve as a biomarker of IBD and may play important roles in methyltransferase. the pathophysiology of IBD and colitis-associated cancer. In order to identify possible mutagenic metabolites (i) the induction of gene mutations by E2 was determined in male Chinese hamster lung fibroblasts (V79 cells) expressing human (h) CYP1A1 and (ii) the metabolite profile of E2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell S57
suspension in the culture medium. (i) Gene mutations were assessed using the out mice compared to wildtype controls. Although it seems that these TRPC proteins are hypoxanthine-guanine phosphoribosyltransferase assay. The promutagen not directly involved in catecholamine release from chromaffin cells induced by benzo[a]pyrene (BaP) served as positive control requiring metabolic activation by acetylcholine application in our hitherto existing experiments, their contribution to the hCYP1A1 and dimethylsulfoxide as solvent control. V79 hCYP1A1 were treated with 100 modulation of catecholamine release by agonists of Gq-coupled receptors still needs to nM E2 for 3 weeks and the resulting 6-thioguanine (6-TG) resistant mutants selected at be analysed. weeks (w) 2 and 3. The frequency of spontaneous 6-TG resistant mutants per 106 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). As expected, 0.25 µM BaP induced a significant increase in mutant frequency (MF, 266 ± 13, w2 and 132 ± 46, w3). Treatment with 100 247 nM E2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in MF, suggesting slight mutagenic activity. Glitazone-like action of glimepiride and glibenclamide in primary human In culture medium of V79 hCYP1A1 treated with 100 nM E2, 2-HO-E2, 2-methoxy- adipocytes (MeO)-E2, 3-O-methyl-2HO-E2 and 4-MeO-E2 (suggesting intracellular formation of 4- 1 1,2 1 1 2,3 HO-E2) were detected. While 4-MeO-E2 concentration remained constant over the Mayer P. , Haas B. , Celner J. , Enzmann H. , Pfeifer A. 1 exposure period, the concentration of the other metabolites increased in a time- Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM), Kurt-Georg-Kiesinger- dependent manner. The maximum concentration increase was reached at w3 for Allee 3, 53175 Bonn, Germany 2 methylcatechols and at w2 for 2-HO-E2, correlating with the maximum increase in MF, Institut für Pharmakologie und Toxikologie, Universität Bonn, Sigmund-Freud-Str. 25, observed after 2 weeks as well. 53105 Bonn, Germany 3 Pharma-Zentrum Bonn, 53113 Bonn, Germany In conclusion, E2 possessed a slight mutagenic potential after hCYP1A1-mediated activation to 2-, 4-HO-E2 and their corresponding methylcatechols. Aims: Sulfonylureas (SUs) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. In addition, SUs have pleiotropic effects on other tissues. Regarding the effects of SUs on adipocytes conflicting findings were reported. We have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. 245 Methods: Primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. Lipid accumulation was assessed by Oil Red O staining and determination of triglyceride content; gene expression was measured by Cumulative effects of three triazole fungicides in a broad dose range in vitro Real-Time PCR and Western blotting. Rieke S., Kneuer C., Bumke Scheer M., Lampen A., Hirsch-Ernst K., Marx-Stoelting P. Results: We initially characterized the genes regulated during human preadipocyte Bundesinstitut für Risikobewertung Chemikaliensicherheit, Max-Dohrn-Str., 10589 differentiation by a global microarray analysis. Treatment with glimepiride and Berlin, Germany glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. Genes involved in lipid metabolism were induced, chemokine Consumers are exposed to multiple residues of different pesticides via the diet. This expression was decreased. Interestingly, the effects of SUs were over all qualitatively raises questions concerning potential cumulative effects, especially for substances and quantitatively similar to pioglitazone. In direct comparison glibenclamide was more causing toxicity by a common mode of action. The aim of this work was to investigate potent than glimepiride in respect to the induction of FABP4 (EC50 0.32 vs. 2.8 µM), an potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol important adipocyte marker gene. SU- induced differentiation was virtually completely and flusilazol for selected parameters in a broad dose range in vitro. Parameters blocked by the PPARγ-antagonist T0070907 but not affected by diazoxide, indicating investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β- PPARγ activation by SUs. Repaglinide, causing insulin liberation like SUs but being hCG), expression of a panel of androgen- or estrogen-responsive genes in the human structurally different, had no effect on adipocytes. placental choriocarcinoma cell-line Jeg-3 and transactivation via estrogen receptors α Conclusions: In primary human pre-adipocytes, glibenclamide and glimepiride strongly and β in stably-transfected HEK 293 cells. induced differentiation, apparently by activating PPARγ . Thus, SUs but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. The ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of Jeg-3 cells. Additionally, the placental peptide hormone β-hCG was measured. While no change in β-hCG and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent 248 decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. Significant activation of ERβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and The role of AT1A and AT1B receptors as mechanosensors in myogenic combined exposure showed additive effects, while no significant activation of ERα was vasoconstriction observed. Based on the data, our findings suggest dose-additivity of triazole pesticides Blodow S.1, Schneider H.1, Wizemann R.2, Storch U.1, Gudermann T.1, Mederos Y with the same mode of action for selected parameters in vitro. No significant effects 1 were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied Schnitzler M. 1 individually nor in combination. LMU München Walther-Straub-Institut, Goethestr. 33, 80336 München, Germany 2 Philipps-Universität Marburg Pharmakologisches Institut, Karl-von-Frisch-Str. 1, 35032 Marburg, Germany
Arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to 246 constrict in response to an elevated intraluminal blood pressure. This physiological reaction is more distinct in small resistance arteries than in large conduit arteries. Understanding the underlying mechanisms should provide useful information for the Transient receptor potential channels as mediators of catecholamine release treatment of diseases like anaphylactic shock and systemic hypertension in which this 1 2 3 1 1 Mathar I. , Vennekens R. , Mannebach S. , Camacho Londono J. E. , Uhl S. , reaction is altered. Whereas the underlying signaling cascade has been extensively 4 2 1 Birnbaumer L. , Voets T. , Freichel M. studied, the molecular identity of the mechanosensory elements still remains elusive. 1Universität Heidelberg Pharmakologisches Institut, Im Neuenheimer Feld 366, 69120 Recent studies at the cellular level suggest a sensory function for a subgroup of G- Heidelberg, Germany protein coupled receptors (GPCRs) coupling to Gq/11-proteins. By determining mRNA- 2KU Leuven Laboratory of Ion Channel Research, O&NI Herestraat 49, 3000 Leuven, expression levels of selected GPCRs in consecutive pairs of resistance and conduit Belgium vessels, we could identify a subset of Gq/11-coupled receptors such as angiotensin II 3 Universität des Saarlandes Experimentelle & Klinische Pharmakologie und Toxikologie, AT1B, vasopressin V1A, endothelin ETA and ETB and α1A adrenoceptor significantly Kirrberger Str. 1, 66421 Homburg, Germany enriched in resistance vessels. By pharmacological blocking of those highly expressed 4NIEHS Research Triangle Park, North Carolina, North Carolina 27709, United States GPCRs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine TRP proteins form cation channels that are regulated through strikingly diverse mesenteric arteries ex vivo. While blocking of V1A receptor and α2A and α2AB mechanisms. Recently, genetic association studies identified many Trp genes including adrenoceptors showed no differences of myogenic tone, blocking of AT1A and AT1B Trpm4 as risk factors for disease states such as arrhythmias, hypertension and receptors by losartan and candesartan, ETA receptor by BQ123 and α1A adrenoceptor by cardiomyopathy. The melastatin TRP channels TRPM4 and TRPM5 have distinct prazosin caused significant reductions of the vascular response. Analyzing the myogenic -/- properties within the TRP channel family; they form non-selective cation channels response of AT1A mice with and without additional blocking of AT1B receptors by activated by intracellular calcium ions and are expressed in heart, aortic endothelial candesartan suggested that especially AT1B receptors play a dominant role for cells, kidney and adrenal gland. Disruption of the TRPM4 gene in mice leads to mechanosensitivity in mice. This was further supported by investigating the myogenic -/- increased basal blood pressure without evidence for impairment of endothelium- or response of AT1B mice. These findings suggest that mechanosensitive Gq/11-protein smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the coupled receptors, especially AT1B receptors, play a dominant role for the development renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. of myogenic vasoconstriction. Instead, TRPM4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. This indicates that TRPM4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. Whether catecholamine release is also regulated by TRPM4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how TRPM4 regulates catecholamine release. Besides TRPM4 we recently identified transcripts encoding additional TRP channels including TRPC5 and TRPC6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. Measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µM) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in TRPC1/C5/C6 triple knock S58
249 vitro, in human vascular smooth muscle cell (VSMC) HAS3 was specifically induced via activation of NFkB by interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNFa) as shown by ChIP assay and utilization of NFkB inhibitor Bay 11-7082. HAS3 was also Frequency of Transient Receptor Potential Melastatin 3 channel (TRPM3) upregulated in a co-culture system by activated macrophages via paracrine release of isoformes in mouse tissues TNFa and IL-1β as verified by neutralizing antibodies. In human atherosclerotic lesions NFkB positive VSMC were frequently detected in close proximity with HA and F4/80 Meiser J., Hasan N., Frühwald J., Philipp S. positive macrophages as shown by immunohistochemistry. To study the effects of HAS3 Institute for experimental and clinical pharmacology and toxicology, 66421 Homburg, mediated HA synthesis in human coronary VSMC, lentiviral overexpression and Germany knockdown of human HAS3 were employed. Overexpression of HAS3 resulted in increased migration and proliferation whereas knock down had the opposite effect. The TRPM3 ion channels are activated by steroidal compounds and noxious heat and are effects of HAS3 were mediated by both PI3K signaling and MAPK signaling via considered to be involved in insulin secretion and pain perception. The expression of the hyaluronan receptors CD44 and RHAMM. TRPM3 gene generates a variety of different transcripts which arise by alternative Conclusion: The present results suggest that HAS3-dependent HA synthesis is induced splicing and the use of different promoters [1]. They encode a substantial variety of in human VSMC by inflammatory cytokines released from activated macrophages. isoformes and so far we have identified more than 20 distinct TRPM3 proteins in mouse Moreover, HAS3-mediated HA production induced phenotypic activation of VSMC. and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24. These variants differ enormously in their biophysical properties. For example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of TRPM3 channels [2], whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant TRPM3 proteins. However, the frequency of these different isoformes in TRPM3 252 expressing tissues is completely unknown. To get insight into the significance of the different TRPM3 isoformes we investigated the abundance of alternative TRPM3 transcripts in different tissues and cell types by Increased arginase activity contributes to pulmonary inflammation, airway Reverse Transcription quantitative PCR (RT-qPCR). We found that the frequency of remodeling and pulmonary hypertension in a guinea pig model of COPD. 1 1 1 1 1 2 2 splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. Maarsingh H. , Pera T. , Zuidhof A. B. , Smit M. , Menzen M. H. , Klein T. , Flik G. , 1 1 Furthermore we analyzed the TRPM3 transcriptome in the choroid plexus of the brain Zaagsma J. , Meurs H. and the pituitary gland, tissues in which TRPM3 transcripts are most abundant. For that 1Department of Molecular Pharmacology, University of Groningen, Groningen, purpose we sequenced more than 120 clones, each. Corresponding to our RT-qPCR Netherlands result, we found a significant number of transcripts lacking exon 13. In cells of the 2Brains On-Line BV, Groningen, Netherlands choroid plexus nearly all (124 /126 clones) carried the short Ca2+ permeable pore. Furthermore, we identified seven variants spliced in exon 20 encoding truncated TRPM3 Pulmonary inflammation and airway remodeling are major features of chronic obstructive proteins. However, the composition of the TRPM3 transcriptome in the choroid plexus lung disease (COPD). In addition, pulmonary hypertension is a common comorbidity, and pituitary gland differed enormously, indicating the importance of alternative splicing which is associated with a poor prognosis of the disease. Recent studies in a guinea pig for TRPM3 function in different tissues. model of allergic asthma have shown that increased arginase activity, which converts L- arginine into L-ornithine and urea and competes with nitric oxide synthases for the [1] Lis, A. et al. (2007) Transcriptional regulation and processing increase the functional common substrate, contributes to allergen-induced airway inflammation, variability of TRPM channels. Naunyn Schmiedebergs Arch. Pharmacol. 371, 315-324. hyperresponsiveness and remodeling. There is evidence that cigarette smoke and [2] Oberwinkler, J. et al. (2005) Alternative Splicing Switches the Divalent Cation lipopolysaccharide (LPS), both involved in the pathogenesis of COPD, increase the Selectivity of TRPM3 Channels. J Biol Chem. 280, 22540-22548 expression of arginase, however, its role in the pathogenesis of COPD is currently unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of LPS-induced COPD. To this aim, guinea pigs 250 were instilled intranasally with LPS or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)S-amino-boronohexanoic acid (ABH) or PBS. The use of TTC values in evaluation and regulation of chemicals and pesticides Repeated LPS exposure increased lung arginase activity, resulting in increased L- ornithine/L-arginine and L-ornithine/L-citrulline ratio’s. Both ratio’s were reversed by ABH Melching-Kollmuss S. treatment. Repeated LPS exposure also induced increased IL-8 levels, neutrophils, BASF SE, 67056 Ludwigshafen, Germany goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by ABH. Moreover, repeated LPS exposure increased right ventricular mass, The concept of "thresholds of toxicological concern" (TTC) defines tolerable dietary indicative of pulmonary hypertension, which was similarly prevented by ABH. intakes for chemicals without toxicity data and is widely applied to chemicals present in In conclusion, increased arginase activity contributes to pulmonary inflammation, airway food in low concentrations such as flavorings. Based on a statistical evaluation of the remodeling and right ventricular hypertrophy in a guinea pig model of COPD, indicating results of many toxicity studies and considerations of chemical structures, the TTC that arginase inhibitors may have therapeutic potential in the treatment of this disease. concept derives a maximum daily oral intake without concern of 1800, 540 or 90 (Supported by MSD). µg/person/day for non-genotoxic chemicals depending on the allocation to so-called Cramer classes I, II or III. For substances with a structural alert for genotoxicity a TTC value of 0.15 µg/person/day might be used. Recently, it has been investigated, whether the TTC values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and 253 developmental, immune effects and endocrine-related effects). Several authors using different specific databases have confirmed that the TTC values derived using Cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental Behavioral abnormalities in HCN3-deficient mice effects. A respective decision tree is going to be presented, also considering substances Michalakis S., Schöll-Weidinger M., Mader R., Cao-Ehlker X., Fenske S., Wahl-Schott or substance classes which shall be excluded from the TTC approach. C., Biel M. There are several areas in which the TTC concept is already used, or a TTC approach is Center for Integrated Protein Science Munich (CIPSM) Department of Pharmacy – considered useful, to assess low-level human exposures, or help in prioritizing Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5- toxicological testing; as for example the assessment of plant metabolites and 13, 81377 München, Germany degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under REACH, residues, metabolites and impurities in plants, HCN3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which chemicals, plant protection products or pharmaceuticals. If no structural alert for is expressed in various brain regions including thalamic, hypothalamic and habenular genotoxicity is given or standard genotoxicity tests are negative the Cramer class III nuclei as well as brain stem and olfactory bulb. In this study we performed a value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to comparative analysis of HCN3-/- and HCN3+/+ mice using a battery of behavioral tests represent a chronic tolerable daily intake of the test substance. Examples for current and and telemetric biopotential measurements to evaluate a potential role of HCN3 in central future uses of the TTC concept in regulatory toxicology are presented. nervous system function. In general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. Telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light 251 phase and the light-dark transition phase. Hippocampus-dependent spatial learning was normal. By contrast, HCN3 knockout mice showed more immobility than control mice on day two of the Porsolt forced swimming test, which could reflect increased depression- like behavior. However, center exploration in the open field test as well as performance Hyaluronan matrix remodeling during atherosclerosis: A key role for hyaluronan -/- synthase 3 in the light-dark transition and the elevated-plus maze tests was normal in HCN3 mice. This suggests that general anxiety was not changed in the knockout mice. In addition, Melchior-Becker A., Kretschmer I., Rabausch B., Twarock S., Dai G., Fischer J. W. HCN3 knockout mice were less active on the second day of the open field test, which Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der supports a habituation phenotype. Finally, HCN3-/- mice had higher burying scores in the Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. Taken together, genetic deletion of HCN3 in mice results in distinct Objective: Hyaluronan (HA), synthesized by three HA-synthases (HAS1, -2, -3), is a behavioral abnormalities related to behavioral despair and expression of repetitive prominent matrix component of atherosclerotic lesions. The aim of the present study behaviors in response to mild stressors. was to identify the HAS isoenzyme that is associated with HA-matrix remodeling in inflammatory regions of atherosclerotic plaques. Furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (VSMC) were addressed. Methods and Results: During atherosclerosis in ApoE deficient mice the peak of macrophage invasion at 14 weeks coincided with HA deposition and induction of HAS3 in aortic root plaques. In human symptomatic carotid artery plaques HAS3 was by far the most prominent HAS isoenzyme as determined by quantitative real time RTPCR. In S59
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Alcohol concentrations in breastfed babies – physiologically based modelling as Protein Kinase G regulates Differentiation and Adipokine Expression in White a decision aid Adipocytes Mielke H.1, Gundert-Remy U.2,1, Partosch F.2, Stahlmann R.2 Kilic A.1, Mitschke M. M.1, Peter M.2, Haas B.2, Scholz D.1, Pfeifer A.1,3 1Bundesinstitut für Risikobewertung Chemikaliensicherheit, Max-Dohrn-Straße 8-10, 1Institute of Pharmacology and Toxicology, Sigmund Freud Strasse 25, 53105 Bonn, 10589 Berlin, Germany Germany 2Charité Universitätsmedizin Berlin Institut für Klinische Pharmakologie und Toxikologie, 2Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175 Luisenstr. 7, 10117 Berlin, Germany Bonn, Germany 3Pharma-Center, Sigmund Freud Str 36, 53105 Bonn, Germany Alcohol consumption when breast feeding is discussed controversially. Some groups recommend breast pumping before alcohol consumption and feeding the stored milk Obesity, the excessive accumulation of white adipose tissue (WAT), has reached instead of breast feeding after drinking alcohol. This study was performed to simulate pandemic dimensions. The factors that determine fat mass are not fully understood, but the blood concentration in the breastfed baby and to assess the health impact. adipocyte hypertrophy and adipokine secretion are thought to be important. In present Method: We established a physiologically based kinetic model. Its parameters were study, we investigated the role of the cyclic GMP (cGMP) signaling pathway focusing on calculated (partition coefficients tissue/blood ; Schmitt, 2008) or taken from literature cGMP-dependent protein kinase I (PKGI) in white adipocytes. (blood flows, tissue weights; IPCR,2002; metabolic clearance: Schipphan et al.,1975, PKGI is expressed in WAT, preadipocytes and differentiated adipocytes as Jones, 2010; milk/blood ratio: da Silva et al.,1993). We simulated 1. the alcohol demonstrated by real-time PCR, western blot and immunochemistry. Differentiation of concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing PKGIfl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment accumulation as evidenced by Oil Red O staining. Deletion of PKGI in PKGIfl/fl during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. adipocytes infected with a Cre lentivirus (LV-Cre, PKGI0/0) exhibited reduced the alcohol concentration during infant´s treatment of bloating by an approved herbal differentiation. Analysis of the triglyceride (TG) content revealed a significant decrease drug containing alcohol. of TG levels by 65% ± 1% in PKGI0/0 as compared to PKGIfl/fl adipocytes. Western blot Results: Peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 L of analysis of white adipocytes showed a significant decrease of C/EBPalpha (19% ± wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old 4.8%), PPARgamma (66% ± 2.9%) and aP2 (37% ± 10.6%) expression in PKGI0/0 cells infant and 0.38 ‰ in the utero/fetal compartment. The peak concentration after herbal as compared to PKGIfl/fl. drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, Treatment of 3T3-L1 cells with cGMP resulted in increased lipid accumulation and respectively. We discuss the results of the simulations and compare it with doses and enhanced expression of fat marker genes. Lentiviral overexpression of PKGI further published concentrations measured in experimental animals or in vitro studies. increased differentiation. Importantly, PKGI significantly induced mitochondrial Conclusions: We conclude that the recommendation „1 to 2 glasses of wine on biogenesis in 3T3-L1 cells. Concomitant activation of PKGI in 3T3-L1 preadipocytes and occasion“ (Agence Nationale d’Accréditation et d‘Évaluation en Santé, ASSANTE 2002) treatment with the demethylating agent 5-aza-deoxycytidine significantly increased is in accordance with the simulation results presented here whereas stricter rules are not expression of uncoupling protein-1 (UCP-1) – a unique protein of brown fat cells. We scientifically sound. found RhoA as major target of PKGI signaling with increased phosphorylation of RhoA at Ser-188 in PKGI overexpressing cells. Moreover, PKGI-dependent phosphorylation References counteracts the effects of RhoA on insulin signaling as well as adipokine expression. ASSANTE (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ Taken together, PKGI is a key player in white adipocyte differentiation that regulates cell breastfeeding_guidelines.pdf size and has an anti-inflammatory effect. PKGI decreases the secretion of pro- da Silva et al. (1993) Braz J Med Biol Res. 26,1097-1103 inflammatory adipokines via inhibition of RhoA signaling. In addition, activation of PKGI ICRP Publication 89. Amsterdam: Elsevier Science.2002 can establish a brown fat cell like phenotype during white adipocyte differentiation if the Jones (2010) Forensic Science International 200: 1-20 UCP-1 promoter is accessible. Schmitt (2008): In Vitro 22, 457-467 Schipphan et al. (1975): Kinderärztliche Praxis 43, 193-201
257 255 Characterization of the functional properties of Rag GTPases Moepps B., Maier J., Koenig C., Pfreimer M., Gierschik P. Adenylyl cyclase type V sensitizes its regulation by inhibitory G proteins University of Ulm Medical Center Institute of Pharmacology and Toxicology, Albert- Milde M., Bünemann M. Einstein-Allee 11, 89081 Ulm, Germany Philipps-Universität Marburg Institut für Pharmakologie und Klinische Pharmazie, Karl- von-Frisch-Str. 1, 35043 Marburg, Germany The Rag GTPases, RagA, RagB, RagC, and RagD form a subfamily GTPases of the Ras-related superfamiliy. Rag proteins are characterized by a modified Ras-like GTP- Adenylyl cyclases (AC) mediate physiological responses in virtually all cells, where their binding domain and a unique C-terminal region lacking a lipid modification motif. regulation through receptors and G proteins results in the modulation of cAMP. In the Interestingly, Rag proteins have been proposed to function as heterodimeric complexes present study we focused on the kinetics of interactions between the alpha-subunit from consisting of RagA or RagB associated with RagC or RagD. Rag GTPases have been inhibitory G protein type 1 (Gαi1) and adenylyl cyclase type V (AC5). These proteins implicated in the control of mammalian target of rapamycin (mTOR) function, in were labeled with CFP and YFP, respectively. The dynamics of their interactions was particular in regulation of the nutrient-stimulated and/or hormone-regulated mTOR monitored by means of high temporal resolution FRET imaging in HEK cells expressing activity. The protein kinase mTOR is found as the catalytic subunit of two larger protein unlabeled a2A-receptor and Gβg subunits. To activate the signaling pathway, we applied complexes referred to as mTOR complex 1 and 2, mTORC1 and 2. Under amino-acid- agonist using a rapid superfusion device. Application of norepinephrine resulted in the rich conditions, activated mTORC1 promotes protein synthesis and inhibits autophagy, development of a FRET signal, indicating interaction between Gai1-CFP and YFP-AC5. while under starvation autophagy inhibition is released. Increasing evidence suggests After withdrawal of agonist the FRET signal recovered with a remarkably slow time that activation of Rag GTPases contributes to mTORC1 function. Thus, Rag proteins course compared to the deactivation kinetics of Gi proteins reported previously were found to be associated with a protein complex termed Ragulator, a major (Bünemann et al. 2003). To further analyze the properties of the dissociation between regulatory protein of mTORC1 function and guanine nucleotide exchange of Rag Gai1 and AC5 we measured in parallel the offset kinetics of the interaction between Gai1- GTPases within the Rag-Ragulator-complex were described to promote mTORC1 YFP and Gβg-CFP (Gi1-FRET) after agonist withdrawal under comparable conditions. In translocation to its functional lysosomal compartment. However, the guanine nucleotide addition we tested to what degree the coexpression of RGS4 accelerated the exchange properties of Rag proteins are poorly characterized, and it is currently deactivation of Gi proteins and the dissociation of Gai1-CFP from YFP-AC5. These unknown, how amino acids promote Rag proteins to facilitate the formation of the active, experiments revealed that in the absence of RGS4 the dissociation of Gai1 from AC5 raptor-binding state of the Rag heterodimers. To characterize the guanine nucleotide after agonist withdrawal takes about 3 times longer than the deactivation of Gi proteins. exchange properties of the Rag GTPases as momomers or heterodimers in more detail, In the presence of RGS4 this difference is even larger due to the pronounced recombinant Rag proteins were expressed in bacteria and purified from this source to acceleration of G protein deactivation. The dissociation of Gai1 from AC5 was only near homogeneity. First, the parameters of GDP/GTP exchange of each of these marginally accelerated by RGS4. These observations lead us to hypothesize, that AC5 proteins were compared using the non-hydrolysable GTP analogon GTPgS. The results might trap activated G protein-subunits and thereby affect the G protein cycle by shifting showed that the Rag isoforms are distinct in their guanine nucleotide exchange the equilibrium towards activated G proteins. If this hypothesis is true, it should result in activities. In particular, nucleotide exchange on RagA and RagC, but not on RagB and a left-shifted dose response curve compared to G protein activation dose response. In RagD, was only observed at low concentrations of GDP and MgCl2 in extraction and support of this hypothesis we found that the concentration response curve for Gai1-AC5 assay buffers, i.e. conditions favoring the GDP/GTP exchange. These findings may interaction was several-fold leftward-shifted compared to the concentration-response indicate that guanine nucleotide exchange on RagA and RagC is controlled by guanine curve of Gi-protein activation under very similar conditions. Influencing the dynamics of nucleotide exchange factors and suggest specific functions of the individual Rag the G protein cycle by effectors may represent a novel and powerful mechanism for fine- GTPases within individual Rag heterodimers. tuning the sensitivity of receptor evoked responses in an effector-specific manner.
References Bünemann M, Frank M, Lohse MJ Proc Natl Acad Sci 2003;100(26):16077-82
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258 Background: MicroRNAs are small non-coding RNAs that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like DNA methylation. MeCP2 (methyl CpG binding protein 2) is Moleculargenetic investigations on the mechanisms mediating the inhibitory a protein that binds methylated DNA CpGs in the promoter region of genes and can thus effect of benzodiazepines on mast cells regulate their expression. Otherwise, MeCP2 is known to be a target gene for several microRNAs including the cluster miR-132/212 in the brain. Recently, our group could Haenisch B.1, Huber M.2, Wilhelm T.2, Molderings G. J.1 1 show that MeCP2 expression is downregulated in human heart failure suggesting that University of Bonn Institute of Human Genetics, Sigmund-Freud-Str. 25, 53127 Bonn, MeCP2 might be involved in cardiac pathogenesis. The aim of this project is to study the Germany 2 upstream regulation of MeCP2 by the cluster miR-132/212 in the heart during cardiac RWTH Aachen University Dep. of Biochemistry and Molecular Immunology, Institute of hypertrophy in-vitro and in-vivo. Biochemistry and Molecular Biology, Pauwelsstraße 30, 52074 Aachen, Germany Methods and Results: To test whether hypertrophic stimuli can induce miR-132/212 expression, we treated cultured NRCMs with 40 µM norepinephrine for 72 hours. This 1,4-Benzodiazepines (BZ) have been shown to be clinically effective in the treatment of induced cardiomyocyte hypertrophy and expression of the hypertrophy marker Nppa, but mast cell mediator-induced symptoms in patients with systemic mast cell activation also of miR-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of miR-212-3p disease. In in-vitro studies on rat and canine mast cells and human mast cell leukemia (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated Mecp2 mRNA and cells HMC1.2 BZ at micromolar concentrations inhibited mediator release which protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). To check whether MeCP2 appeared to be related to an inhibition of the intracellular cAMP pathway. In order to downregulation also occurs by direct miR-132/212 activation we increased levels of miR- identify potential targets on/in mast cells at which BZ may cause an inhibitory effect on 132 and miR-212 in cardiac myocytes by transfecting precursor miR-132 and miR-212- mast cell activation, the 1,4-BZ flunitrazepam (flu), clonazepam (clo) and 4- 3p molecules. Again, we observed NRCM hypertrophy, Nppa mRNA upregulation and chlorodiazepam (4-cd) were selected because of their different affinity and selectivity Mecp2 mRNA and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after miR- to/for the GABA-A-receptor and the translocator protein (TSPO): flu and clo bind with 132 overexpression. Similar results were obtained by overexpression of miR-212-3p. nanomolar affinity to GABA-A receptors, whereas 4-cd is a selective ligand at TSPO with To test the effects of adrenoceptor activation on the miR-132/212-MeCP2 axis in-vivo, nanomolar affinity to TSPO but only micromolar affinity to GABA-A receptors. Flu also wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 possesses nanomolar affinity to TSPO, whereas clo has no or only micromolar affinity to mg/kg/day each). After 7 days, cardiac ventricles were analyzed. Nppa gene expression TSPO. After incubation of HMC1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to (2.81 ± 0.32 -fold of control animals), miR-132 and miR-212-3p levels (3.67 ± 0.5 and 712 genes were significantly differently expressed in a substance-specific and time- 2.62 ± 0.4 –fold of control animals, p<0.01 and p<0.05, respectively) were increased dependent manner. Comparison of the genes differently expressed at 6 hours revealed while MeCP2 protein levels decreased to 77% (p<0.05) that the expression of 217 genes was regulated by both flu and clo but only 8 genes Conclusion: These results suggest that in-vitro and in-vivo adrenoceptor stimulation were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression leads to the activation of miR-132/212 expression and to downregulation of MeCP2 in by acting at a target site different from that of 4-cd. The difference between the gene cardiac myocytes in-vitro and in-vivo. regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. Since it was conceivable that the beneficial effects of the 1,4-BZ could be mediated by the recognition sites targeted by the 2,3-BZ, i.e. the GRIA2-encoded ionotropic glutamate receptor AMPA2, we investigated by quantitative PCR whether HMC1.2 cells express GRIA2, TSPO, the genes encoding the subunits of 261 the GABA-A receptor and the GABA-forming enzyme glutamic acid decarboxylase. TSPO, GABRA3, GABRB3, GABRE and GABRD were moderately expressed. In addition, there was a week or very week expression of GABRA2, GABRA4, GABRB2, Rosiglitazone-to-pioglitazone comparison on the increase of the prejunctional GABRG3 and GABRR2. Expression of GRIA2 was not detectable. Taken together, it effect of angiotensin II in the rat left ventricle 1 2 3 1 1 1 1 cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on Moura D. , Jäger J. , Posch B. , Ferreira I. , Pereira L. , Silva F. , Bastos R. , Pinheiro 1 mast cell activation is due to an action at TSPO or at GABA-A receptors of a novel H. subunit composition. 1University of Porto Department of Pharmacology and Therapeutics, Faculty of Medicine and Institute of Molecular and Cellular Biology, Alameda Hernani Monteiro, 4200-319 Porto, Portugal 2Medizinische Universität, Innsbruck, Austria 259 3Leopold-Franzens-Universität, Innsbruck, Austria
AT-1 receptor antagonists block the angiotensin II-enhancing effect on noradrenaline Sulfotransferases activate the food carcinogens 5-hydroxymethylfurfural and release from sympathetic neurons. In a cell-free assay the binding affinity of the AT-1 furfuryl alcohol to DNA-reactive metabolites in vitro and in animal models in vivo receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferator- activated receptor (PPARγ) is close to that of the PPARγ selective agonists Monien B. H., Glatt H. thiazolidinediones (TZDs). We tested whether the TZDs rosiglitazone and pioglitazone German Institute of Human Nutrition (DIfE) Department of Nutritional Toxicology, Arthur- would also modify the prejunctional facilitatory effect of angiotensin II. Scheunert-Allee 114-116, 14558 Nuthetal, Germany Left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. The negative logarithm of the drug concentration that caused a 5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are common constituents of 30% increase of control (pEC30%) was calculated. foodstuffs in which they are formed by heat- and acid catalyzed reactions from Angiotensin II caused a concentration-dependent increase of tritium overflow induced by carbohydrates. HMF and FFA have been reported to induce the formation of electrical stimulation [pEC30%=8.6±0.2 (mean±SEM, n=18); maximum increase=110±8%]. hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, Neither rosiglitazone nor pioglitazone (0.3-3 µM) had a direct effect. The concentration- respectively. We studied whether the carcinogenic effect of these hydroxymethyl- response to angiotensin II in the presence of fixed concentrations of rosiglitazone was substituted furans may originate from sulfotransferase (SULT)-catalyzed formation of shifted to the left with increase of the maximum (pEC30%=8.8±0.2, 9.2±0.2 and 9.3±0.3; electrophilic esters. HMF was inactive in in vitro mutagenicity tests using standard maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µM activating systems. In contrast, it was mutagenic in V79 cells genetically engineered for of rosiglitazone, respectively, n=4-6, each). In contrast, pioglitazone in concentrations up to expression of human SULT1A1 suggesting that HMF is converted into the reactive 5- 3 µM had no effect on the release-enhancing effects of angiotensin II. sulfooxymethylfurfural (SMF). Following incubation of mutagenic SMF with porcine liver Results show that rosiglitazone but not pioglitazone potentiates the noradrenaline- DNA in vitro, specific methylfurfural adducts were detected using liquid chromatography release enhancing effect of angiotensin II. This action might contribute to the risk for tandem mass spectrometry (LC-MS/MS), i.e., N6-((5-formylfuran-2-yl)methyl)-2'- 6 2 2 myocardial infarction from rosiglitazone use but not from pioglitazone use. deoxyadenosine (N -FFMdA) and N -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (N - FFMdG). These adducts were also detected in DNA from V79-SULT1A1 cells incubated References with HMF. In order to determine sulfo conjugation of HMF in mice in vivo, we conducted Niessen SE, Wolsky K (2010) Rosiglitazone revisited: an updated meta-analysis of risks pharmacokinetic measurements showing that about 500 ppm of the HMF dose was for myocardial infarction and cardiovascular mortality. Arch Intern Med 170:1191-1201 converted to SMF and reached the circulation. Like HMF, FFA was negative in the standard Ames test and various other in vitro genotoxicity tests. We showed that FFA is mutagenic in Salmonella typhimurium TA100 engineered for expression of human SULT1A1. The putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver DNA, in which various nucleoside adducts were found. The main 262 adducts, N2-((furan-2-yl)methyl)-2'-deoxyguanosine (N2-MFdG) and N6-((furan-2- yl)methyl)-2'-deoxyadenosine (N6-MFdA) were synthesized, and LC-MS/MS quantification methods were devised. N2-MFdG and N6-MFdA were detected in DNA of The transcriptional coactivators Megakaryoblastic Leukemia 1/2 (MKL1/2) mediate FFA-exposed Salmonella strain TA100-SULT1A1 and in DNA of liver, lung and kidney of tumorigenesis upon loss of the tumor suppressor Deleted in Liver Cancer 1 (DLC1) 1 1 2 1 3 1 1 FVB/N mice that had received about 390 mg FFA/kg body weight per day via the Hampl V. , Khalid S. , Singer S. , Frank N. , Prywes R. , Gudermann T. , Muehlich S. drinking water for 28 days. In summary, both furan derivatives form mutagenic sulfate 1Ludwig-Maximilians-Universität München Walther-Straub-Institut für Pharmakologie und esters in vitro and in vivo. In the future, we will use genetically engineered mice to Toxikologie, Goethestr. 33, 80336 München, Germany characterize the role of single murine and human SULT forms in the bioactivation of the 2Uniklinikum Heidelberg Molekulare Hepatopathologie, Im Neuenheimer Feld 220/221, furan derivatives and the contribution to tumor induction. 69120 Heidelberg, Germany 3Columbia Universität Department of Biological Sciences, 1212 Amsterdam Avenue, New York City, New York 10027, United States
260 Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. Despite its significance, the molecular mechanisms that drive cancerous transformation upon DLC1 loss remain unclear. MicroRNA-132/212 - mediated regulation of MeCP2 in the heart We found that the transcriptional coactivators Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are constitutively localized to the nucleus in hepatocellular and mammary Monroy-Ordoñez E. B., Weiss S., Beetz N., Hein L. carcinoma cells that lack DLC1. Moreover, DLC1 loss and MKL1 nuclear localization Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstr. 25, correlated in primary human hepatocellular carcinoma. Nuclear accumulation of MKL1 in 79104 Freiburg, Germany DLC1-deficient cancer cells was accomplished by activation of the RhoA/actin signaling pathway and concomitant impairment of ERK-mediated MKL1 phosphorylation. DLC1 S61
loss led to constitutive activation of the MKL-dependent, tumor-relevant target genes After myocardial infarction (MI) inflammatory cells and cardiac fibroblasts (CF) determine CTGF, Cyr61, Myl9 and Myh9. Furthermore, we identified a novel target gene, Integrin the remodeling response. Interleukin-6 (IL-6) is induced in the ischemic myocardium and a5, with a key role in cell migration and metastasis, that exhibited a DLC1- and MKL- is known to stimulate the differentiation of fibroblasts to myofibroblasts. Hyaluronan (HA) dependent regulation. Depletion of MKL1/2 suppressed not only cell migration, but also is an extracellular matrix component synthesized by HA-synthase isoenzymes (HAS 1-3) cell proliferation and anchorage-independent cell growth induced by DLC1 loss. and is also known to control fibroblast phenotypes. However, it is presently unknown Our data provide insight into the mechanism by which DLC1 loss initiates tumorigenesis. whether IL-6 participates in the remodeling of the HA-matrix or whether the HA-matrix As MKL1 and 2 have a key role in this process, this pathway may provide promising modulates the responses to IL-6. pharmacological targets for cancer therapy. Therefore, the aim of the present study was to elucidate whether IL-6 regulates the expression and function of HA-matrix in CFs. Cells were isolated from C57Bl/6J mice and used during passage 2-3 for experiments. CFs were stimulated with IL-6 or Hyper-IL-6 which is a fusion protein of IL-6 and soluble 263 IL-6 receptor (sIL-6R). After 10 and 30 min signal transducer and activator of transcription 3 (STAT3) was phosphorylated in response to Hyper-IL-6 but not in response to IL-6. RT-PCR revealed rapid upregulation of HAS 1 (5.94 ± 2.49 fold of Development of Potent and Selective Ligands for P2 Purine and Pyrimidine unstimulated control, 1h) in response to Hyper-IL-6. HAS2 was induced to a lesser Nucleotide Receptors degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas HAS3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). In contrast, IL-6 had no effect on transcript Müller C. E. levels of HAS isoenzymes. In turn, expression of HAS1 and HAS2 in response to Hyper- Universität Bonn, Pharma-Zentrum Bonn Pharmazeutisches Institut, Pharm. Chemie I, IL-6 was inhibited by AG490, which indicates the involvement of STAT signaling. An der Immenburg, 53121 Bonn, Germany Interestingly, despite induction of HAS1 and HAS2 the amount of secreted HA as determined by an ELISA-like assay was not affected by Hyper-IL-6. This may indicate Membrane receptors activated by purine and/or pyrimidine nucleotides (“P2 receptors”) that IL-6 regulates the cell surface associated HA-matrix of CFs. are widely distributed in the body and constitute novel (potential) drug targets. They are In conclusion, the present data demonstrate that cardiac fibroblasts respond to IL-6 subdivided into G protein-coupled P2Y receptors (P2Y1,2,4,6,11,12,13,14), and homo- or trans-signaling (Hyper-IL-6) via the soluble IL-6R and subsequent STAT3 signaling with heterotrimeric ligand-gated ion channel or P2X receptors (subunits: P2X1-7). We have increased HA-synthesis. The fact that IL-6 had no significant effect suggests that the been interested in the identification and development of potent and subtype-selective expression of the non-signaling membrane-bound IL-6 α-receptor (IL-6R) in cultured ligands - as tool compounds and potential drugs - for the various P2Y and P2X receptor murine cardiac fibroblasts is not sufficient to induce HAS 1 and -2 gene expression. subtypes. Our strategy involves (i) establishment of a proprietary compound library Therefore, IL-6 trans-signaling mediated by IL-6 and the circulating sIL-6R might be consisting of synthetic small molecules and natural products; (ii) development of necessary to mediate the IL-6-induced HAS expression in vivo. screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. This approach has led to new biological tools for several targets, including P2Y and P2X receptors [1-6]. 266 References 1Baqi Y, Müller CE. Nat. Protoc. 2010, 5, 945-953. 2El-Tayeb A, Qi A, Nicholas RA, Müller CE. J. Med. Chem. 2011, 54, 2878-2890. MRGPRD receptor endogenously expressed in dorsal root ganglia: evidence for 3Baqi Y, Atzler K, Köse M, Glänzel M, Müller CE. J. Med. Chem. 2009, 52, 3784-3793. an activation by 3-aminoisobutyric acid 4Hoffmann K, Baqi Y, Morena MS, Glänzel M, Müller CE, von Kügelgen I. J. Pharmacol. Müller S., Hoffmann K., von Kügelgen I. Exp. Ther. 2009, 331, 648-655. Universität Bonn Institut für Pharmakologie und Toxikologie, Sigmund-Freud-Straße 25, 5Hillmann P et al. J. Med. Chem. 2009, 52, 2762-2775. 53127 Bonn, Germany 6Baqi Y, Hausmann R, Rosefort C, Rettinger J, Schmalzing G, Müller CE. J. Med. Chem. 2011, 54, 817-830. The GPCR MRGPRD (mrgD) is highly expressed in small diameter dorsal root ganglion (DRG) neurons and has been implicated to play a role in nociception. The receptor was previously shown to respond to β-alanine. In the present study we searched for agonistic activity of structural analogues of β-alanine. 264 Human MRGPRD was stably expressed in CHO Flp-In (Chinese hamster ovary) cells. For further characterization of the receptor we used Fura-2 fluorimetry, a NFAT luciferase reportergene assay and the determination of the inhibition of forskolin-induced 3 Measurement of particulate matter emissions from indoor environmental tobacco cAMP production ([ H]-cAMP affinity assay). First, we confirmed the activation of the smoke (ETS) receptor by β-alanine and GABA. In reportergene experiments we then identified 3-DL- 1 2 1 aminoisobutyric acid as an agonist, with similar potency but weaker affinity when Müller D. , Schulze J. , Groneberg D. compared to β-alanine (Ec 165 µM). Fura-2 fluorimetry showed an increase in 1 50 Goethe Universität Frankfurt/Main Institut für Arbeits-, Sozial und Umweltmedizin, intracellular Ca2+ levels by 3-DL-aminoisobutyric acid (300 µM). Moreover, 3-DL- Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany aminoisobutyric acid reduced the forskolin-induced cAMP production by up to 65 % 2 Goethe Universität Frankfurt/Main Dekanat, Theodor Stern Kai 7, 60590 (Ec50 195 µM). In addition to 3-DL-aminoisobutyric acid, we identified 3-DL-aminobutyric Frankfurt/Main, Germany acid as a weak agonist acting at the MRGPRD. Other closely related substances failed to show significant responses. Fine particles in particulate matter (PM) are effective vehicles to transport toxicants into the Next to the agonists we further characterized antagonists inhibiting the response to β- lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. alanine mediated by MRGPRD. Chlorpromazine shifted the concentration-response In recent years the PM fraction PM2.5 has especially been correlated with both pulmonary curve of β-alanine to the right with an apparent pKB of 4.9 (NFAT assay), thioridazine and cardiovascular diseases. In order to better characterize PM emission and distribution of with an apparent pKB of 5.4 (NFAT assay) and 5.3 (cAMP assay) and rimcazole with an environmental tobacco smoke (ETS) from cigarettes (reference cigarette (RC), brand apparent pKB of 5.2 (NFAT assay) and 5.2 (cAMP assay). cigarette (BC)) we have developed an ETS emitter to simulate human smoking emission In conclusion we show for the first time that 3-DL-aminoisobutyric acid is an agonist at the and measured PM2.5 concentration in a telephone booth (1,75 m3 volume) as an example MRGPRD and that the structure-activity relationship of agonists at MRGPRD is very close. for small indoor spaces like cars. Fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. For the PM2.5 concentration the following values were calculated: cumulative PM2.5 concentration as AUC-PM2.5 (µg/m3/sec), peak PM2.5 concentration as cmax- PM2.5 (µg/m3) and average PM2.5 concentration Cmean-PM (µg/m3). In closed door 267 condition both cigarettes produced particulate AUC-PM2.5 values of 59 000 ± 15 000 µg/m3/sec (RC) to 85 000 ± 31 000 µg/m3/sec (BC). When opening the door circulation reduced the AUC-PM2.5 by about 90% for both cigarettes (RC: 5 550 ± 3 942 µg/m3/s, BC: Homologous and heterologous phosphorylation of the CXCR4 C-terminal domain 7 186 ± 2 330 µg/m3/s). The cmax-PM2.5 values showed a similar reduction by 80% in Müller W., Schulz S., Stumm R. open door condition (RC: 185 ± 130 µg/m3; BC: 245 ± 84 µg/m3) compared to closed door Jena University Hospital - Friedrich Schiller University Jena Institute of Pharmacology condition (RC: 1 035 ± 227 µg/m3; BC: 1 536 +/- 513 µg/m3, p>0,05). This method and Toxicology, Drackendorferstrasse 1, 07747 Jena, Germany measures smoking exposure in confined spaces like cars; the results will allow quantification of PM exposure in relation to spatial parameters and allow a better estimation The SDF-1-chemokine receptor CXCR4 plays a key role during embryogenesis and of toxic burden in e.g. cars or small offices. The method may also be modelled to other regulates functions of immune and stem cells in adult life. Furthermore, CXCR4 is microenvironments, which currently are not amenable to direct measurements. involved in disease states including inflammation and cancer. It is well established that SDF-1-stimulated CXCR4 receptors activate Gi protein-dependent signal transduction pathways and undergo C-terminal phosphorylation and internalization. Because the CXCR4 C-terminal domain contains 15 serine and 3 threonine residues, it is 265 incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of CXCR4. Here, we analyzed the phosphorylation pattern of CXCR4 at 3 C-terminal sites after stimulation of the receptor Interleukin-6 trans-signaling induces hyaluronan matrix remodeling in cardiac with SDF-1 and after PMA-induced activation of the PKC pathway as a model for fibroblasts heterologous receptor phosphorylation. Using phospho-specific antibodies against 1 1 2 3 2 1 S324/325, S338/339 and S346/347 in immunoblot analyses, we showed that the 3 sites Müller J. , Kretschmer I. , Garbers C. , Rose-John S. , Scheller J. , Fischer J. W. were phosphorylated after stimulation with SDF-1 or PMA. Stimulation with EGF or 1 Institut für Pharmakologie und Klinische Pharmakologie Universitätsklinikum der forskolin did not induce phosphorylation at these sites. SDF-1-induced phosphorylation Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany at S324/325, S338/339 and S346/347 was reversible after wash out of the ligand. Time 2 Institut für Biochemie und Molekularbiologie II Universitätsklinikum der Heinrich-Heine- course analyses revealed that phosphorylation occurred first at S346/347 and then at Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany S324/325 and S338/339. Taken together, these results indicate that the C-terminus of 3 Biochemisches Institut Christian-Albrechts-Universität zu Kiel, Olshausenstraße 40, CXCR4 is phosphorylated at multiple sites by homologous and heterologous pathways 24098 Kiel, Germany and that phosphorylation at the different sites may be hierarchically organized.
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268 [3] Muñoz, K, Blaszkewicz, M, Degen, G.H. 2010. J Chromatogr B 878, 2623-2629 [4] Muñoz K, Campos V, Blaszkewicz M, Vega M, Alvarez A, Neira J, Degen GH. 2010. Mycotoxin Research 26: 59-67 Lactational Transfer of the Mycotoxin Ochratoxin A in Humans Munoz K.1, Campos V.2, Blaszkewicz M.1, Vega M.2, Degen G. H.1 1Leibniz-Institut für Arbeitsforschung an der TU Dortmund (IfADo), Ardeystr. 67, 44139 Dortmund, Germany 270 2University of Concepcion, Faculty of Pharmacy Department of Food Sciences, Barrio Universitario s/n, Concepcion, Chile The Bioactivation of the Major Infertility Drug Clomiphene Depends on CYP2D6 Human milk represents the best form of nutrition for infants early in life. However, it can Polymorphism 1 1 1,2 1 1 3 1 also contain toxic contaminants that may adversely affect infant’s development. The Mürdter T. , Kerb R. , Turpeinen M. , Schroth W. , Ganchev B. , Böhmer G. , Igel S. , 1 1 1 1,3 nephrotoxin ochratoxin A (OTA) is present in human milk (Tab. 1 in [1]), but information Schaeffeler E. , Zanger U. , Brauch H. , Schwab M. on transfer from maternal blood to milk is scarce: Published data [2] indicate that levels 1Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University of OTA in milk are roughly one tenth (0.1) of those in blood. But, the efficiency of the Tübingen, Auerbachstr. 112, 70376 Stuttgart, Germany OTA-transfer at various stages of breastfeeding may vary since studies in animals 2University of Oulu Institute of Biomedicine Pharmacology and Toxicology, Aapistie 5B, revealed that transfer of OTA is apparently time- and dose-dependent. Thus, the aim of 90220 Oulu, Finland this study was to assess the OTA transfer from blood to milk at different stages of 3University Hospital Tübingen Department of Clinical Pharmacology, Ottfried-Müller Str. breastfeeding in humans. 45, 72076 Tübingen, Germany In a small Chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. These samples were collected on four different occasions within the first A mixture of (E)- and (Z)-Clomiphene citrate is the first line therapy of female infertility. months after delivery and analyzed using HPLC with fluorescence and/or tandem mass However, up to 30% of patients do not respond. (E)-Clomiphene is structurally closely spectrometric detection [1]. The transfer of OTA from blood to milk was quantitatively related to another selective estrogen receptor modulator, tamoxifen which is frequently used assessed by measuring the milk to plasma ratio (M/P). for the treatment of hormone receptor-positive breast cancer. Like tamoxifen, clomiphene is The average OTA level in blood plasma was 235 ± 129 ng/L, and no major variations extensively metabolised by the cytochrome P450 system. Using the estrogen receptor were observed over time (p = 0.19). On the other hand, OTA levels found in colostrum response assay (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene (EC50: (85 ± 60 ng/L) were higher than in mature milk (p < 0.05). In line with these data, higher 2.5 and 1.4 nM, respectively) turned out to be 100 times more active at the ER compared to M/P ratios (Table) were obtained with samples collected in the first six days after the parent drug isomers and de-ethylated metabolites. Using recombinant expressed delivery. human CYP isoforms and inhibitory antibodies, CYP2D6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. N-deethylation was Table: Transfer of OTA from blood to milk in a four months follow up study catalysed by CYPs 3A4/5, 2D6, 2C19 and 2C8. Rates of 4-hydroxylation in microsomes Day 1 to 6 15 to 30 days 2 months 4 months from 30 human liver donors correlated with the number of functional CYP2D6 genes. These M/P 0.43 ± 0.26 0.15 ± 0.13 0.09 ± 0.07 0.24 ± 0.17 in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. In carriers of two non-functional CYP2D6 genes This study showed that the transfer of OTA from blood to milk was more efficient with (poor metabolizers) Cmax of (E)-4-hydroxyclomiphene and 4-hydroxy-N- colostrum (M/P 0.43 ± 0.26) than with mature milk. Thus, a higher exposure to OTA can desethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects be expected for neonates than for infants at later stages of breastfeeding. Moreover, the with at least one fully functional CYP2D6 allele. In contrast, half-life of (E)-clomiphene and lactating women have lower average OTA levels in plasma than non lactating women (E)-N-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. from Chile [3], indicative of milk as additional excretion route. Our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. Acknowledgement: This work has been supported by a stipend from CONICYT/DAAD to KM. Supported by the Robert Bosch Foundation (Stuttgart, Germany), the BMBF and the IZEPHA (Germany) and the Academy of Finland (Helsinki, Finland) References [1] Muñoz K, Campos V, Blaszkewicz M, Vega M, Alvarez A, Neira J, Degen GH. 2010. First biomonitoring results in human milk (colostrum) from Chile. Mycotoxin Research 26: 59-67 271 [2] Breitholtz-Emanuelson A, Olsen M, Oskarsson A, Palminger I, Hult K, 1996. Ochratoxin A in cow’s milk and in human milk with corresponding human blood samples. J AOAC Int.76: 842-846 Toxic effects of gold-(I)-complexes in Hct 116 human colon carcinoma cells [3] Muñoz K, Vega M., Rios G., Muñoz S., Madariaga R. (2006) Preliminary study of Nakhla A.1, Ackermann D.1, Havermann S.1, Kunz P. C.2, Braun M.2, Fritz G.1, Wätjen Ochratoxin A in human plasma in agricultural zones of Chile and its relation to food 1 consumption. Food and Chemical Toxicology 44 (11), 1884 – 1889 W. 1 Heinrich-Heine-University Düsseldorf Institute of Toxicology, P.O. Box 101007, 40001 Düsseldorf, Germany 2Heinrich-Heine-University Düsseldorf Institute of Inorganic Chemistry and Structural 269 Chemistry I, Universitätsstraße 1, 40001 Düsseldorf, Germany
Gold-(I)-complexes are interesting for pharmaceutical research due to their antiproliferative properties. Several classes of gold-based drug candidates have been Non-invasive biomonitoring to assess infant exposure to ochratoxin A 1 1 2 2 1 described previously. Distinct complexes could be candidates for the development of Munoz K. , Blaszkewicz M. , Cramer B. , Humpf H. - U. , Degen G. H. new metal-based anticancer drugs. Recently, we developed four imidazol-2-yl-gold-(I)- 1 + Leibniz-Institut für Arbeitsforschung an der TU Dortmund (IfADo), Ardeystr. 67, 44139 complexes with the general structure [L2NHC-Au-NHCL2] (NHC: imidazol-2-yl, L: ligand Dortmund, Germany in position 4 or 5): 1: L = 2 COOEt, 2: L = COOEt, 3: L = 2 Cl, 4: L = coffeine. These 2Westfälische Wilhelms Universität Münster Institute of Food Chemistry, Corrensstraße gold-(I)-complexes showed a cytotoxic potential in Hct 116 colon carcinoma cells. 45, 48149 Münster, Germany Among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µM. Compounds 2 and 3 showed Exposure of infants to ochratoxin A (OTA) deserves particular attention since OTA is significant toxic effects at a concentration of 5 µM. The compound 4 showed no toxicity nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1]. up to a concentration of 100 µM. All derivatives 1, 2 and 3 have a EC50 between 10 Moreover, infants may be more vulnerable to the toxic effects of contaminants than and 15 µM. We further proved the induction of apoptosis by Apo-ONE assay (caspase adults. OTA-levels in plasma of infants are indicative of an early exposure in life [2]. But 3/7 activity) and life/dead-assay (fluorescence microscopy). In conclusion, these gold blood sampling is an invasive method not readily applicable for breastfed infants. Thus, complexes exhibit an example of interesting potential candidates for future anticancer the aim of this study was to implement a non invasive biomonitoring method to assess pharmaceuticals due to relatively high cytotoxicity. OTA-exposure in this group. To assess the exposure to OTA, breast milk and infants’ urine specimens were collected, from two different cohorts: Chile (n= 28) and Turkey (n=10, only urine). Analysis of the samples was performed using enzymatic hydrolysis prior to extraction 272 and HPLC-MS/MS [3]. The magnitude of infants’ exposure was assessed by calculating the OTA-daily intake with human milk and relating it also to urinary OTA levels. Calculations of the daily intake with human milk [4] showed that infants may be exposed Gene regulating effects in mouse liver subsequent to treatment with selected to OTA at high levels, exceeding the tolerable daily intake (TDI) of 5 ng/Kg-bw/day set dioxin-like compounds and PCB 153 using whole genome microarray analysis for adults [1]. In both cohorts, most of the urine samples tested positive for OTA (Chile Neser S.1, Lohr C.1, van Ede K. I.2, Andresen K.3, van Duursen M. B. M.2, van Den Berg 72%, Turkey 80%). OTA levels observed in urine samples from the Turkish infants 2 4 1 (range: 116 -1,361 ng/L) were 10 fold higher than levels found in Chilean samples M. , Andersson P. L. , Schrenk D. 1 (range positive samples: 17 – 320 ng/L). Further analysis of phase II metabolites in urine University of Kaiserslautern Food Science and Toxicology, Erwin-Schroedinger-Str. 52, confirm the excretion of OTA as conjugate (glucuronide) in highly exposed infants. 67663 Kaiserslautern, Germany 2 In conclusion, OTA exposure of infants early in life was documented. Given that OTA- Utrecht University Institute for Risk Assessment Sciences, PO Box 80176, 3508 TD intake by several infants exceeded the TDI for adults, further biomonitoring in this Utrecht, Netherlands 3 vulnerable group is advised including also suitable biomarkers of effect. Institute of Biotechnology and Drug Research, Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany 4 References Umeå University Department of Chemistry, SE-901 87 Umeå, Sweden [1] Kuiper-Goodman T, Hilts C, Billiard SM, Kiparissis Y, Richard I, Hayward S, 2010. Food Add and Contam 27: 212-240 Toxic responses associated with certain polychlorinated biphenyls (PCBs), dibenzo-p- [2] Hassan A, Sheashaa H, Abdel Fattah M, Ibrahim A, Gaber O, Sobh M, 2006. Pediatr dioxins (PCDDs), and dibenzofurans (PCDFs) include dermal toxicity, immunotoxicity, Nephrol 21: 102-105 carcinogenicity, as well as adverse effects on reproduction, development, and endocrine functions. The ‘dioxin-like’ (DL) compounds reveal their biological and toxic effects via S63
interaction with the aryl hydrocarbon receptor (AhR), with 2,3,7,8-tetrachlorodibenzo-p- 275 dioxin (TCDD) being the most potent congener amongst the AhR agonists. Recent risk assessments have employed the toxic equivalency factor (TEF) concept. The current EU-project SYSTEQ aims at developing, validating, and implementing human systemic Impact of genetic and non-genetic factors for interindividual variability of TEFs as indicators of toxicity for DL-compounds. At present, the best known parameter SLC22A7/OAT2 expression in human liver of AhR mediated effects is the induction of cytochrome P450 isoenzymes (CYPs), i.e., Resch C.1, Schaeffeler E.1, Zanger U.1, Schwab M.1,2, Nies A.1 CYP1A1, 1A2, and 1B1. One of the major objectives of the SYSTEQ project is the 1 identification of novel quantifiable biomarkers. In a three day study, female C57BL/6 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany mice were treated with single doses of six DL-congeners (TCDD, 1-PnCDD, 4-PnCDF, 2 PCB 118, PCB 126, and PCB 156), and the 'non-dioxin-like' (NDL) PCB 153. Quality Institute of Experimental and Clinical Pharmacology and Toxicology, University Hospital tested (Agilent® Bioanalyzer) mRNA isolated from livers was analyzed using the Agilent® Tübingen Department of Clinical Pharmacology, Otfried-Müller-Straße 45, 72076 mouse whole genome array (4x44K) system. The quantity of genes affected (≥ 2fold) Tübingen, Germany was highly heterogeneous amongst the DL-compounds. Whereas TCDD-treatment up- regulated 89 genes, and down-regulated 66, 4-PnCDF-treatment had impact on 3208 Introduction: Human organic anion transporter 2, OAT2 (SLC22A7), is abundantly (up), and 2396 (down). Treatment with PCB 118 led to marginal numbers of 16 up and 6 expressed in kidney and liver and mediates the sodium-independent uptake of clinically down-regulated genes. With 64 (up), and 38 (down) genes shared, the most extensive relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. overlap occurred between TCDD- and 1-PnCDD-treatment. No overlap was found due While immunohistochemical studies have localized human OAT2 to the basolateral to treatments with the NDL PCB 153 (21 up, 1 down) and TCDD. When comparing the membrane of kidney proximal tubules, its hepatic localization is currently unknown. We, effects of all DL-congeners, minor numbers of genes of 19 up, and 5 down-regulated therefore, firstly determined OAT2 localization in human liver. Because interindividual remain, most of them being related to drug metabolism. While PCB 118 regulated only variability of OAT2 expression may affect hepatobiliary drug uptake and elimination, we genes involved in drug metabolism, omission of PCB 118-regulated genes resulted in next systematically investigated the influence of genetic and non-genetic factors on consistently 51 (up), and 24 (down) regulated among DL-compounds. In conclusion, our hepatic OAT2 expression. findings suggest that the pattern of gene regulation in mouse liver elicited by PCB 153 was strictly different from TCDD, while a very limited coincidence of genes was found Methods: An expression profile of OAT2 for 20 human tissues was determined by real- even among DL-compounds. Comparison of these ‘core’ genes with data from human time quantitative polymerase chain reaction (TaqMan). OAT2 mRNA expression was models is required with respect to determination of novel biomarkers. analyzed in well-characterized human liver samples from 150 Caucasians that were accompanied by detailed demographic and clinical data. OAT2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic OAT2 protein levels were determined. Resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single 273 nucleotide polymorphism microarray technology served to genotype variants in the SLC22A7 gene region.
Patient need for sufficient information regarding systemic antibiotics – incidence Results: OAT2 mRNA was expressed in several human tissues, including liver. and reason for enquiries at a drug information service for patients Moreover, a new alternatively spliced variant of OAT2 was identified in human liver. 1 1 1 2 Neubauer P. , Tuttas K. , Goltz L. , Kirch W. Hepatic expression of full-length OAT2 mRNA and OAT2 protein varied 37-fold and 41- 1Institut für Klinische Pharmakologie, TU Dresden Arzneimittelberatungsdienst, fold, respectively. OAT2 mRNA and protein levels did not correlate with each other. Fiedlerstraße 27, 01307 Dresden, Germany OAT2 was localized to the sinusoidal membrane of human hepatocytes. No novel 2Institut für Klinische Pharmakologie, TU Dresden, Fiedlerstraße 27, 01307 Dresden, variants in the 9 exons, the 5’-flanking region, or the 3’-untranslated region of the Germany SLC22A7 gene were identified. Univariate analysis showed that OAT2 mRNA is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. Introduction: Proper use of antibiotics is essential with regard to effective treatment of bacterial infections. Providing adequate information for patients can contribute to Conclusions: Whereas the influence of genetic variants on hepatic OAT2 expression achieve this aim. appears to be limited, cholestasis significantly contributes to the variable interindividual Materials and methods: Data was collected from the relational database of the drug OAT2 expression. This indicates consequences for hepatic drug elimination of and information service at Dresden University of Technology. The patients, who used the response to OAT2 drug substrates such as paclitaxel or tetracycline. service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. Possible contact paths were Supported by the Robert-Bosch Foundation, Stuttgart, Germany, the BMBF grant phone, e-mail or letter. In the present evaluation, all enquiries from the years 2009 and 03IS2061C (Berlin, Germany) and the FP7-grant PITN-GA-2009-238132. 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. Results: In the evaluated period, 4454 enquiries were registered in total. In 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 276 50.9% of those antibiotics were found to be the direct reason for the enquiry. Most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-drug- Interaction of bispyridinium compounds with the orthosteric binding site of interactions (19.4%). The majority of the requesting patients (61.5%) was born before human α7 nicotinic acetylcholine receptors (hα7 nAChRs) 1970. A correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. Niessen K. V., Seeger T., Thiermann H., Worek F. Conclusion: Mainly patients aged 40 years or more seem to need or search for further Institut für Pharmakologie und Toxikologie der Bundeswehr, Neuherbergstr. 11, 80937 information about antibiotic medication. Advice is required especially regarding adverse München, Germany drug reactions and diagnosis or treatment. In order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. Introduction The life threatening toxicity of organophosphorus (OP) nerve agents is caused by the inhibition of the acetylcholine esterase (AChE). Oximes were shown to be potent reactivators of inhibited AChE, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. For such cases, an alternative new strategy may be 274 the intervention at nicotinic acetylcholine receptors (nAChR). Previous studies with the bispyridinium non-oxime MB327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct Active secretion of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) in interaction with nAChRs [1]. We investigated the interaction of MB327 and several the colon structure analogous at the orthosteric binding site of human α7 nAChR (hα7 nAChRs), a 1 2 2 1 1 von Keutz A. , Schröder B. , Breves G. , Steinberg P. , Nicken P. subtype which appears to be widespread in the human body, and compared the results 1Tierärztliche Hochschule Hannover Lebensmitteltoxikologie und Chemische Analytik, with data obtained from Torpedo-nAChRs, which show a high degree of homology with Bischofsholer Damm 15, 30173 Hannover, Germany human muscle-type nAChRs. 2Tierärztliche Hochschule Hannover Physiologisches Institut, Bischofsholer Damm 15, 30173 Hannover, Germany Methods Interaction of compounds with the orthosteric binding site of hα7 nAChRs were Colon cancer is one of the most frequent cancers in the industrialized nations. investigated with radioligand binding experiments performed as high-throughput method Epidemiological studies show a correlation between highly processed meat and the [2]. Membrane preparations of GH4C1 cells stably expressed hα7 nAChRs were development of colorectal tumours. It is assumed that the risk of developing colorectal incubated with the nAChR agonist [³H] epibatidine and appropriate concentrations of the cancer, among various different factors, is related to the uptake of toxic substances unlabelled competitors e.g. bispyridinium compounds. After incubation, bound and free contained in food such as heterocyclic aromatic amines that arise during the processing [³H] epibatidine were separated by rapid vacuum filtration. Ki values of the competing of fish and meat. PhIP is the most abundantly formed heterocyclic compound, and compounds were calculated with nonlinear regression. therefore has the biggest impact. In a previous study, we measured the absorption of PhIP in different intestinal segments Results of the rat. In the present study we focussed on the potential mechanisms by which PhIP Three bispyridinium compounds, MB442, MB456 and MB770 exhibited Ki values at is reabsorbed. The unidirectional PhIP transport from the mucosal to the serosal micromolar concentrations while three other compounds, MB327, MB583 and the compartment (Jms) and in the opposite direction (Jsm) was examined using the Ussing pharmacological inactive MB424 (negative control) did not show any interaction with the 14 chamber technique and C-PhIP as a radiotracer. The proximal jejunum and distal orthosteric binding site of hα7 nAChRs. With Torpedo-nAChRs, Ki values were in similar colon of male Fischer 344 rats in short-circuit current chambers was clamped, so that orders of magnitude – except MB442 which indicated significant subtype selectivity. mucosal and serosal compartments were built. The PhIP flux rates were determined at Interestingly, the affinity of monomeric pyridinium derivates did not correlate with their defined intervals over 120 min. The experimental conditions were selected in such a way bispyridinium structure analogues. that negative net flux rates (Jnet = Jms-Jsm) were indicative of an active secretion. Both intestinal segments showed large differences. While in the jejunum Jms and Jsm of PhIP were not significantly different, there was an active secretion in the colon. In a next step the transport proteins involved in this process should be examined. S64
Conclusion 279 Obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. Role of Gα proteins in immune function and infection i Novakovic A.1, Wiege K.2, Beer-Hammer S.1, Gessner J. E.2, Nürnberg B.1 References 1 [1] S.R. Turner et al. Toxicol. Lett. 206 (2011), 105-111 Institut für Pharmakologie und Toxikologie Abteilung für Pharmakologie und Experimentelle Therapie, Wilhelmstr. 56, 72074 Tübingen, Germany [2] K.V. Niessen et al., Toxicol. Lett. 206 (2011), 100-104 2 Klinik für Immunologie und Rheumatologie, Medizinische Hochschule Hannover, Carl- Neuberg-Str. 1, 30625 Hannover, Germany
Most chemoattractants, including chemokines, complement C5a, fMLP, and leukotriene 277 B4 are signaling through heterotrimeric G proteins of the Pertussis toxin (PTx)-sensitive Gi family. The functional inactivation of all Gαi proteins with PTx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single Chloroethylnitrosourea induced genotoxicity is counteracted by DNA double- Gα coding gene results in mild phenotypes in mice. We are mostly interested in the non- strand break repair i neuronally found Gαi2 and Gαi3 isoforms and their redundant and specific roles in Nikolova T., Hennekes F., Bhatti A., Kaina B. immune function and infection. For this purpose cellular in vivo and ex vivo models and Universitätsmedizin der Johannes-Gutenberg Universität Institut für Toxikologie, Obere- in vivo infection model with Listeria monocytogenes are being used. Zahlbacher Str. 67, 55131 Mainz, Germany Macrophages were isolated from the peritoneal cavity of wild type (wt) and Gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. We In this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug confirmed previous observations that in Gαi2-deficient mice the migration of nimustine (ACNU) in cells deficient in repair proteins involved either in homologous macrophages into the peritoneal cavity was reduced after induction of peritonitis. recombination (HR) or non-homologous end-joining (NHEJ). We show that HR mutants Regarding the expression levels of Gαi and Gβ isoforms in the lavage samples, the are extremely sensitive to ACNU as measured by the induction of apoptosis and colony predominant Gαi isoform Gαi2 was upregulated in Gαi3-deficient macrophages. Vice formation as well as the induction of chromosomal aberrations. The NHEJ mutants were versa Gαi3 was upregulated in Gαi2-deficient macrophages. Concerning Gβ isoforms, slightly sensitive to ACNU and differed in their sensitivity, with the Ku80 mutants being both Gβ1 and Gβ2 were strongly reduced in the Gαi2-deficient macrophages which moderately sensitive and the DNA-PKcs mutant resistant, comparable to the wild-type resulted in a reduced total amount of Gβ. Surprisingly, the Gαi3-deficient macrophages (wt). Cell death was mostly executed via the caspase-dependent apoptotic pathway with showed reduced Gβ1 protein levels only which caused a change in the Gβ2/ Gβ1 quotient involvement of caspase-3 and -7, and necrosis was also induced. Further, we in favour of Gβ2. investigated the kinetics of DNA double-strand break (DSB) formation that resulted from We are currently establishing an in vivo infection model with L. monocytogenes in Gαi2- the repair of ACNU-induced interstrand cross-links by means of γH2AX and 53BP1 foci and Gαi3-deficient mice. Our previous in vitro infection studies in mice embryonic analysis in wt and mutant cells. Cells mutant in HR did not repair DSB and went into the fibroblasts provided us with information about possible distinct roles of these two apoptotic or necrotic pathway, whereas wt cells were able to repair most of the DSB. isoforms as far as the uptake of L. monocytogenes in the cells is concerned. Challenging Cells deficient in Ku80 formed at early times after ACNU treatment less γH2AX and the immune system of Gαi-deficient mice with this pathogenic organism will give us new 53BP1 foci compared to the corresponding wt, which might be due to a reduced capacity insights into the systemic immune response in these mice upon bacterial infection. of recognising DSB. At later times after treatment, Ku80 mutant cells show foci levels Our data indicate that we may surmount the redundancy between these two isoforms similar to the wt indicating restitution of H2AX phosphorylation. We also analysed and focus on their distinct and specific roles in pathogen defense. whether DSB formation after ACNU treatment was replication-dependent using synchronised cells. We determined the formation of γH2AX and other DSB marker in wt cells that passed through the first cell cycle after demecolcine synchronization. The level of γH2AX foci increased significantly in the S-phase and remained at a high level during 280 G2 where a fraction of cells remained arrested. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX. These foci were ameliorated significantly in S- and G2-phase, which was similar to the time course of γH2AX foci FRET-based β-Arrestin2 biosensors reveal conformational changes upon binding formation. In western blots, we confirmed a higher phosphorylation level of ATM and to the β -adrenergic receptor in real time and living cells ChK2 and less phosphorylation of CHK1 in HR mutants. The data indicate that ACNU- 2 induced DNA cross-links give rise to cyto- and genotoxicity via the formation of DSBs Nuber S., Zabel U., Ziegler N., Hoffmann C., Lohse M. J. that activate the cellular DNA damage response. Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacherstr.9, 97078 Würzburg, Germany References 1. Nikolova T, Ensminger M, Lobrich M and Kaina B. Homologous recombination β-Arrestins are multifunctional adapter proteins that regulate seven transmembrane- protects mammalian cells from replication-associated DNA double-strand breaks arising spanning receptor (7TMR) signaling and initiate also alternative signaling pathways. in response to methyl methanesulfonate. DNA Repair (Amst) 2010; 9:1050-63. Studies have shown that β-Arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. To investigate these conformational changes in living cells we constructed FRET based biosensors of β-Arrestin2, in which CFP was fused to the C-terminus and the FlAsH- 278 binding motif (CCPGCC) was inserted to different positions within the N- or C-domain of β-Arrestin2. Upon β2-adrenergic receptor (β2AR) stimulation we observed a decrease of the intramolecular FRET signal between CFP and FlAsH at the N-domain (β- Arrestin2FlAsH2), indicating a conformational change moving the C-terminus and the N- Insights into CB1 Molecular Mechanism and Its Role in Pain Perception domain of β-Arrestin2 relative to each other. Kinetic analysis revealed that this 1 1 2 1 Njoo C. , Agarwal N. , Lutz B. , Kuner R. conformational change immediately follows β-Arrestin2/β2AR interaction on a timescale 1 Pharmakologische Institut Universität Heidelberg AG Kuner, INF 584, 69120 of seconds. A β2AR mutant that was previously shown not to interact with β-Arrestin2 Heidelberg, Germany was utilized as control and did not induce a conformational change in the β-Arrestin2 2Institute of Physiological Chemistry University Medical Center of the Johannes molecule. Our data provide evidence that β-Arrestin2 changes it`s conformation upon Gutenberg University Mainz, Mainz, Germany binding to the activated β2AR in living cells. The β-Arrestin2FlAsH2 sensor could serve as universal biosensor for GPCR activation. The endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. Cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. However, in human the activation of cannabinoid 281 receptors is associated with side effects. For clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without Studies on the physiological role of annexin A4 in the heart attenuating their analgesics effect. In animal studies, the anti-nociceptive efficacy of 1 1 1 2 2 1 cannabinoids has been unequivocally demonstrated in several models of inflammatory Nunes F. , Husser X. , Schulte K. , Kaetzel M. A. , Dedman J. R. , Schmitz W. , Müller F. U.1 and neuropathic pain. However, there are marked inconsistencies between different 1 reports with respect to the locus of these pain-protective effects. WWU Münster, Institut für Pharmakologie und Toxikologie, Domagkstraße 12, 48149 Münster, Germany We are working towards establishing the contribution of CB1 receptors expressed on the 2 peripheral terminals of nociceptors to cannabinoid-induced analgesia. Using CB1 University of Cincinnati, Genome Research Institute, Galbraith Rd, Cincinnati, 45237, globally knock-out animal as background, we induce the expression of CB1 specifically United States in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. Our results support the The calcium binding protein annexin A4 has been examined in the context of heart development of peripherally acting CB1 analgesic agonist with reduced central side failure in the past. Annexin A4 expression level was found to be elevated in ventricles of effects. Furthermore, we are utilizing proteomics approach to identify protein complexes human failing heart in comparison to expression levels in non-failing ventricles. that interact with CB1 receptor which hold promise in understanding cannabinoid Furthermore the intracellular localization pattern in atrial cardiomyocytes was found to signaling in health and disease. be altered in the failing human heart (Moravec and Matteo, Cardiovasc Res 2000). In order to gain insight into the possible physiological significance of these findings we References utilized an annexin A4 gene trap model (GT) in which the annexin A4 protein content Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, Rubino T, was not detectable in ventricles and atria. Measurements of sarcomere shortening and Michalski CW, Marsicano G, Monory K, Mackie K, Marian C, Batkai S, Parolaro D, calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged Fischer MJ, Reeh W, Kunos G, Kress M, Lutz B, Woolf CJ, Kuner R (2007) calcium transient decay at stimulation frequencies of 0.5 Hz, 1Hz and 2 Hz as well as an Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in increased sarcomere shortening at 1 Hz and 2 Hz in anxa4 gene trap animals in nociceptors. Nat Neurosci 10:870–879 comparison to wild type (WT) (Table; *= p<0.05 vs. WT; n=57-60/6).
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0.5 Hz 1 Hz 2 Hz drugs the corresponding HPLC method was selected: a Purospher RP18 column (55 mm x 2 mm; 4 µm, MERCK) and a mobile phase with a steep acetonitrile gradient. WT GT WT GT WT GT Results: The great advantage of having analytes with different molecular masses and sarcomere 0.077 0.070 0.082 0.102 0.068 0.095 similar retention times in combination with MS/MS detection enabled us to aim at a shortening [µm] ± 0.006 ± 0.005 ± 0.006 ± 0.007* ± 0.006 ± 0.006* minimum separation that might remove some salts or matrix components that can transient time to 0.22 0.27 0.16 0.18 0.12 0.13 suppress or interfere with the analyses from the target components, while maintaining baseline [ms] ± 0.006 ± 0.006* ± 0.003 ± 0.003* ± 0.002 ± 0.002* good sample throughput. The method was validated. The assay is precise, accurate, fast, sensitive, and selective. The effects of the β-adrenoreceptor agonist isoprenaline (Iso) on the shortening of Discussion: The developed method is suitable for therapeutic drug monitoring of ventricular cardiomyocytes was increased in GT as compared to WT (0,2±0.013 vs. retigabine. The correlation of the serum concentration and the effect of the drug and 0.17±0.012, *=p<0.05 vs. WT; N=14-18/5). Western blot analyses indicated that the thus the necessity of TDM have to be tested. expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) and the phosphorylation status of its regulator protein phospholamban (PLB) did not differ between groups (n=7). However, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with HAX1, which acts as a repressor of SERCA2a (n=4). We 284 performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10-9M–10-5M). The positiv inotropic effect -4 of isoprenaline was significantly increased in GT atria (rel. force at 10 M Iso [%]: WT: Targeting inflammatory T lymphocytes with conditional chemokine receptor 433±76; GT: 699±63 *= p<0.05 vs WT; n=8-10). In conclusion, annexin A4 contributes to antagonist expression for a tissue-specific therapy of chronic inflammatory the regulation of cardiomyocyte contractility. The anxa4 up-regulation might therefore disorders contribute to diminished cardiac performance in heart failure. Ogrissek N., Giegold O., Pfeilschifter J., Radeke H. H.
References Uniklinikum der Goethe-Universität pharmazentrum / ZAFES, Theodor-Stern-Kai 7, Matteo RG, Moravec CS. Immunolocalization of annexins IV, V and VI in thefailing and 60590 Frankfurt am Main, Germany non-failing human heart. Cardiovasc Res. 2000 Mar;45(4):961-70.PubMed PMID: 10728422. Chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. Several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. In previous experiments our group identified three promising antagonists. CXCL11(4-79) 282 has antagonistic function for CXCR3, CXCL12(P2G2) is able to inhibit CXCR4 and the herpesvirus 8 encoded protein vMIP-II interferes with CCR1, -2 and -5 as well as with CXCR3, -4 and CX3CR1. Their expression and secretion was confirmed in Pichia Structural and Functional Characterisation of Amphibian Constitutive Androstane pastoris and antagonistic function has been proven by a reduction of T cell migration. Receptor The aim of this project is to develop a cell-based therapy for chronic inflammation with a 1 1 1 1 1 1 Nußhag C. , Mathäs M. , Qiu H. , Gödtel-Armbrust U. , Römer K. , Wojnowski L. , treatment that is based on the collective effect of CXCL11(4-79), CXCL12(P2G2) and 2 Windshügel B. vMIP-II. With targeting of stable transduced memory T cells these antagonists should 1Universitätsmedizin Mainz Institut für Pharmakologie, Obere Zahlbacher Straße 67, be conditional expressed and secreted directly in the centre of inflammation, resulting in 55131 Mainz, Germany inhibition of further inflammatory T cell accumulation. 2Universität Hamburg Zentrum für Bioinformatik, Bundesstraße 43, 20146 Hamburg, To realize this project we first cloned constitutive lentiviral constructs containing these Germany antagonists and optimized transduction of T cells, such as the OVA-specific memory Th- 1 cell clone IF12 with the potential to initiate antigen specific nephritis in SCID mice. Background: Pregnane X receptor (PXR) is considered the most important sensor of Next we investigated expression and secretion of CXCL11(4-79), CXCL12(P2G2) and natural and anthropogenic xenobiotics in vertebrates. In contrast, the amphibian ortholog vMIP-II with PCR, Western Blot and ELISA. is involved in neural development and irresponsive to xenobiotics. Instead, the Xenopus At the moment we want to measure the inhibition efficiency of T cell migration in vitro laevis constitutive androstane receptor (CAR) was recently found to possess PXR-like with chemotaxis and flow chamber assays. Construction of an inducible lentiviral vector properties, featuring low basal activity and a pronounced ligand spectrum. Thus a plasmid to ensure expression of the antagonists only upon T cell activation, is also part structural and functional characterisation of X. laevis CAR may provide further insights of our current work. Finally we would like to test the chemokine receptor antagonists in into human CAR basal and ligand-induced activity. vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. Methods: The time-point of origin of CAR genes was determined by macrosynteny # § analyses of CAR, PXR, and VDR (vitamin D receptor) gene loci, which form the NR1I Supported by the DFG graduate school GRK1172 and Merck KGA subfamily of nuclear receptors. Based on a 3-dimensional protein model of Xenopus laevis CAR, docking studies with structurally diverse agonists were conducted. Protein- ligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human CAR. The organ response to CAR activators 285 was determined in Xenopus laevis using RNA microarrays. Results: CAR emerged together with PXR and VDR from an ancestral NR1I gene in early vertebrates via two whole-genome duplications. This was followed by losses of Impact of Single Nucleotide Polymorphisms located in the promoter of SHP-1 on CAR from the fish lineage and of PXR from Sauropsida (reptiles and birds). Amino acids SHP-1 transactivation important for ligand binding were identified. Structural features responsible for the Olbert M.1, Hentschel K.1, Böttcher K.1, Völzke H.2, Kroemer H. K.1, Meyer zu pronounced basal activity in human constitutive androstane receptor are not present in Schwabedissen H.1 X. laevis CAR. In human PXR the inter-helical loop in front of helix 3 is part of the ligand- 1 binding pocket and supposed to be responsible for the wide substrate spectrum. In Ernst Moritz Arndt University Department of Pharmacology, Felix-Hausdorff-Str. 3, 17489 Greifswald, Germany amphibian CAR this inter-helical loop plays no role in ligand binding. CAR agonists 2 resulted in a pronounced induction of antimicrobial peptides in the ovary. Ernst Moritz Arndt University Department of Community Medicine, Walter-Rathenau- Conclusions: CAR emerged already in early vertebrates and it is conserved in land Str. 48, 17475 Greifswald, Germany vertebrates, whereas xenosensing PXR is found only in the fishes and mammals. We provide a comprehensive modeling and mutational analysis of this first reported Small Heterodimer Partner 1 (SHP-1) is a member of the superfamily of nuclear amphibian xenosensor. The induction of antimicrobial peptides by CAR activators receptors (NRs). In contrast to other NRs this orphan receptor lacks the DNA binding suggests a link between xenosensing and innate immunity. The latter one may play a domain. However, SHP-1 is known to inhibit activity of several NRs by direct protein- previously unrecognized role in the amphibian reproduction. protein interaction. Importantly several of the interacting NRs have been shown to directly regulate SHP-1 expression, suggesting that SHP-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid- and drug metabolism and glucose homeostasis. Recently binding sites for NRs were identified in the promoter region of SHP-1, including HNF4α, LRH1, LXR, FXR, SREBP1c and 283 PPARγ. The aim of our study was to identify single nucleotide polymorphisms (SNPs) in the promoter region of SHP-1 and to determine their impact on the transactivation of SHP-1. Determination of retigabine in serum of patients with LC/MS/MS 120 DNA samples from subjects of the population based cohort Study of Health in 1 1 2 1 Oertel R. , Sebastian D. , Junghanns S. , Kirch W. Pomerania were analyzed by Sanger sequencing, thereby we identified four SNPs 1TU Dresden Institut für klinische Pharmakologie, Fiedlerstraße 27, 01305 Dresden, namely -594T>C (rs71636795), -413G>C, -423 C>T (rs78182695) and del-195CTGA Germany (rs145613139). Subsequently those polymorphisms were tested for their functional 2Universitätsklinikum Carl Gustav Carus Klinik für Neurologie, Fetscherstraße 74, 01305 consequence performing cell based reporter gene assays testing all above mentioned Dresden, Germany modulators (LRH1, LXR, FXR, SREBP1c and PPARγ) of SHP-1 expression. Only the transactivation by HNF4α was decreased in the presence of the -423 C>T polymorphism Background: Retigabine belongs to a novel class of potent anticonvulsant drugs and is to 69% and the -413G>C polymorphism to 75%. currently being investigated in clinical routine. The therapeutic range of retigabine serum In conclusion we described SNPs with impact on transactivation. It will be aim of future concentration is unknown. A therapeutic drug monitoring (TDM) is used for most other studies to determine the potential impact on physiological processes or disease anticonvulsant drugs. The aim of this study was to develop a method for the development. determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. Method: A HPLC method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. Solid-phase extraction (SPE) of 250 µl serum with Oasis HLB cartridges allowed a reliable quantification down to 5 ng/ml. In order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. To implement the determination of retigabine in a second step in the routine TDM of anticonvulsant S66
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Characterisation of adenylyl cyclase isoforms in PCK rats, a model of autosomal Development of novel Bid inhibitors for therapeutic approaches in models of recessive polycystic kidney disease neuronal cell death Ollenschläger K.1, Sandner P.2, Seifert R.1 Oppermann S.1, Elsaesser K.1, Schrader F.2, Krasel C.1, Bünemann M.1, Klebe G.2, 2 1 1Medizinische Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Str. 1, Schlitzer M. , Culmsee C. 30625 Hannover, Germany 1Phillips-Universität Institut für Pharmakologie und klinische Pharmazie, Karl-von- 2Bayer HealthCare Global Drug Discovery, Aprather Weg 18a, 42096 Wuppertal, Frischstr.1, 35033 Marburg, Germany Germany 2Phillips-Universität Institut für Pharmazeutische Chemie, Marbacher Weg 6-10, 35032 Marburg, Germany Autosomal recessive polycystic kidney disease (ARPKD) is a rare genetic disease, afflicting about 1 in 20.000 individuals. ARPKD is characterized by cystic fusiform In neurodegenerative diseases, such as Alzheimer´s disease and Parkinson´s disease, dilatations of the renal collecting ducts leading to massive enlargement of the kidneys mitochondrial pathways of apoptosis are considered as major features of the underlying and ultimately loss of renal function. In addition, the patients suffer from congenital neuronal cell death. Such mitochondrial mechanisms of apoptosis are mediated by the hepatic fibrosis (CHF), possibly leading to portal hypertension and liver enlargement. BH3-only protein Bid, a member of the Bcl-2 family that triggers mitochondrial So far, there is no cure for ARPKD. Therapy is focussing on controlling the disease permeabilization and the subsequent release of death-promoting proteins into the symptoms [1]. cytosol. The pivotal role of Bid in apoptotic cascades of neuronal cells has been shown Mutations in the PKHD1 gene cause ARPKD. More than 300 different mutations in this in our previous studies showing a neuroprotective effect of Bid siRNA and small gene have been reported, all leading to the same phenotype, though there are molecule Bid inhibitors such as BI6c9 in vitro. In vivo, however, the available Bid- differences regarding the severity of the disease [2]. inhibitors failed to protect brain tissue likely because the compounds were not In animal models of autosomal dominant polycystic kidney disease (ADPKD) as well as bioavailable or did not cross the blood brain barrier. Therefore, chemical modifications of ARPKD elevated levels of cAMP were shown [1-3]. In isolated kidney cells cAMP BI-6c9 were generated resulting in new structures and molecules with different stimulates Cl- secretion and activates the B-raf /MEK/ERK pathway. These both are pharmacophors. The aim of the present study is to identify novel potent Bid inhibitors important factors for cyst development and disease progression [2,3]. available for applications in model systems of brain damage in vivo. Intracellular cAMP regulation is based on conversion of ATP to cAMP by adenylyl For the first screening of 80 compounds we used a model of glutamate toxicity in cyclases (ACs) and degradation by phosphodiesterases . Referring to this, we asked the immortalized mouse hippocampal neurons (HT- 22 cells). In this model system, question if there are differences in the activation and expression pattern of ACs in PCK glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase rats, an animal model of ARPKD [4] and in Sprague Dawley rats. activity and enhanced formation of toxic reactive oxygen species (ROS). To investigate Therefore, we examined membranes in a radioactive AC activity assay using various the compounds’ ability to prevent glutamate induced cell death, we first analyzed the cell stimulatory compounds, e.g. forskolin, a direct AC activator, or hormones like glucagon viability by the MTT assay. In addition, we examined the cell survival by using real time and vasopressin to characterize ACs. Furthermore, we examined AC isoform expression monitoring of cell impedance (xCELLigence System) to determine the neuroprotective on the mRNA level via RT-PCR. potency of the new structures. We observed that in PCK rats AC activity was decreased in general in comparison to Using these assays, we identified 10 novel molecules that significantly prevented Sprague Dawley rats. In future experiments we are aiming to obtain further knowledge glutamate- induced toxicity in HT-22 cells. Further we were able to express and to purify about the influence various hormones exhibit on PCK rat ACs and to biochemically recombinant Bid in a high amount. In the ongoing study the purified Bid protein will be characterize ACs. used for co-crystallization with the identified neuroprotective structures for further optimization of novel Bid inhibitors for therapeutic applications in experimental models of References neurodegenerative diseases in vivo. 1. Takiar V, Caplan MJ, Polycystic kidney disease: Pathogenesis and potential therapies, Biochim Biophys Acta, 1812:1337–1343, 2011 2. Harris PC, Torres VE, Polycystic Kidney Disease, Annu Rev Med, 60:321–37, 2009 3. Wallace DP, Cyclic AMP-mediated cyst expansion, Biochim Biophys Acta , 289 1812:1291–1300, 2011 4. Katsuyama M, Masuyama T, Komura I, Hibino T, Takahashi H, Characterization of a novel polycystic kidney rat model with accompanying polycystic liver, Exp Anim 49:51- Polymorphic enzymes, urinary bladder cancer risk and structural change in the 55, 2000 local industry
Ovsiannikov D.1, Selinski S.2, Lehmann M. - L.2, Blaszkewicz M.2, Moormann O.3, Haenel M. W.4, Hengstler J. G.2, Golka K.2 1St.-Marien Hospital Department of Urology, Altstadtstr. 23, 44534 Lünen, Germany 287 2Leibniz Research Centre for Working Environment and Human Factors, Ardeystr. 67, 44139 Dortmund, Germany 3St.-Josefs-Hospital Dortmund-Hörde Department of Urology, Wilhelm-Schmidt-Str. 4, Contribution of the CROP domain of Clostridium difficile TcdA to toxin uptake 44263 Dortmund, Germany Olling A., Goy S., Frenzel E., Tatge H., Just I., Gerhard R. 4Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an Medizinische Hochschule Hannover Toxikologie, Carl-Neuberg-Str. 1, 30625 Hannover, der Ruhr, Germany Germany In the 1990s, an uncommonly high percentage of glutathione S-transferase M1 (GSTM1) The major pathogenicity factors TcdA and TcdB from Clostridium difficile negative bladder cancer cases (70 %) was reported in the greater Dortmund area (Golka monoglucosylate and thereby inactivate small GTP-binding proteins of the Rho et al., 1997). The question arose whether this uncommonly high percentage of GSTM1 subfamily after entering host cells via receptor-mediated endocytosis. Although the negative bladder cancer cases was due to environmental and occupational exposure intracellular mode of action of the toxins is well understood, far less is known about decades ago. Thus, 15 years later, another study on bladder cancer was performed in binding structure and internalization pathway of TcdA and TcdB. Since antibodies the same area after the coal, iron and steel industries had finally closed in the 1990s. In directed against the C- terminal located clostridial repetitive oligopeptides (CROPs) are total 196 bladder cancer patients from the St.-Josefs-Hospital Dortmund-Hörde and 235 able to neutralize toxin cytotoxicity the CROP domain is acknowledged to mediate controls with benign urological diseases were investigated by a questionnaire and receptor binding. However, we recently demonstrated that CROP deletion mutants of genotyped for GSTM1, GSTT1 and the N-acetyltransferase 2 (NAT2) tag SNP TcdA (TcdA1-1874) and TcdB (TcdB1-1852) enter host cells and exhibit full cytopathic rs1495741. The frequency of the GSTM1 negative genotype was 52 % in bladder cancer potency though lacking the proposed receptor binding domain. We therefore refute the cases and thus much lower, compared to a previous study performed from 1992-95 in accepted opinion of a solely CROP- mediated toxin uptake and re-evaluate the role of the same area (70%). NAT2 genotypes were distributed equally among cases and the CROPs in toxin endocytosis. TcdA1-1874 and TcdB1-1852 induced time and controls (63% slow acetylators). Less GSTT1 negative genotypes were present in cases concentration dependent cell rounding and Rac1-glucosylation. However, depending on (17%; controls 20%). the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. The observed difference in toxin potency References might reflect the recognition of different receptor structures or the use of various 1. Golka K. et al.: N-Acetyltransferase 2 (NAT2) and glutathione S-transferase µ endocytotic routes. Interestingly, pre-incubation of cells with the isolated CROP domain (GSTM1) in bladder cancer patients in a highly industrialized area. Int. J. Occup. enhances binding as well as cytotoxicity of subsequent applied truncated TcdA1-1874 Environ. Health 3, 105-110 (1997) indicating that the CROPs primarily determine toxin uptake. In fact, competition experiments revealed that TcdA and TcdA1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by TcdA. Different routes for cellular uptake might enable the toxins to enter a broader 290 repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile. Thus, characterization of alternative endocytotic pathways used by the C. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as Neuroprotection mediated by AIF-deficiency - a preconditioning effect? therapeutic option. Öxler E., Culmsee C. Philipps-Universität Marburg Klinische Pharmazie und Pharmakologie, Karl-von-Frisch- Str.1, 35033 Marburg, Germany
Apoptosis inducing factor (AIF) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. AIF is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale DNA fragmentation. Previous studies revealed that AIF deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. However, AIF also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. S67
Thus, AIF deficiency may significantly alter mitochondrial functions and metabolic mosaic synaptic defects most probably caused by random X-chromosomal inactivation homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. of the healthy allele. Electroretinography revealed a loss of scotopic and photopic The present study addresses this hypothesis and investigates whether neuroprotection photoreceptor function. This loss of retinal network function resulted in impaired by AIF depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial performance of Cav1.4 knockout mice in a water maze-based behavioral test of rod and function and integrity. As model system we use glutamate induced oxytosis in cone function. immortalized mouse hippocampal HT-22 neurons. In conclusion, loss of Cav1.4 channels strongly impairs rod and cone retinal function and Silencing of AIF expression by siRNA (20nM) protected mitochondrial morphology and vision in mice. integrity against glutamate induced damage. Microscopy analysis of the mitochondrial morphology revealed that AIF siRNA prevented mitochondrial fission. Furthermore, FACS analysis confirmed that mitochondrial membrane potential was stable in cells with AIF silencing. This protection of mitochondrial morphology and integrity by AIF depletion 293 was associated with preserved ATP levels and inhibition of cell death as detected by an MTT assay. Pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by AIF siRNA. These LSR is the host cell receptor for clostridial iota-like toxins protective effects of AIF siRNA were highly similar to effects obtained with low doses of rotenone (20nM), which was applied as an inhibitor of complex I and mediated Papatheodorou P., Aktories K. comparable preconditioning effects in the HT-22 cells. Albert-Ludwigs-Universität Freiburg Institut für Experimentelle und Klinische Overall, these findings support the conclusion that AIF depletion mediates a Pharmakologie und Toxikologie, Albertstr. 25, 79104 Freiburg, Germany preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. In order to link these preconditioning effects to complex I functions, protein expression and The human enteric pathogen Clostridium difficile is the most serious cause of antibiotic- functional analysis of complex I are being analysed to identify the molecular associated diarrhea and pseudomembranous colitis. Hypervirulent strains of the mechanisms of AIF dependent control of neuronal life and death. pathogen, associated with more severe disease and increased death rates, produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase) in addition to the Rho glucosylating toxins A and B. CDT is member of the family of clostridial iota-like toxins, including C. spiroforme toxin (CST) and the eponym C. perfringens iota toxin. The toxins induce depolymerization of the actin cytoskeleton and the formation of microtubule- 291 based cell protrusions that increase adherence and colonization of Clostridia. Using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (LSR) as the target molecule for entry of CDT into host cells. In addition, we present evidence that Der Arylhydrocarbonrezeptor (AhR) und das Metabolische Syndrom – LSR is shared as a cell entry point by all members of the iota-like toxin family. Untersuchungen an AhR-gendefizienten Mäusen Identification of the toxin receptor provides a most valuable basis for antitoxin strategies. 1 1 1 1 2 3 4 Padberg E. , Scholz R. , Kadow S. , Martiensen B. , Diel P. , Burkart V. , Albrecht C. , 1 Esser C. References 1Leibniz-Institut für Umweltmedizinische Forschung Molekulare Immunologie, Auf´m Papatheodorou P., et al. (2011) Lipolysis-stimulated lipoprotein receptor (LSR) is Hennekamp 50, 40225 Düsseldorf, Germany the host receptor for the binary toxin Clostridium difficile transferase (CDT). Proc 2Deutsche Sporthochschule Inst. für Kreislaufforschung und Sportmedizin, Carl- Natl Acad Sci U S A 108(39):16422-7. Diemweg 6, 50927 Köln, Germany 3Leibniz-Zentrum für Diabetesforschung Inst. für klinische Diabetologie, Auf´m Hennekamp 65, 40225 Düsseldorf, Germany 4Leibniz-Institut für Umweltmedizinische Forschung Partikelforschung, Auf´m Hennekamp 50, 40225 Düsseldorf, Germany
Mit dem Begriff „metabolisches Syndrom“ (MetS) werden Stoffwechselstörungen insbesondere des Glukose- und Lipidstoffwechsel bezeichnet. Typ2 Diabetes und koronare Herzerkrankungen können Folge sein. Die Ursachen des MetS und die an seiner individuellen Ausprägung beteiligten Faktoren sind unbekannt. Vorbote ist häufig eine periphere Insulinresistenz. Der Arylhydrocarbonrezeptor (AhR) ist ein potentieller Regulator für die Glucosehömeostase und des Lipidstoffwechsel. Epidemiologische Studien zeigten eine Assoziation des AhR mit Typ2 Diabetes und Glukose-Homöostase. Wir untersuchten die Rolle des AhR für Parameter des MetS in Mäusen, denen der AhR Cell intoxication: fehlt (AhR-KO). AhR-KO hatten verglichen mit Wildtyp-Tieren eine stark verkürzte Ectopic expression of LSR (+LSR) in LSR-deficient cells (w/o LSR) restores Lebenserwartung und eine verminderte Glukosetoleranz, jedoch einen normalen HbA1c sensitivity toward CDT Wert. Altersdifferenzierende Untersuchungen zeigten, dass Glukoseintoleranz bereits in jungen Tieren bestand und bis hohe Alter (>2 Jahre) anhielt. Darüber hinaus zeigten orale Glukosetoleranztests, dass in alten AhR-KO der Insulinspiegel im Serum nach Induktion stärker als in Wildtyp-Kontrollen anstieg, und eine gestörte Kinetik aufwies. Eine Adipositas der Tiere konnte hingegen nicht beobachtet werden, trotz Zugang zu Futter ad libitum. Größe und Zahl der Langerhans´schen Inseln waren in AhR-KO nicht 294 verändert. Die AhR-KO Mäuse zeigten signifikant erhöhte Triglyceride, und HDL-Werte im Serum. Cholesterin und LDL-Werte waren unverändert im Normbereich. Unsere Daten zeigen erstmals einen Einfluss der AhR auf einige, aber nicht alle typischen UDP-glucuronyltransferase 2B15 polymorphism and Bisphenol A concentrations Parameter des MetS, Interessanterweise führen diese Störungen weder zu Adipositas in blood noch zu Typ2 Diabetes, auch nicht in sehr alten AhR-KO Mäusen. Partosch F.1, Mielke H.2, Gundert-Remy U.1,2 1Charité Universitätsmedizin Berlin Institut für Klinische Pharmakologie und Toxikologie, Luisenstr. 7, 10117 Berlin, Germany 2Bundesinstitut für Risikobewertung Chemikaliensicherheit, Max-Dohrn-Straße 8-10, 292 10589 Berlin, Germany
Bisphenol A (BPA) is a chemical of high interest due to its endocrine activity.
Functional characterization of the visual phenotype of Cav1.4-deficient mice Controversy exists concerning the blood concentration due to normal exposures. Some Paparizos C.1, Shaltiel L.1, Michalakis S.1, Sothilingam V.2, Garcia Garrido M.2, Koch authors claimed to have measured concentrations in the ng/ml range which is in contrast S.1, Rötzer K.1, Tanimoto N.2, Seeliger M.2, Biel M.1, Wahl-Schott C.1 to kinetic properties of BPA. BPA is excreted in the urine as glucuronide and sulfate 1 metabolites. Recently, data on the in vitro metabolism of BPA by recombinant UDP- Center for Integrated Protein Science Munich Department of Pharmacy - Center for glucuronyltransferase 2B15 enzymes (UGT2B15) revealed that UGT2B15.2 and Drug Research, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 UGT2B15.5 had markedly lower intrinsic clearance as compared to UGT2B15.1 München, Germany 2 (Hanioka et al., 2011). Using the in vitro metabolism data, we scaled the k and V University of Tuebingen Ocular Neurodegeneration Research Group Centre for M- max- values in an established human physiologically based toxicokinetic (PBTK) model Ophthalmology Institute for Ophthalmic Research, Schleichstr. 4/3, 72076 Tübingen, (Mielke and Gundert-Remy, 2009, Mielke et al., 2011) to the values of the variants. For Germany oral doses at relevant exposure levels, the maximum blood concentration (Cmax) for the
2+ UGT2B15.2 variant (V2) was 5 fold and those of the UGT2B15.5 variant (V5) was 7 fold Retinal network activity crucially depends on Ca influx through presynaptic Cav1.4 2+ higher than that of the UGT15.1 variant. With dermal exposure at a relevant exposure voltage-gated L-type Ca channels. These channels are unique among the high 2+ 2+ level, the Cmax values were 1.4 (V2) and 1.6 fold (V5) of UGT15.1 variant. A combined voltage-activated Ca channel family because they completely lack Ca -dependent exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, inactivation and display very slow voltage-dependent inactivation. Both properties are of resulted in 2.4 fold (V2) and 3.2 fold (V5) higher Cmax values as compared to UGT15.1 crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where 2+ variant. The values for the area under the blood concentration time curve (AUC) were for sustained Ca influx through Ca 1.4 channels is required to couple slowly graded v a relevant oral dose 5.7 fold (V2) and 8.6 fold (V5), for relevant dermal exposure 1.4 fold changes of the membrane potential with tonic glutamate release. Mutations in the gene (V2) and 1.6 fold (V5), and for combined exposure 1.9 fold (V2) and 2.5 fold (V5) of coding for Cav1.4 cause severe impairment of retinal circuitry function and have been UGT15.1 variant. From the results we conclude: (1) Polymorphism of UDP- linked to congenital stationary night blindness type 2A (CSNB2), Aland Island eye glucuronyltransferase (2B15.2 and 2B15.5) has an impact on the blood concentrations disease (AIED) and cone-rod dystrophy type 3 (CORDX3). The clinical phenotypes of which, however, is less than 10 fold for Cmax and for AUC. The effect is more these eye diseases vary substantially regarding the ratio of rod to cone functional pronounced for oral as compared to dermal or combined exposure. (2) Polymorphism of impairment. The reasons for this variability are not known. To gain more insights into the metabolism does not explain the blood/plasma concentrations in the ng/ml range pathophysiology caused by loss of Ca 1.4 function we analyzed the visual phenotype of v measured by some authors. Ca 1.4-deficient mice. To this end, we combined immunohistochemistry, v electroretinography (ERG) and vision-dependent behavioral testing.
Immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in Cav1.4-deficient mice. Heterozygous Cav1.4 mice showed S68
References 297 Hanioka N, Oka H, Nagaoka K, Ikushiro S, Narimatsu S. Effect of UDP- glucuronosyltransferase 2B15 polymorphism on bisphenol A glucuronidation. Arch Toxicol. 2011, 85(11):1373-81. DEPHOSPHORYLATION OF G PROTEIN-COUPLED RECEPTORS: different Mielke H, Gundert-Remy U. Bisphenol A levels in blood depend on age and exposure. dephosphorylation patterns of sst2- and MOR-receptor Toxicol Lett. 2009 8;190(1):32-40. Mielke H, Partosch F, Gundert-Remy U. The contribution of dermal exposure to the Peuker K., Pöll F., Doll C., Schulz S. internal exposure of bisphenol A in man. Toxicol Lett. 2011 204(2-3):190-8. Friedrich Schiller Universität Jena, Universitätsklinikum Jena Institut für Pharmakologie und Toxikologie, Drackendorfer Str. 1, 07747 Jena, Germany
Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. Although the regulation of agonist-induced 295 phosphorylation has been studied in detail for many GPCRs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. Recent studies have shown that phosphatase inhibitors, such as okadaic acid and cGMP kinase I, cardiac hypertrophy and PDE inhibition calyculin A, can block the dephosphorylation of a number of GPCRs including the ß2 1 1 2 3 1 Patrucco E. , Domes K. , Lukowski R. , Rybalkin S. , Hofmann F. adrenergic receptor, D1 dopamine receptor, parathyroid hormone receptor 1, 1TU München 1FOR923, Institut für Pharmakologie und Toxikologie, Biedersteiner Str. thromboxane A receptor and the vasopressin receptor 1. However, a specific 29, 80802 München, Germany phosphatase has not been identified so far. In present studies, we have examined the 2Universitätsklinikum Tübingen 2Institut für Pharmakologie und Toxikologie, Abteilung mechanism and function of receptor dephosphorylation using the sst2A somatostatin Pharmakologie und Experimentelle Therapie, Tübingen, Germany receptor and the µ-opioid receptor (MOR) as models. Within those analyses, we have 3University of Washington Pharmacology Department, Seattle, United States identified protein phosphatase 1beta (PP1ß) as the phosphatase for the cluster of phosphorylated threonines (353TTETQRT359) within the sst2A somatostatin receptor The heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic carboxylterminus using siRNA knock down screeening. Those phosphorylation sites signals that contrast and dampen the onset and development of hypertrophy. Cyclic mediate ß-Arrestin binding. We have also identified protein phosphatase 1gamma guanosine monophosphate (cGMP) and its downstream effector cGMP kinase I (cGKI) (PP1γ) as MOR phosphatase that catalyzed T370 and S375 dephosphorylation at or have been suggested to be an important anti-hypertrophic signaling pathway (1). near the plasma membrane within minutes after agonist removal. Here, we show the Intracellular levels of cGMP can be raised by the action of nitric oxide (NO) and different activated phosphatases with functional selective mutants. We examined tail- natriuretic peptides (ANF, BNF), or by inhibiting cGMP-degrading phosphodiesterases swap mutants which specify the different phosphatase activities. Therefore we produced (PDE). A growing body of evidence suggests that the PDE5 specific inhibitor Sildenafil a MOR-rsst2A chimera with the rsst2A C-terminal tail and a rsst2A-MOR chimera with a (Sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice MOR C-terminal tail. subjected to thoracic aorta constriction (TAC) by elevating cGMP levels and cGKI activation (1). In contrast, using a mouse model that lacks cGKI expression in every cell type except smooth muscle cells (βRes mice; see ref.2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by TAC or 298 isoproterenol infusion (2). Sil is believed to increase cardiac cGMP levels, although it is unclear, if its target (PDE5) is expressed in CM (2). Sil may act on other PDEs, such as PDE1C which is abundant in Detoxification by conjugation of glutathione? Formations of DNA adducts of CMs. It is also unclear if Sil effects are mediated by other cardiac cell types, in particular patulin activated by glutathione by cardiofibroblast. To answer these questions, we are currently investigating whether Sil is able to prevent Pfenning C., Lehmann L. hormone induced cardiac hypertrophy in the absence of cGKI in CM. University Wuerzburg, Institute of Pharmacy and Food Chemistry Section of Food Preliminary results on βRes mice show that even in the case of chronic AngII infusion, Chemistry, Am Hubland, 97074 Wuerzburg, Germany lack of cGKI In CM does not alter the Induction of hypertrophic response, at least in the initial phase (7days of AngII infusion at 2mg/kg/day). Interestingly, βRes mice showed As a frequent contaminant in apple juice, the mycotoxin patulin (PAT) has shown impaired cardiac function, as indicated by decreased Fractional Shortening. mutagenic potential in cultured mammalian cells at concentrations which are equivalent Sil was able to partially block the onset of cardiac hypertrophy in WT animals, but not in to those found in marketable foods. This fact is in contrast with the assumption that βRes mice, indicating a requirement of cGKI in this process. In particular, Sil was able to conjugation to the major intracellular nucleophile glutathione (GSH) leads to block the transcription of pro-fibrotic genes such as TGFβ, CTGF, CollagenI and detoxification of the electrophile PAT. Although PAT reacts readily with GSH, previous Fibronectin. studies showed that co-incubation of PAT with model thiols and amine compounds increased the reactivity of PAT towards amines forming mixed-type adducts. Thus, we References hypothesise that the potential to react with DNA bases after being activated by GSH 1. Takimoto, E., Champion, H. C., Li, M., Belardi, D., Ren, S., Rodriguez, E. R., Bedja, might contribute to the mutagenicity of PAT. D., Gabrielson, K. L., Wang, Y., and Kass, D. A. (2005) Nat Med 11(2), 214-222 Adduct formation of DNA bases (adenine, guanine or cytosine) with PAT in the presence 2. Lukowski, R., Rybalkin, S. D., Loga, F., Leiss, V., Beavo, J. A., and Hofmann, F. and absence of GSH was studied under neutral conditions. Liquid chromatography (2010) Proc Natl Acad Sci U S A 107(12), 5646-5651 coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. Besides published as well as hitherto unknown PAT-GSH adducts, several PAT-DNA base adducts were formed both in presence and absence of GSH. In addition, with each of the three DNA bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed 296 thiol-amine adduct was detected. Based on the fragment ions of adducts formed with GSH and chemically modified derivatives, we postulate a cyclic structure of the PAT- GSH-DNA base adducts, resulting from the reaction of the α-amino group of the Phosphorylation of carboxyl-terminal threonine 333 regulates rapid internalization glutamic acid residue with the C7-carbonyl function of PAT. The exocyclic amino group and recycling of the human somatostatin receptor 5 of the DNA base is linked to C1 of the PAT backbone by an amid bond. Petrich A., Schulz S. Thus, the present study demonstrates the reactivity of PAT towards DNA bases and the Uniklinikum der FSU Jena Pharmakologie und Toxikologie, Drackendorfer Straße 1, participation of GSH in adduct formation. The postulated structures of DNA adducts 07747 Jena, Germany could be used as biomarkers for the determination of the internal exposure to PAT in humans. The overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. Whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known 299 about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333- or the Different indications, contraindications, warnings and precautions for the same T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation drug – a comparison of international prescribing information for commonly used occurs at T333, whereas T347 is constitutively phosphorylated in the absence of psychiatric drugs agonist. We further demonstrate that the pan-somatostatin analog pasireotide and the Pfistermeister B., Fromm M. F., Maas R. sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a clearly detectable T333 phosphorylation. However, none of these compounds was able Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Lehrstuhl für Klinische Pharmakologie und Klinische Toxikologie, Fahrstraße 17, 91054 Erlangen, to stimulate T333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and Germany selectively mediated by G protein-coupled receptor kinase 2 (GRK2). Like that observed Prescribing information detailed in the Summary of Product Characteristics (SPC) forms for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike the officially approved basis for safe prescribing of drugs. In a project funded by the that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete German Federal Ministry of Education and Research (BMBF) we aimed to derive an within minutes. We also identify protein phosphatase 1g (PP1g) as sst5 receptor phosphatase. Together, we provide direct evidence for agonist-selective phosphorylation internationally valid data set for safe prescribing of psychiatric drugs and therefore of carboxyl-terminal T333. In addition, we identify GRK2-mediated phosphorylation and analyzed and compared the content of internationally available prescribing information. PP1g-mediated dephosphorylation of T333 as key regulators of rapid internalization and Methods: A team of pharmacists and clinical pharmacologists performed an in-depth comparison recycling of the human sst5 receptor. of the German, Swiss, British and US-American SPCs of 10 top prescribed psychiatric drugs. For 7 drugs (of identical pharmaceutical form) the SPCs from the same manufacturer were available for all countries, whereas for three drugs SPCs were only available from different companies. In these cases the most recent prescribing information from each country was included in the comparison. Results: S69
In 40 SPCs 2220 individual data points (55.5±17.4 per individual SPC) were compared. Results and Conclusion Between countries the timeliness of prescribing information for an individual drug varied Differential expressions of 20 phosphoproteins were found in CB-treated cells while an by a median of 18.5 (range: 6-134) months. The respective SPCs covered on average altered expression of 33 phosphoproteins was observed in CB+-treated cells. The 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range MALDI-ToF analysis revealed proteins involved in the regulation of the endothelial 15.4-100%) of all mentioned contraindications. The warnings and precautions section of permeability and the cellular plasticity such as vimentin, actin and transitional an individual SPC covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned endoplasmic reticulum ATPase. Further, the invasion assay supported these results as warnings and precautions for that drug. The variation observed was only marginally the CB-exposed cells showed a high invasive potential as compared to control. improved when restricting the analysis to the 28 SPCs of the 7 drugs available in all four countries from the same manufacturer. References Conclusion: [1] Pink M. et. al., Precipitation by lanthanum ions: A straightforward approach to isolate Across countries, the Summary of Product Characteristics of individual psychiatric drugs phosphoproteins, J.Protomics, 75, 2011, 375-383 show substantial variation in crucial prescribing information. As different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of Summary of Product Characteristics. 302 This project is supported by the German Federal Ministry of Education and Research (BMBF), project grant No. 01 EX1015B. Application of the TTC approach in cosmetics Platzek T. Bundesinstitut für Risikobewertung, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany 300 Regulatory toxicologists in Europe have been discussing the TTC approach since more than a decade, e.g. the previous Scientific Committee on Food in 1996. Since then, the Identification of new inhibitors of protein kinase CK2 subunit interaction concept was further developed and is now applied in the EU for the assessment of 1 2 3 4 1 3 Raaf J. , Neundorf I. , Herzig S. , Issinger O. - G. , Niefind K. , Pietsch M. flavouring substances by EFSA. Two committees are discussing possible applications: 1Institute of Biochemistry, Department of Chemistry, University of Cologne, Otto-Fischer- The EFSA Scientific Committee prepared an opinion exploring options for the application Str. 12-14, 50674 Cologne, Germany in food and feed, e.g. for impurities of food additives, thermal reaction products, food 2Institue of Biochemistry, Department of Chemistry, University of Cologne, Zülpicher Str. contact materials, contaminants etc. In addition, an EU non-food expert Committee 47, 50674 Cologne, Germany consisting of members of SCCS, SCHER and SCHENIR discussed the TTC concept in 3Department of Pharmacology, University Hospital of Cologne, Gleueler Str. 24, 50931 general as well as additional possible fields of application with the focus on cosmetics Cologne, Germany and an opinion was already published for public consultation. The proposal to apply the 4Institute for Biochemistry and Molecular Biology, University of Southern Denmark, TTC approach also for cosmetic ingredients was introduced by a paper published in Campusvej 55, 5230 Odense, Denmark 2007 by a COLIPA (The European Cosmetics Association) supported working group. Major aspects to be considered are the following: Protein kinase CK2 (former name ‘casein kinase 2’) is a highly conserved 1. Applicability domain. The chemical space of the TTC dataset (> 600 compounds) serine/threonine kinase, which acts as a component of regulatory networks implicated in has to be compared with that of cosmetic ingredients (> 10 000 compounds). many cellular processes but is also linked to various types of human cancer [1,2]. 2. Route to route extrapolation. Since no adequate dermal toxicity database is Elevated CK2 activity has been associated with aggressive tumor behavior and results available both data on oral intake used in the TTC approach and on dermal exposure to in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells cosmetic ingredients have to be transfomed to internal exposure figures. Gastrointestinal [3]. CK2 is a heterotetramer consisting of two catalytic subunits (CK2α) attached to a and dermal bioavailability as well as route specific differences in metabolism have to be dimer of regulatory subunits (CK2β) [4]. CK2β stabilizes CK2α against denaturation and integrated. modulates the substrate specificity of the catalytic subunit [5]. Due to the relatively small 3. Exposure. The reliability of exposure estimation is the second pillar of the TTC and hydrophobic CK2α–CK2β interface (832 Ų) [4], low molecular weight inhibitors are approach. Compared to food, data on exposure to substances in cosmetics and able to interfere with the CK2 subunit interaction and thus affect the kinase activity [6]. consumer products is scarce. Such inhibitors might exhibit an increased specificity in comparison to those compounds A pragmatic step forward is the comparison of TTCs and NOAEL-derived safe exposure interacting with the conserved ATP binding site [3]. levels for cosmetic ingredients. This work was already done with substances in food We have developed an ELISA-based CK2α–CK2β binding assay using recombinant contact materials and chemicals from the ELINCS list. For cosmetic ingredients a similar human CK2 subunits. Different blocking reagents were analyzed to minimize non- European project is ongoing. Further refinement of the TTC approach is needed taking specific binding. The optimized binding assay was then applied to screen for inhibitors of into account the up-to-date toxicological knowledge. With cosmetics specific problems the CK2 subunit interaction. Primary hits were further characterized by determination of may arise in praxi: according to the new EU cosmetic legislation the safety of cosmetic the parameters IC50 and Ki as well as by comparing the results from the binding assay products available on the market has to be assessed by the manufacturer or importer with literature-known or recently obtained crystal structures. and also assessors with limited toxicological experience may apply the TTC approach, e.g. by running the TOXTREE software. References [1] Litchfield, D.W. Biochem. J. 2003, 369, 1-15. [2] Guerra, B.; Issinger, O.-G. Curr. Med. Chem. 2008, 15, 1870-1886. [3] Prudent, R.; Cochet, C. Chem. Biol. 2009, 16, 112-120. 303 [4] Niefind, K.; Guerra, B.; Ermakowa, I.; Issinger, O.-G. EMBO J. 2001, 20, 5320-5331. [5] Meggio, F.; Boldyreff, B.; Marin, O.; Pinna, L.A.; Issinger, O.-G. Eur. J. Biochem. 1992, 204, 293-297. Cytochrome P450 1A1/1B1-activities and anti-benzo[a]pyrene-7,8-diol-9,10- [6] Prudent, R.; Sautel, C.F.; Cochet, C. Biochim. Biophys. Acta 2010, 1804, 493-498. epoxide DNA-adducts in A549 lung carcinoma cells after B[a]P-incubation
Plöttner S., Marczynski B., Käfferlein H. U., Welge P., Groth H., Engelhardt B., Schmitz K., Erkes A., Brüning T. Institut für Prävention und Arbeitsmedizin der Deutschen Gesetzlichen 301 Unfallversicherung - Institut der Ruhr-Universität Bochum (IPA) Toxikologie, Bürkle-de- la-Camp-Platz 1, 44789 Bochum, Germany
Phosphoproteomic analysis of endothelial cells after exposure to ultra-fine Polycyclic aromatic hydrocarbons (PAHs) comprise several hundred compounds with particles different carcinogenic potentials, typically occurring in complex mixtures. Due to a lack of Pink M., Stein C., Rettenmeier A. W., Schmitz-Spanke S. data risk estimations for PAH mixtures are usually based on those for benzo[a]pyrene Universitätsklinikum Essen Institut für Hygiene und Arbeitsmedizin, Hufelandstr. 55, (B[a]P). The aim of our present study was to explore the suitability of a permanent 45122 Essen, Germany human lung cell line as tool for future studies on genotoxicity of PAH mixtures. In this pilot study we investigated the time- and concentration-dependent generation of specific Introduction anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE)-DNA adducts as well as Numerous epidemiological studies have shown associations between exposure to ultra cytochrome P450 (CYP) 1A1/1B1 enzyme activities after B[a]P-incubation in vitro. fine particles and an increase in cardiovascular diseases such as atherosclerosis, We used metabolically competent A549 lung carcinoma cells which display several coronary heart disease and myocardial infarction. Ultra-fine particles have an characteristics of alveolar epithelial type II cells. After 24 h and 48 h incubations with aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition different B[a]P-concentrations cytotoxic effects were assessed with the neutral red assay metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). and CYP1A1/1B1 activities using luminescent tests. The formation of specific anti- Posttranslational modification (PTM) of proteins, particularly phosphorylation, is a key BPDE-DNA adducts was determined by HPLC with fluorescence detection. element in the regulation of cell function and any disturbance can lead to multiple A time- and concentration-dependent formation of anti-BPDE-DNA adducts was diseases. The present study focused on the proteomic-based identification of observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-BPDE/108 phosphorylated proteins to understand the mechanism behind ultrafine particle exposure nucleotides after incubation with 1 µM (24 h) and 3 µM B[a]P (48 h), respectively. and cardiovascular disease development. As one of the major sources for UFP However, the mean adduct rates decreased at higher B[a]P-concentrations. The emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (CB) reduction was more pronounced after 24 h than after 48 h. Increased CYP1A1/1B1 and benzo[a]pyrene loaded carbon black (CB+) were used in the present study. activities were observed at > 0.1 - 1 µM (24 h) and > 0.1 - 3 µM (48 h). A clear decrease Methods of enzyme activities was observed at higher concentrations for both incubation times. In Cells of the endothelial cell line EA.hy926 were exposed for 14 days to 100 ng/mL CB the neutral red assay no more than 10% cytotoxicity in relation to the negative control and CB+. Phosphoprotein extraction of whole cell lysates was carried out by the method were found after 24 h incubation with ≥ 10 µM B[a]P and after 48 h with ≥ 1 µM B[a]P. developed in our lab1. The obtained proteins were then separated by two-dimensional Overall, incubation of A549 cells with B[a]P resulted in a time- and concentration-dependent gel-electrophoresis followed by MALDI-ToF-MS (matrix-assisted laser increase of CYP-activities and anti-BPDE-DNA adducts. This clearly shows that A549 cells desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed are able to generate mutagenic DNA-adducts. Thus, the in vitro model used in the present proteins. To further validate the results invasive potential of cells were monitored by work appears suitable for genotoxicity studies of individual PAHs and PAH mixtures, and plating exposed cells for 24 hrs on top of Matrigel-coated inserts. therefore may be a useful tool for research on syncarcinogenesis.
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Mutation of potential phosphorylation sites in the cardiac Cav1.2 channel complex The initial recruitment of Gi to the Alpha2A-adrenergic receptor is affected by G- and β-adrenergic regulation protein-coupled receptor kinases and arrestins Domes K.1, Poomvanicha M.1, Blaich A.1, Brandmayr J.1,2, Moosmang S.1,3, Wegener Prokopets O. S., Krasel C., Bünemann M. 1 J. Philipps-Universität Marburg Institut für Pharmakologie und Klinische Pharmazie, Karl- 1Institut für Pharmakologie und Toxikologie, TUM Forschergruppe 923 Carvas, von-Frisch-Str. 1, 35043 Marburg, Germany Biedersteinerstr. 29, 80802 München, Germany 2Institut für Pharmakologie und Toxikologie, TUM, Biedersteinerstr. 29, 80802 München, Most G-protein-coupled receptors undergo homologous desensitization after agonist Germany stimulation. In this process, agonist-activated receptors are phosphorylated by G- 3Institut für experimentelle und klinische Pharmakologie und Toxikologie der Universität protein-coupled receptor kinases (GRKs), followed by binding of arrestins to the still des Saarlandes, Universitätsklinikum 46, 66421 Homburg, Germany agonist-occupied, phosphorylated receptors. It is assumed that arrestin competes with heterotrimeric G-proteins for the receptor molecule and thereby causes desensitization The β subunit of the Cav1.2 channel complex has been shown to play a key role in of G-protein-mediated responses. We tested this idea by investigating the effect of Cav1.2 channel trafficking and channel characteristics like opening probability. GRKs and arrestins on the recruitment of G-proteins by the alpha2A-adrenergic receptor. Furthermore, the last exon of the CACNB2 gene coding for Cavβ2, exon 14, contains HEK293T cells were transfected with YFP-tagged alpha2A-adrenergic receptor and the 2+ several potential phosphorylation sites, e.g. for protein kinase A or Ca /calmodulin three subunits of Gi1. The G(beta) subunit was CFP-tagged. Upon stimulation with dependent CaMKII. PKA-dependent phosphorylation mediates β-adrenergic stimulation noradrenaline, a very rapid, robust increase in FRET between the receptor and the of Cav1.2. Potential phosphorylation sites are Ser478 and Ser479 (in the β2 subunit in G(beta) subunit was observed, confirming previous observations that the alpha2A- a rat, Perez-Reyes et al. 1992 ) and Ser1928 in the C-terminus of the poreforming α1C adrenergic receptor recruits Gi with a half-life of around 150 milliseconds. When b 2+ subunit (Lemke et al. 2008 ). In cardiomyocytes CaMKII regulates Ca release and arrestin3 and GRK2 were also co-transfected, the half-life of Gi recruitment was reuptake from and into the SR and is involved in the facilitation of the calcium channel. substantially delayed, increasing to around 2 seconds. There seemed to be no effect of Potential interaction sites between the Cav1.2 channel complex and CaMKII are Thr498 GRK2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the c in the β2 subunit (in rat, Grueter et al. 2006 ) and Ser1512 and Ser1570 in the C- extent were altered compared to the first stimulation. Interestingly, GRK2 alone seemed d terminus of the α1C subunit (Blaich et al. 2010 ). However, the exact pathways remained to cause a similar but slightly less pronounced delay of G(beta) recruitment to the widely unclear up to now. alpha2A-adrenergic receptor. In corresponding experiments, we measured the To clarify these mechanisms and to identify the relevant phosphorylation sites for PKA recruitment of arrestin3-CFP to YFP-tagged alpha2A-adrenergic receptors in the and CaMKII we established a mouse line carrying a stop codon in exon 14 after aa presence of GRK2. Upon stimulation with noradrenaline, we observed a robust increase Pro501. This mutation prevented translation of the Cavβ2 C-terminus containing the in FRET between the receptor and arrestin3, confirming previous observations that the corresponding potential phosphorylation sites mentioned above (Cavβ2stop mouse). alpha2A-adrenergic receptor recruits arrestin3. Co-transfection of the three subunits of These mice were viable, showed unaltered expression of the truncated of Cavβ protein Gi1 had no effect on the kinetics of arrestin3 binding to the alpha2A-adrenergic receptors. and unchanged ECG and echocardiography. Electophysiological analysis of isolated Based on previous results with other receptors, an attenuation of Gi recruiting to the cardiomyocytes showed no differences in current density, the effect of isoproterenol, the alpha2A-adrenergic receptor is quite unexpected. Our data suggest that the relation time course of inactivation and the facilitation property when compared to cells isolated between receptors, G-proteins, GRKs and arrestins may be more complex than from littermate controls. For further investigations we bred the Cavβ2stop mice with previously postulated. S1928A mice (Lemke et al. 2008b) lacking the PKA phosphorylation site S1928 in the d α1C subunit (S1928Aβ2stop mouse) or with SF mice (Blaich et al. 2010 ) lacking the CaMKII phosphorylation sites S1512 and S1570 in the α1C C-terminus (SFβ2stop). Both mouse lines were viable and showed unchanged echocardiography recordings 307 compared to their control littermates and unaltered ECG for S1928Aβ2stop mice. Electrophysiological investigations on cardiomyocytes of S1928Aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. Role of urothelium in b-adrenoceptor mediated relaxation in human detrusor These results suggest that β adrenergic regulation of the cardiac Cav1.2 channel is not muscle mediated by these phosphorylation sites. 1 2 3 1 2 Propping S. , Wuest M. , Kaumann A. J. , Wirth M. P. , Ravens U. 1 References Universitätsklinikum Carl Gustav Carus Klinik für Urologie, Fetscherstrasse 74, 01309 a Perez-Reyes et al.(1992) J Biol Chem 267(3), 1792-1997 Dresden, Germany 2 b Lemke et al. (2008) J Biol Chem., 283 (50), 34738-34744 Technische Universität Dresden Institut für Pharmakologie und Toxikologie, c Grueter et al. (2006) Mol Cell 23 (5), 641-650 Fetscherstrasse 74, 01309 Dresdeb, Germany 3 d Blaich et al. (2010) Proc Natl Acad Sci U S A, 107 (22), 10285-10289 University of Cambridge Department of Physiology, Downing Street, Cambridge, CB2 3EG, Great Britain
Introduction: Human urinary bladder expresses mRNA of the three known b- 305 adrenoceptor (b-AR) subtypes (b1, b2, b3) in detrusor and urothelium. We have shown previously that only b3-AR is involved in human detrusor relaxation. To investigate the urothelium-induced modulation of b-AR-mediated relaxation, we have examined systematically whether other b-AR subtypes are involved. Cardiac fibroblasts as potential therapeutic targets in chronic atrial fibrillation Poulet C., Lu L., Christ T., Wettwer E., Ravens U. Materials and Methods: Human detrusor tissue samples were obtained from patients Medizinisch Theoretisches Zentrum Department of Pharmacology and Toxicology, undergoing radical cystectomy for the treatment of bladder cancer. Detrusor strips were Fetscherstr, 74, 01307 Dresden, Germany studied with and without an intact mucosa layer. Muscle strips were precontracted with 1 µM carbachol and relaxation was studied in response to the b-AR agonist NE. a-AR Introduction. Chronic atrial fibrillation (cAF) is marked by increased fibrosis which mediated processes were blocked with the a-AR antagonists phentolamine and contributes to the perpetuation of the disease. In addition to the role of fibrosis in prazosin. Selective b-ARs antagonists were used to investigate b-AR mediated structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via relaxation. At the end of each experiment 10 µM forskolin was used to determine gap junction thereby altering the electrophysiological properties of the later and maximum cAMP-mediated relaxation. potentially participating in atrial electrical dysfunction. In order to understand the importance of fibroblasts in the pathophysiology of cAF, we compared the electrical Results: Cumulatively increasing concentrations of NE relaxed human detrusor muscle. properties of atrial fibroblasts isolated from patients in sinus rhythm (SR) and cAF. In urothelium-denuded strips the b-AR agonist was more potent (-logIC50[M] 6.41±0.09 Methods. Fibroblasts were isolated by outgrowth culture from right atrial biopsies and (n=24) versus 5.64±0.12 (n=20). Also, NE relaxed urothelium-free detrusor slightly more cultivated in medium containing 10% fetal calf serum. We used whole-cell patch clamp (71±4% versus 63±3% of forskolin). The selective b1-AR blocker CGP 20712A did not techniques to investigate ion currents and membrane potential. influence the NE induced relaxation. In the presence of the b2-AR antagonist ICI Results. SR and cAF fibroblasts showed similar capacitance (SR: 43.6 ± 4.6 pF, n=33; 118,551 NE was more potent (-logIC50[M] 6.39±0.16 (n=4) and effective (79±5%) in cAF: 54.7 ± 5.1 pF, n = 17) and membrane potential (SR: -21.0 ± 4.3 mV, n = 14; cAF: - intact muscle strips. The selective b3-AR antagonist L748,337 shifted the CRC for NE to 27.4 ± 4.8 mV, n = 16). In both groups, we observed fast activating outward currents higher concentrations without effecting maximum relaxation. Calculation of apparent with a mean threshold at -20 mV. Interestingly, current amplitude was significantly larger affinity values (pKB[M]) for L748,337 using Schild plot analysis (slope=1) yielded in SR than cAF cells (SR: 23.8 ± 4.2 pA/pF, n = 15; cAF: 6.1 ± 1.0, n = 6; p < 0.05). 7.79±0.05 for denuded and 7.63±0.07 for intact detrusor strips. When maintained in culture for 3-5 weeks, cells from both groups developed Na+ currents. Surprisingly, the fraction of cAF cells displaying such currents was larger than Conclusion: The presence of intact urothelium reduces potency but not effectivity of NE the SR counterpart (cAF: 38%; SR: 15%). Furthermore, Na+ current amplitude was indicating involvement of urothelium-mediated processes not only during detrusor significantly larger in cAF fibroblasts (SR: 6.1 ± 2.0 pA/pF, n = 5; cAF: 17.4 ± 4.4 pA/pF, contraction but also during relaxation. NE-mediated detrusor relaxation is mediated + n = 6; p < 0.05). Na currents were not altered by 100 nM Tetrodotoxin (TTX), but 10 µM through b3-AR in the absence of urothelium. But in intact detrusor strips b2-ARs seem to TTX reduced current amplitude to 42% of control, suggesting that the channel involved have an additional inhibitory effect. Affinity of the selective b3-AR antagonist L748,337 is is the cardiac TTX-resistant isoform Nav1.5. unchanged, therefore the intact urothelium does not interact with the function of b3-ARs. Conclusion. In the context of cAF, fibroblasts undergo electrophysiological changes which need to be thoroughly described. Understanding whether those changes contribute to the AF substrate might provide new therapeutic targets for the treatment of cAF.
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Aldosterone causes oxidative stress and DNA damage independent of blood Microarray gene expression profiling reveals up-regulation of the cardiac lipid pressure in vivo metabolic process at the onset of heart failure Queisser N., Schupp N. Fu X., Abd Alla J., Quitterer U. Universität Würzburg Institut für Toxikologie, Versbacherstr. 9, 97078 Würzburg, ETH Zürich Molekulare Pharmakologie, Winterthurerstrasse 190, 8057 Zürich, Germany Switzerland
Background: An inappropriate increase of the mineralocorticoid aldosterone (Ald) can Atherosclerosis and chronic pressure overload are major cardiovascular risk factors for be induced by a stimulated renin-angiotensin-aldosterone system. Epidemiological the development of heart failure in patients. To mimic those risk factors in experimental studies exploring the connection between hypertension and cancer found higher cancer models we used atherosclerosis-prone apolipoprotein E (ApoE)-deficient mice, and mortality and an increased risk to develop kidney cancer in hypertensive individuals. We chronic pressure overload was imposed by abdominal aortic constriction (AAC). Cardiac recently showed that Ald produces oxidative stress, activates transcription factor NF-kB function was monitored by echocardiography. Severe atherosclerosis in aged ApoE- and is genotoxic in kidney tubule cells. deficient mice or chronic pressure overload induced signs of heart failure as evidenced Objectives: This study investigated the capacity of Ald to induce oxidative/nitrosative by a significantly reduced cardiac ejection fraction (<30%). To investigate stress, DNA damage, DNA repair, apoptosis, cell proliferation and the activation of NF- pathomechanisms underlying the development of heart failure, microarray gene κB in rat kidneys. expression analysis and QT-RT-PCR were performed of failing heart tissue relative to Methods: Mineralocorticoid-dependent hypertension was induced by Ald/salt in Sprague age-matched controls. Gene ontology analysis of the microarray data revealed that the Dawley rats. DNA damage and oxidative/nitrosative stress markers were detected onset of heart failure, in two different experimental models, was characterized by a immunohistochemically. strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. Results: Ald/salt treatment caused increased blood pressure compared to untreated Lipid metabolism genes were involved in lipid synthesis, storage and oxidation. The rats. Tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the renin-angiotensin-aldosterone system, could lower the blood pressure, while the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. Taken mineralocorticoid receptor (MR) antagonist spironolactone was administered in a together the data support that the onset of experimental heart failure is characterized by subtherapeutical dose not lowering the blood pressure. Ald/salt treatment caused a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. oxidative and nitrosative stress, structural DNA damage, double strand breaks, DNA repair and NF-κB activation. Spironolactone decreased these markers significantly. Tempol was also able to reduce these markers, while hydralazine had no effect. Ald/salt- treated kidneys showed a tendency to lower apoptosis and to increased cell proliferation 311 compared to control rat kidneys. Discussion: This study provides a first hint of blood pressure-independent effects of Ald. The MR and the production of ROS seem to be crucial for the damaging effects of Cardinal symptoms of Metabolic Syndrome are improved in diet induced obese Ald. An aberrant or long-term activation of NF-κB by persistently high Ald levels could rats by chronic AT -receptor blockade support resistance to apoptosis and the survival of cells with damaged DNA, and 1 increase cell proliferation. These actions could contribute to the increased cancer Hübel N., Müller-Fielitz H., Stölting I., Raasch W. incidence in hypertension by initiating carcinogenesis. Universität Lübeck Institut für experimentelle und klinische Pharmakologie und Grant support by the DFG is gratefully acknowledged. Toxikologie, Ratzeburger Allee 160, 23538 Lübeck, Germany
AT1-receptor blockers (ARBs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by ARBs. In the past years evidence increased that body weight may also be lessened particularly in rats and mice. However, less data 309 are available whether ARBs still reduce weight, when treatment was initiated not until animals became obese by diet. Prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with Higher expression of the CREB regulating transcriptional coactivator 1 in murine chow but also with a cafeteria diet (CD) to develop obesity. Controls received only chow and human hypertrophied heart (CON ). CD-fed SHR were treated for 3 months with telmisartan (TEL 8mg/kg/d) or 1 2 2 3 4 chow Quentin T. , Steinmetz M. , Wichmann H. , Schöndube F. , Schlossarek S. , amlodipine (AML, 12 mg/kg/d), whereas controls received vehicle (CON ). Systolic 4 4 5 5 6 1 CD Eschenhagen T. , Carrier L. , Kaul A. , Hasenfuß G. , Zimmermann W. - H. , Oetjen E. blood pressure (SBP), feeding behaviour, body weight, abdominal fat mass (by MRT) 1Universitätsklinikum Hamburg-Eppendorf Institut für Klinische Pharmakologie und and energy expenditure (by indirect calorimetry) was monitored. Leptin sensitivity was Toxikologie, Martinistraße 52, 20246 Hamburg, Germany assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of 2Universitätsklinikum Göttingen Pädiatrische Kardiologie und Intensivmedizin, Robert leptin. Insulin sensitivity was functionally determined by glucose and insulin tolerance Koch Straße 40, 37075 Göttingen, Germany tests. 3Universitätsklinikum Göttingen Thorax-, Herz- und Gefäßchirugie, Robert Koch Straße Due to CD feeding body weight was increased after 6 months by more than 60 g. TEL 40, 37075 Göttingen, Germany normalized SBP whereas it remained >200 mmHg in CONCD and CONchow. TEL 4Universitätsklinikum Hamburg-Eppendorf Institut für Experimentelle Pharmakologie und additionally reduced CD-induced increase of body weight and abdominal fat mass. Food Toxikologie, Martinistraße 52, 20246 Hamburg, Germany intake was diminished during the first 4 weeks, but raised beyond control levels during 5Universitätsklinikum Göttingen Kardiologie und Pneumologie, Robert Koch Straße 40, the last 4 weeks of treatment. The shift of the respiratory index to lower levels indicated 37075 Göttingen, Germany improved energy expenditure. In response to exogenous leptin, the food intake of 6 Universitätsklinikum Göttingen Pharmakologie, Robert Koch Straße 40, 37075 CONCD was higher compared to CONchow, indicating a leptin resistance. This assumption Göttingen, Germany is further supported by high triglyceride concentrations of CONCD. After TEL, leptin- induced food intake was reduced and energy expenditure was increased compared to CREB regulating transcriptional coactivator 1 (CRTC1) is a transcriptional coactivator of CONCD, indicating that leptin sensitivity was at least partially restored. Accordingly, the transcription factor CREB. We have recently shown its expression in cardiomyocytes triglycerides were reduced. Compared to CONCD, the insulin sensitivity was improved by and its activation by beta-adrenergic signaling. Beta-adrenergic signaling contributes to TEL since maximal increases in plasma concentrations of glucose and insulin in the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the response to glucose challenge were reduced, but glucose response to insulin challenge therapeutic success of the beta-adrenoceptor antagonists. In order to investigate if was diminished. Even though reduction in blood pressure was almost similar between CRTC1 is involved in this process, we investigated the expression of CRTC1 in TEL and AML, metabolic and antiobese efficacies of AML were markedly attenuated. hypertrophied myocardium from mice and humans. We conclude that telmisartan reveals wide efficacies in improving all symptoms of the Methods: Protein lysates from mouse and human samples were investigated for CRTC1 Metabolic Syndrome. The pleiotropic effects are not related to the hypotensive action of protein expression. We distinguished between an acquired and an inherited form of TEL. cardiac hypertrophy. Acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. In mice this kind of hypertrophy can be evoked by Transverse aortic constriction (TAC). The inherited form of cardiac hypertrophy is caused by mutations in genes coding for 312 proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (HCM). As a model for HCM, transgenic mice with a mutation in the Mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein C (cMyBP-C), were A β2-adrenoceptor – cAMP mediated, immediate stimulation of β2-adrenoceptor used. These transgenic mice were characterized by a hypertrophied heart. In case of gene expression in human lung fibroblasts is opposed by a delayed up-regulation human samples we distinguished between patients with an acquired form of hypertrophy of inhibitory factors due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (HOCM), a special form of HCM. Racké K., Lamyel F., Kämpfer N., Schütz I., Warnken M. Results: In the TAC mice and in the MYPPC3 mutant mice the expression of CRTC1 Univ. Bonn Dept. Pharmacol. &Toxicol., Sigmund-Freud-Str. 25, 53105 Bonn, Germany was significantly higher than in the controls (2.1- and 1.9-fold, respectively; n=3). In both forms of human hypertrophy, we found a significantly 5-fold upregulation of CRTC1 Based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential protein level in comparison to human samples from non-failing myocardium or samples elements in the treatment of bronchial asthma and COPD. However, treatment with from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). long-acting β2-adrenoceptor agonists has been associated with possible worsening of Conclusions: CRTC1 is upregulated in cardiac hypertrophy with acquired or genetic- airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. Therefore, based origin. The evaluation of the role of CRTC1 in the heart may help to elucidate the the molecular regulation of β2-adrenoceptor expression was addressed here. role of beta- adrenergic signaling in the development of cardiac hypertrophy. MRC-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mRNA determination by qPCR. β2-Adrenoceptor mRNA decreased with a half-life of 25 min after inhibition of mRNA synthesis with actinomycin D (30 µM), but increased by 333±85%, 502±52% and 640±165% (means±SEM) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µM). The β2-adrenoceptor agonists formoterol and olodaterol (1-100 nM) induced a rapid increase in β2-adrenoceptor mRNA (maximally within 1 h by 100±19% and 110±19% at 10 nM, resp.). However, after 4 h exposure to S72
10 nM formoterol or olodaterol a reduction in β2-adrenoceptor mRNA by 59±8% and H9c2 cells showed almost identical hypertrophic responses to those observed in human 58±6%, resp., was observed. Both, the stimulatory and inhibitory effects of β2- ventricles and rat hearts. This finding validates H9c2 cells as a model for in vitro studies adrenoceptor agonists were mimicked by forskolin (10 µM, increase by 88±14% and of cardiac hypertrophy. In further studies we will investigate the consequences of a inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by knock-down of DPP6 and DPP10 in doxorubicin-induced hypertrophic H9c2 cells. In 77±7%). The formoterol-induced up-regulation of β2-adrenoceptor mRNA was blocked preliminary experiments specific short-hairpin RNA, targeting DPP6 and DPP10, has by actinomycin D, but not by cycloheximide. Moreover, in presence of cycloheximide, β2- been designed and tested in heterologous expression systems. adrenoceptor agonist induced inhibition was converted into a marked stimulation. In conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. The observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified 315 suppressors. Although both, the time-dependent up- and down-regulation of the β2- adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase - cAMP signalling, only the stimulatory effect appears to be a direct The nonsynonymous c.521T>C germline genetic variation in the liver-specific action on the β2-adrenoceptor gene. organic anion transporter SLCO1B1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia Radtke S.1, Zolk O.2, Renner B.3, Stanulla M.4, Möricke A.4, Langer T.1 1Kinder- und Jugendklinik des Universitätsklinikums der Friedrich-Alexander Universität 313 Erlangen-Nürnberg LESS-Studienleitung, Loschgestraße 15, 91054 Erlangen, Germany 2Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Friedrich- Alexander Universität Erlangen-Nürnberg Lehrstuhl für Klinische Pharmakologie und β2-Adrenoceptors and muscarinic receptors mediate opposing effects on Klinische Toxikologie, Fahrstraße 17, 91054 Erlangen, Germany endothelin-1 (ET-1) expression in human lung fibroblasts 3Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Friedrich- 1 2 1 Ahmedat A. , Juergens U. R. , Racké K. Alexander Universität Erlangen-Nürnberg Lehrstuhl für Pharmakologie und Toxikologie, 1Univ. Bonn Dept. Pharmacol. & Toxicol., Sigmund-Freud-Str. 25, 53105 Bonn, Fahrstraße 17, 91054 Erlangen, Germany Germany 4Klinik für allgemeine Pädiatrie des Universitätsklinikums Schleswig-Holstein ALL BFM 2Univ- Hospital Dept. Pulmonology, Sigmund-Freud-Str. 25, 53105 Bonn, Germany Studienzentrale, Schwanenweg 20, 24105 Kiel, Germany
ET-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, Background: Methotrexate (MTX) plasma concentration is related to its clinical effect. but also in fibrotic remodeling associated with chronic obstructive airway diseases. Since Transport proteins, such as ABCC2, SLCO19A1, and SLCO1B1, have been implicated human lung fibroblasts (hLF) are a source of ET-1 and have been shown to be in the disposition of MTX. Here we investigated whether common reduced-function controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and β- variants in ABCC2, SLCO19A1, and SLCO1B1 contribute to the interindividual variability adrenergic modulation of ET-1 expression in hLF was explored. in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (ALL). MRC-5 hLF were cultured for up to 24 h in absence or presence of test substances, followed by prepro-ET-1 (ppET-1) mRNA determination by qPCR. Methods: We analyzed MTX pharmacokinetics (MTX plasma concentration at the end The muscarinic agonist oxotremorine (10 µM) induced an increase in ppET-1 mRNA by of infusion C24h, MTX AUC24-48h, and MTX clearance Cl) in an unselected population of 180%, an effect prevented by 10 nM tiotropium. The β2-adrenoceptor agonist olodaterol 419 children with ALL from the ALL-BFM 2000 trial (ClinicalTrials.gov: NCT00430118) (up to 100 nM) caused a reduction of ppET-1 mRNA expression by 45%. The effect of who received 1676 courses of MTX at 5 g/m2 as 24 h infusions. The contribution of 10 nM olodaterol was prevented by ICI 118,551 (1 µM), but not affect by CGP 20712 (3 genes (genetic component, rGC) to the interindividual variability in MTX pharmacokinetics µM). The PKA agonist 6-Bnz-cAMP (500 µM) caused a reduction in ppET-1 mRNA was estimated according to the method of Kalow et al. (1998). ABCC2 c.-24C>T expression by 65%, whereas the Epac agonist 8-CPT-2’-O-Me-cAMP (100 µM) caused (rs717620), SLCO19A1 c.80G>A (p.His27Arg, rs1051266), SLCO1B1 c.521T>C only a marginal inhibition by 22%. Olodaterol (10 nM) strongly opposed the stimulatory (p.Val174Ala, rs4149056) and SLCO1B1 388A>G (p.Asn130Asp, rs2306283) genotypes effect of 10 µM oxotremorine. An increase in ppET-1 mRNA expression by 185% caused were analyzed by TaqMan polymerase chain reaction. by 0.3 ng/ml TGF-β was effectively opposed by 10 and 100 nM olodaterol, resulting in an inhibition comparable to that in absence of TGF-β. However, the increase in ppET-1 Results: There was substantial interpatient variability in average (± SD) MTX C24h mRNA caused by a maximally effective concentration of TGF-β (1 ng/ml, increase by (50.95 ± 24.15 µmol/l), AUC24-48h (57.44 ± 37.52 h*mg/l), and Cl (390.72 ± 223.95 620%) was not significantly affected by 10 or 100 nM olodaterol. Likewise, the PKA- ml/min/m²). The rGC values of C24h, AUC24-48h, and Cl ranged from 0.61-0.71 suggesting agonist 6-Bnz-cAMP (500 µM) opposed the increase in ppET-1 mRNA expression that variation in MTX pharmacokinetics has a substantial genetic component. After caused by 0.3 ng/ml TGF-β, but not that caused by 1 ng/ml TGF-β. TGF-β caused, with adjustment for age and sex by multiple regression, the SLCO1B1 c.521T>C SNP was an IC50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mRNA expression, significantly associated with C24h (P<0.001), AUC24-48h (P<0.001), and Cl (P=0.011) of maximally by 90% within 6 h. MTX. Compared with the wildtype genotype, in patients with the TC genotype C24h and ET-1 expression in hLF is stimulated by muscarinic receptors and inhibited by β2- AUC24-48h increased by 18% (P=0.009) and 28% (P=0.003), respectively, whereas Cl adrenoceptors. The effect of β2-adrenoceptors may be mediated via PKA. ET-1 significantly decreased by 15% (P=0.012). Pharmacokinetic variables significantly expression in hLF is markedly up-regulated by TGF-β, but only effects of sub-maximally changed with increasing number of variant c.521T>C alleles (P<0.02, Jonckheere- effective concentrations of TGF-β are opposed by the β2-adrenoceptor - PKA pathway, Terpstra), suggesting a per allele effect consistent with a co-dominant model of in part because of TGF-β-induced down-regulation of β2-adrenoceptors. Since ET-1 can association. In contrast, the ABCC2 c.-24C>T, SLCO19A1 c.80G>A, and SLCO1B1 promote pro-fibrotic features in hLF, inhibition of ET-1 expression could contribute to 388A>G polymorphisms did not show an association with MTX pharmacokinetics. long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. Conclusions: The nonsynonymous c.521T>C polymorphism in SLCO1B1 contributes to the variability of MTX pharmacokinetics in this study of high-dose MTX in pediatric ALL.
This project is supported by the Johannes und Frieda Marohn-Stiftung. 314
Cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like 316 proteins in human and rat Radicke S., Hutschenreuther A., Schaefer M. Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr. Induction but not inhibition of COX-2 confers human lung cancer cell apoptosis 16-18, 04107 Leipzig, Germany by celecoxib 1 2 1 1 1 Ramer R. , Linnebacher M. , Walther U. , Borchert P. , Hinz B. Cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and 1Institut für Toxikologie und Pharmakologie, Universität Rostock, Schillingallee 70, mortality. Functional down-regulation of K+ currents is a prominent feature of cells 18057 Rostock, Germany isolated from failing ventricles. A marked decrease in the transient outward potassium 2Abteilung für Molekulare Onkologie und Immuntherapie, Universität Rostock, + current Ito has been shown in various models. Changes in the K channel expression Schillingallee 69, 18057 Rostock, Germany differ depending on the species, and the mechanism of induction of heart failure. To study the regulation of Ito channel subunits we compared the hypertrophic responses in The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in celecoxib is still a matter of debate. Using human lung cancer cell lines (A549, H358, rats and in H9c2 embryonic rat cardiac cells. Specifically, we quantified mRNA H460), the present study investigates the contribution of COX-2 and peroxisome expression of the pore-forming subunits Kv4.3 and Kv4.2, the cytosolic β-subunit proliferator activated receptor γ (PPARγ) to apoptosis elicited by celecoxib. Celecoxib KChIP2 and the transmembrane subunits DPP6 and DPP10 using RT-PCR. was found to cause apoptotic cell death in a concentration-dependent manner (10 - 50 Treatment with doxorubicin (2 µM) induced hypertrophy and increased the mRNA µM), whereas structurally-related COX-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) expression of the hypertrophy marker genes ANF, BNP and beta-MHC in H9c2 cardiac were inactive in this respect. Apoptotic cell death by celecoxib was suppressed by myoblasts. While Kv4.3 was detected in H9c2 cells and hearts from human and rat, preincubation of tumor cells with the selective COX-2 inhibitor NS-398, the PPARγ Kv4.2 mRNA was only expressed in adult rats. During hypertrophy Kv4.3 was down- antagonist GW-9662 and by siRNA targeting COX-2 or PPARγ. Celecoxib-induced regulated in human tissue as well as in doxorubicin-treated H9c2 cells compared to the apoptosis was paralleled by a time- and concentration-dependent upregulation of COX-2 controls. In rat hearts Kv4.2 expression was increased after doxorubicin treatment. and PPARγ at both mRNA and protein level. Using an established COX-2 activity assay Interestingly, Kv4.2 was also found to be up-regulated in rat heart tissues and H9c2 cells monitoring immediate conversion of exogenously added arachidonic acid to the after treatment with doxorubicin. As previously shown, KChIP2 mRNA expression was respective prostaglandins (PGs), NS-398 was shown to suppress celecoxib-induced significantly reduced in tissues of failing hearts. In contrast, KChIP2 mRNA was up- COX-2 activity when added prior to arachidonic acid. Among the COX-2-dependent PGs 12,14 regulated in hypertrophic rat hearts and H9c2 cells. The expression of DPP6 and DPP10 analyzed, PGD2 and its dehydration product 15-deoxy-∆ -PGJ2 were found to induce was observed only in human hearts. But mRNA levels of both were significantly cytosol-to-nucleus translocation of PPARγ as well as PPARγ-dependent apoptosis. increased in failing tissues. DPP6 and DPP10 were not expressed in the adult rat heart Celecoxib-elicited translocation of PPARγ was inhibited by preincubation of cells with or H9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in NS-398 which itself did not alter celecoxib-induced total PPARγ protein expression. doxorubicin-treated H9c2 cells, the mRNA of DPP6 and DPP10 was up-regulated. Finally, a COX-2- and PPARγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer S73
patients. Together, our data demonstrate a proapoptotic mechanism of celecoxib 319 involving initial upregulation of COX-2 and PPARγ and a subsequent nuclear translocation of PPARγ by COX-2-dependent PGs.
Differences in Histamine-H2-Receptor-Mediated Increases in cAMP Concentration and Reactive Oxygen Species Formation in Human Eosinophils and Neutrophils Reher T.1, Brunskole I.2, Burhenne H.3, Neumann D.1, Seifert R.1 317 1Medizinische Hochschule Hannover Pharmakologie, Carl Neuberg Straße 1, 30625 Hannover, Germany 2Universität Regensburg Pharmazeutische und medizinische Chemie, 93040 Direct inhibitors of thrombin and factor Xa prevent mitogenic and inflammatory Regensburg, Germany reponses in vascular smooth muscle cells exposed to blood clots generated in 3Medizinische Hochschule Hannover Core Unit Mass spectrometry/metabolomics, Carl vitro Neuberg Straße 1, 30625 Hannover, Germany 1 1 2 Fender A. C. , Schrör K. , Rauch B. H. 1Klinikum der Heinrich-Heine-Universität Düsseldorf Institut für Pharmakologie und Human eosinophil and neutrophil granulocytes are cells of the innate immune system. Klinische Pharmakologie, Moorenstraße 5, 40225 Düsseldorf, Germany They both express formyl peptide receptors (FPR) und histamine H2 receptors (H2R). 2 Ernst-Moritz-Arndt-Universität Greifswald Institut für Pharmakologie, Friedrich-Loeffler- Activation of FPR leads to a release of reactive oxygen species (ROS). H2R activation Str. 23d, 17487 Greifswald, Germany results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) concentration and inhibition of FPR-mediated ROS release via adenylyl cyclase Aim: Purified thrombin and active factor X (FXa) stimulate proliferation of cultured activation. In this study we compared the effects of various H2R ligands on cAMP vascular smooth muscle cell (SMC) at low-mid nanomolar concentrations. The clinical accumulation and formyl peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)- relevance of these observations is commonly questioned. We investigated how much induced ROS release in isolated eosinophils and neutrophils. cAMP concentration was thrombin and FXa is generated during clotting and if this is sufficient to influence determined by HPLC/tandem mass spectrometry, and ROS release was assessed by adjacent SMC. monitoring superoxide dismutase-inhibitable reduction of ferricytochrome C. Methods: Clots were generated from platelet poor plasma (PPP) and incubated in PBS In eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies for up to 2h. Thrombin and FXa content was determined in clots, clot-conditioned PBS and efficacies with regard to cAMP accumulation and inhibtion of ROS release. In and in remaining uncoagulated PPP. For functional studies, clots were placed onto marked contrast, in human neutrophils, we observed dissociations in potencies and cultured human aortic SMC synchronised by serum-deprivation and pretreated ± efficacies of ligands at increasing cAMP accumulation and inhibition of ROS production. inhibitors (DX-9065a, hirudin, argatroban, rivaroxaban). Other SMC were stimulated with Our data suggest that in human eosinophils, but not neutrophils, cAMP mediates remaining unclotted PPP (native or boiled to inactivate protease activity). DNA synthesis inhibtion of ROS production. In a broader context our data provide a compelling was assessed by [3H] thymidine incorporation, inflammatory gene expression by example of the context-dependency of the pharmacological properties of G-protein- realtime PCR and apoptosis by flow-cytometry. coupled-receptors. Specifically, one has to be cautious when extrapolating experimental Results: Thrombin was initially detected in clots at approximately 90 nM, at 2h 40 nM observations from one cell type to another, even when they are very closely related to remained. Meanwhile clot-conditioned PBS accumulated up to 25 nM thrombin. each other. Uncoagulated PPP contained 40-60 nM thrombin throughout. FXa was initially Supported by Research Training Group (GRK 1441) "Allergic response in lung and skin" measured in clots at 10 nM. This level declined over time while clot-conditioned PBS of the Deutsche Forschungsgemeinschaft (DFG) accumulated FXa. Exposure of human aortic SMC to clots or native unclotted PPP for 24h only marginally influenced SMC apoptosis but increased mitogenesis over 15-fold. This was reduced by all 4 inhibitors. Clot-stimulated induction of TNFα and interleukin-6 mRNA was also attenuated by the inhibitors. Denatured PPP (no protease activity) 320 increased SMC mitogenesis to a level seen in SMC exposed to clot and combined hirudin + DX9065a, reflecting the well-known mitogenic actions of serum alone. Conclusion: Coagulation of human plasma generates nanomolar amounts of thrombin Chemical analysis of released components from polymerized dental composites and FXa, sufficient to stimulate the proliferative and inflammatory properties of adjacent 1,2 1 1 1 1 3 2 SMC. Our observations validate the use of purified thrombin and FXa at nanomolar Reichl F. - X. , Durner J. , Styllou M. , Styllou P. , Heym R. , Schweikl H. , Maier M. , Dettinger K.2 concentrations for in vitro studies, and support the individual and coagulation- 1 independent roles of these proteases in cell proliferation and inflammation. Zahnklinik München LMU Parodontologie und Zahnerhaltung, Goethestr. 70, 80336 Antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of München, Germany 2Walther-Straub-Institut LMU, Nussbaumstr. 26, 80336 München, Germany clot-derived thrombin and FXa, while normal anti-platelet therapy would not. This aspect 3 should be considered in the clinical use of these agents, specifically in healing Zahnklinik der Universität Regensburg Zahnerhaltung und Parodontologie, Franz-Josef- processes after vessel injury. Strauß-Allee 11, 93042 Regensburg, Germany
Comonomers and monomers are used as dental restorative materials (e.g. in dental composites). Unconverted compounds can be released from dental composites and can enter the body in humans. Comonomers can induce various side effects in humans. This 318 study was evaluated to qualify and to quantify eluted compounds from various dental composites. Following composites were tested (producer in parentheses): Els extra low shrinkage (Saremco), Synergy Duo Shade (Coltène), Grandio (VOCO), Tetric Evo RhoGEF17 mediates cGMP/cGK induced adherence and relaxation of vascular Ceram (Vivadent), Venus (Kulzer), Gradia (G.C.), and Premise (Kerr).Polymerized smooth muscle cells composites (100 mg) were incubated in GC vials with 1 ml dest. water or 1 ml methanol, 1 1 2 1 Rauch J. , Stephan-Schnatz K. , Lutz S. , Wieland T. each at 37 °C for 72 hours. Aliquots were taken, and eluted compounds were analyzed 1Med. Fakultät Mannheim der Universität Heidelberg Experimentelle Pharmakologie, with the method of gas chromatography/mass spectrometry (GC-MS) and liquid Maybachstraße 14, 68169 Mannheim, Germany chromatography/mass spectrometry (LC-MS).From all composites 18 different 2Universitätsmedizin Göttingen Pharmakologie, Robert-Koch-Straße 40, 37075 compounds were found. Following comonomers were quantified (µg/ml; mean ± s.d.; Göttingen, Germany n=4)(fig. 1).Following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): Venus > Gradia > Synergy Duo The guanine nucleotide exchange factor RhoGEF17 is the only GEF known so far to be Shade > Tetric Evo Ceram Premise > Grandio > Els Extra low shrinkage. directly activated by cGMP-dependent kinase. It is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. In a Table 1 previous investigation we showed that the knockdown of RhoGEF17 by a shRNA detected comonomers detected comonomers approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. We now provide evidence composites dest. water methanol that the expression level of RhoGEF17 influences the re-attachment of cultured rat aortic HEMA TEGDMA HEMA TEGDMA smooth muscle cells (RASMC) to a surface after detachment. Although RhoGEF17 Els extra low depleted RASMC were still able to adhere and spread, their cell surface area remained n.d.* n.d.* n.d.* n.d.* considerably smaller than that of control cells in the first 24 hours after seeding. Cell shrinkage counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was Synergy Duo n.d.* 104 ± 16 n.d.* 126 ± 23 significantly lower in the RhoGEF17 knockdown group compared to the control group. Shade These data indicate a delay in attachment. Interestingly, the knockdown of RhoGEF17 Grandio n.d.* 36 ± 5 n.d.* 68 ± 12 was paralleled by a loss in RhoA and cadherine expression. As RhoGEF17 mediated a cGMP-induced activation of the small GTPase RhoA in RASMC, we studied the effect of Tetric Evo Ceram n.d.* 57 ± 12 496 ± 77 n.d.* a stable cGMP analogon (8-pCPT-cGMP) on the adhesion process. In accordance with Venus n.d.* 197 ± 26 n.d.* 76 ± 7 previously published data, cGMP treatment accelerated the attachment of RASMC to the surface within the first 12 hours. In contrast, the adhesion of RASMC after Gradia n.d.* 123 ± 18 500 ± 66 62 ± 2 RhoGEF17 knockdown was no longer stimulated by cGMP. As these data indicate that Premise n.d.* 48 ± 7 n.d.* 51 ± 9 RhoGEF17 mediates cGMP-dependent signalling in a physiological process we * n.d. = not detectable (below limit of detection). Triphenylstibane was detected in Tetric wondered whether this protein might also play a role in the regulation of cGMP- Evo Ceram (5 ± 2 µg/ml). dependent relaxation of vascular smooth muscle cells. Thus, we performed a collagen- based contraction assay with RhoGEF17 depleted RASMC. While the contraction in response to serum was not affected by the depletion of RhoGEF17, we observed a slight decrease in basal contractility. Interestingly, cGMP was not able to counteract the serum-driven contraction of RhoGEF17 knockdown cells. There was no cGMP-induced relaxation in these cells. We conclude that RhoGEF17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. Its activation is required to mediate cGMP-induced signalling in terms of vascular smooth muscle cell adherence and relaxation.
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Reevaluation of CXCR4 chemokine receptor expression in human normal and Probing the substrate-specificity of human phosphodiesterase 3A with MANT- neoplastic tissues using the rabbit monoclonal antibody UMB-2 substituted purine and pyrimidine cyclic nucleotides Reimann C., Lupp A., Schulz S. Reinecke D.1, Schwede F.2, Seifert R.1 Institut für Pharmakologie und Toxikologie Universitätsklinikum Jena, Drackendorfer Str. 1Medizinische Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Str. 1, 1, 07747 Jena, Germany 30625 Hannover, Germany 2BIOLOG Life Science Institute, Flughafendamm 9a, 28199 Bremen, Germany Objective: CXCR4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. In the adult organism CXCR4 is Cyclic nucleotide phosphodiesterases (PDEs) are classified into eleven families and are physiologically expressed on various cell types, in particular on lymphocytes. With respect essential for second messenger metabolism in human cells.1 Recently, we have shown to neoplastic tissues, in the current literature an over-expression of CXCR4 is described in that several human PDEs possess a much broader substrate-specificity than previously different types of tumors, especially in breast and prostate cancer. Additionally, an assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine involvement of CXCR4 in tumor metastasis is discussed. Subsequently, detection of 3’,5’-monophosphate (cAMP) and cyclic guanosine 3’,5’-monophosphate (cGMP), but CXCR4 expression in a given human tumor sample would provide a valuable predictive also pyrimidine nucleotides such as cyclic uridine 3’,5’-monophosphate (cUMP).2 These information on disease prognosis and possible therapeutic intervention. However, previous data were obtained using a highly sensitive HPLC mass spectrometric assay which is attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit quite expensive and whose technical requirements are available only in few laboratories. polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic In our present study we developed a fluorimetric PDE activity assay using 2'-O-(N'- staining, but did not result in the identification of cell surface receptors. Thus, the aim of the methylanthraniloyl) (MANT)-substituted cyclic purine and pyrimidine nucleotides that can present study was to reassess the CXCR4 expression in a panel of formalin-fixed and be used more broadly in the scientific community. paraffin-embedded human normal and neoplastic tissue samples by means of Human PDE3A is important for the regulation of platelet aggregation, oozyte maturation, immunohistochemistry using the well characterized novel rabbit monoclonal anti-CXCR4 vascular smooth muscle relaxation and contractility of cardiac myocytes.1 Moreover, this antibody UMB-2. PDE shows a broad substrate-specificity, hydrolyzing cAMP, cGMP, cUMP and cyclic Methods: In comparison to negative and positive control samples (CXCR4-knockout and inosine 3’,5’-monophosphate (cIMP).2 Using this enzyme, here, we demonstrate that wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue various MANT-substituted cyclic nucleotides are substrates of PDE3A and undergo a specimens was scored from zero (no expression) to three (high expression). significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative Results: CXCR4 was found to be expressed in all neoplastic tissue entities analyzed. In analysis of catalysis via fluorescence detection. In fact, not only native cUMP but also many cases, the receptor was predominantly localized at the plasma membrane of the MANT-cUMP is a substrate of PDE3A. This finding is consistent with data published by tumor cells. However, in all CXCR4-expressing tumor entities a huge interindividual Hardman and Sutherland3 who described a cUMP-degrading PDE activity in variability both in the percentage of positive cells and in the intensity of staining was noted homogenates from beef and dog heart, leading to the assumption that the PDE activity which strongly differed also between the various types of cancer. The most intense (score described there could be attributed to PDE3A. As cUMP has furthermore been proven to three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of be present in mammalian cells4, to differentially activate cAMP- and cGMP-dependent pheochromocytoma. Additionally, lymphatic organs such as lymph nodes, spleens and protein kinases5 and to be synthesized from UTP by mammalian soluble guanylyl tonsils were CXCR4 positive, with mainly B cells displaying a distinct staining of the plasma cyclase6, our present study supports the hypothesis that this cyclic nucleotide could play membrane. an important role in cell metabolism. The newly established fluorescence assay with Conclusion: The rabbit monoclonal antibody UMB-2 may prove to be of great value in the MANT-cUMP facilitates future studies on PDE3A and the assumed second messenger assessment of CXCR4 expression in different human tumor entities and of the mechanisms function of cUMP. underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. References [1] Bender AT, Beavo JA (2006) Pharmacol Rev 58:488-520 [2] Reinecke D, Burhenne H, Sandner P, Kaever V, Seifert R (2011) FEBS Lett. 585:3259-3262 322 [3] Hardman JG and Sutherland EW (1965) J. Biol. Chem. 240:3704–3705 [4] Burhenne H, Beste KY, Spangler CM, Voigt U, Kaever V, Seifert R (2011) Naunyn- Schmiedeberg’s Arch. Pharmacol. 383 (Suppl.):P096 .- [5] Wolter S, Golombek M, Seifert R (2011) Biochem. Biophys. Res. Commun. (in press) Link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced O2 production in human neutrophils and ADRB2-polymorphisms [6] Beste KY, Burhenne H, Kaever V, Stasch JP, Seifert R (2011) Biochemistry (in 1 1 2 2 1 press) Reinartz M. T. , Kälble S. , Wetzke M. , Kabesch M. , Seifert R. 1Hannover Medical School Institute of Pharmacology, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 2Hannover Medical School Clinic for Paediatric Pneumology and Neonatology, asthmagene.de, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 324
β2-Adrenoceptor (ADRB2)-agonists are in daily use for asthma therapy. Although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics Toxin-induced RhoA activity mediates CCL1-triggered STAT signaling 1 1 2 1 1 remains difficult to treat. The ADRB2 gene contains a total of 49 polymorphisms. This Reipschläger S. , Orth J. , Kubatzky K. , Aktories K. , Schmidt G. variability could cause part of the ~70 % genetically-determined differences in therapy 1Universität Freiburg Pharmakologie und Toxikologie, Albertstr. 25, 79104 Freiburg, response. Genetic and corresponding functional data on ADRB2 can help to understand Germany the complex disease and, in cases of severe asthma, optimize therapy with ADRB2- 2Universität Heidelberg Mikrobiologie und Hygiene, Im Neuenheimer Feld 324, 69120 agonists for each individual. (Ortega et al. 2007; Chung et al. 2011) Heidelberg, Germany Our present study connects sequence data with pharmacological data of prototypical ADRB2-ligands, namely, (R)-isoproterenol, (R,R)- and (S,S)-fenoterol, (R)- and (S)- RhoA is reportedly involved in STAT-dependent transcription. However, the pathway salbutamol and (R,R)-formoterol. As a pathophysiologically relevant cell type, we analysed connecting the GTPase and STAT signaling has not been characterized. We made use .- human neutrophils from peripheral blood of healthy volunteers. Formyl-peptide-induced O2 of bacterial toxins, which directly activate Rho GTPases to analyse this pathway. production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. Cytotoxic necrotizing factors (CNFs) are produced by pathogenic Escherichia coli Characteristic pharmacological values (pIC50, Emax) are obtained for each individual. The strains and by Yersinia pseudotuberculosis. They activate small GTPases of the Rho data-set for each individual is supplemented by a differential blood cell analysis and an family by deamidation of a glutamine, which is crucial for GTP hydrolysis. asthma-related questionnaire. Most importantly, each volunteer’s ADRB2-sequence variant We show that RhoA activation leads to phosphorylation and activation of STAT3 and is determined by Sanger-sequencing. Complete determination of a 1,490 bp sequence, identify signal proteins involved in this pathway. RhoA-dependent STAT3 stimulation including the entire ADRB2 exon and part of the flanking 5’- and 3’- untranslated regions, requires ROCK and JunKinase activation as well as AP1-induced protein synthesis. The allows mapping of the most common, but also of new or rare polymorphisms. First data secretion of one or more factor/s activate/s the JAK-STAT pathway in an auto/paracrine demonstrate the inter-day and intra-individual robustness of the functional data. In the next manner. We identify CCL1/I-309 as an essential cytokine, which is produced and step, we will link sequence variants and functional differences within a population of sixty secreted upon RhoA activation and which is able to activate STAT3-dependent signaling volunteers with sufficient statistical power. Collectively, this study represents a straight- pathways. The knowledge about the connection between RhoA and STAT signaling is forward approach to link functional and genetic data of a clinically relevant receptor. crucial for understanding several deseases, especially cancer.
References Ortega VE, Hawkins GA, Peters SP, Bleecker ER (2007) Pharmacogenetics of the beta 2-adrenergic receptor gene. Immunol Allergy Clin North 325 Am 27: 665-684 Chung LP, Waterer G, Thompson PJ (2011) Pharmacogenetics of β2 adrenergic receptor gene polymorphisms, long-acting β- Acid sphingomyelinase-deficient mice are protected from the lethal agonists and asthma. Clin Exp Allergy 41:312-326 cardiovascular effects in TNF-induced septic shock Reiss L. K.1, Adam D.2, Uhlig S.1 1Universitätsklinikum der RWTH Aachen Institut für Pharmakologie und Toxikologie, Wendlingweg 2, 52074 Aachen, Germany 2Christian-Albrechts Universität Institut für Immunologie, Michealisstrasse 5, 24105 Kiel, Germany
Introduction: The cytokine tumor necrosis factor (TNF) is a mediator of septic shock. Sepsis is a major cause of acute respiratory distress syndrome (ARDS), a Data-set of a proband: heterogeneous lung disease with a mortality of about 50%. The present study was Collected data-set of clinical and genetic data with corrosponding cellular and designed to investigate the effects of high systemic TNF-levels on the lung and on the functional data -/- systemic circulation in wildtype and acid sphingomyelinase-deficient (ASM ) mice. The S75
enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a We conclude that the consumption of high concentrations of CGA via coffee might critical role in edema formation and vasodilatation. influence the gastrointestinal transit time and consequently affect CGA bioavailability. Material and Methods: ASM-/- and wildtype mice were ventilated mechanically at -1 VT=8mL/kg and f=180min with FiO2=0.3 and PEEP=2cmH2O while lung mechanics This study was supported by the Nestlé Research Centre (Lausanne, Switzerland). were followed. Half of the mice received 50µg of murine TNF intravenously. Blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°C to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after 328 TNF application. Results: TNF induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and -/- Interaction of antagonists with the ATP binding pocket at the human P2X3 ion reflex tachycardia. Interestingly, ASM mice were protected from the TNF-induced channel cardiovascular effects and mortality. In the present study, circulating TNF failed to cause lung injury. Lung mechanics stayed stable during ventilation in all groups and also Helms N., Riedel T., Illes P. pulmonary histopathology, cytokine levels and microvascular permeability were Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16-18, 04107 unaffected. Leipzig, Germany Conclusion: Circulating TNF alone is not sufficient to cause acute lung injury. We conclude that the cardiovascular effects in TNF-induced septic shock are partly The homomeric P2X3 receptor (P2X3R) is a rapidly activating and desensitizing cation mediated by acid sphingomyelinase. channel, gated by extracellular ATP. It consists of three homomeric subunits. This representative of the P2X receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. Therefore, the development of selective antagonists for P2X3 receptors and knowledge about the binding of these antagonists are of great significance for future 326 pain therapy and therapy of urge incontinence. To simulate the shape of the rapidly desensitizing agonist-induced current responses via P2X3 receptors, we created a specific Markov model to describe the binding of agonists and competitive antagonists. Cyclophilin A siRNA provides mitoprotection and prevents AIF-dependent This model can be used to prove the competitive character of inhibition and to calculate neuronal cell death the association and dissociation constants of the antagonists. Furthermore we use this 1 1 2 2 3 Reuther C. , Culmsee C. , Doti N. , Ruvo M. , Plesnila N. model to fit current responses at P2X3 wild type receptors and their mutants to α,β- 1Philipps University of Marburg Institute of Pharmacology and Clinical Pharmacy, Karl- methylene ATP in the presence of different antagonists. Whole-cell patch-clamp von-Frisch-Str. 1, 35033 Marburg, Germany recordings were performed on HEK 293 cells, heterologously expressing the human 2Institute of Biostructure and Bioimaging-CNR, Via Mezzocannone 16, 80147 Naples, P2X3 receptor, to determine the concentration-response relationship of different Italy antagonists. By applying increasing concentrations, differences of antagonist potency 3Universitiy of Munich Medical School Institut of Stroke and Dementia Research, could be observed at the wild type receptor. Afterwards, we chose amino acid residues Heiglhofstr. 55, 81377 München, Germany for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, Cyclophilin A (CypA) is a peptidyl-prolyl-cis-trans isomerase which is localized in the developed from the zebrafish P2X4R crystal structure and previous mutagenesis cytosol. Recent data suggested that neuronal cell death involved cytosolic CypA studies. We intend to identify those amino acids which are important for competitive translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis antagonist binding by monitoring the altered antagonist potency on the mutated receptor inducing factor (AIF). This CypA-AIF complex induced caspase-independent chromatin when compared with the wild-type receptor. condensation, DNA degradation and cell death in various paradigms of apoptosis. On the basis of these data, the selective inhibition of the AIF-CypA complex was proposed as a potential strategy to prevent AIF-dependent cell death in neurons. Therefore, the aim of this study was to determine effects of CypA silencing in a model of 329 glutamate toxicity in immortalized hippocampal HT22 neurons. First, we addressed the interaction of AIF and CypA by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. After Analysis of the P2X3 agonist binding site by double mutant cycles exposure of HT-22 cells to glutamate the translocation of AIF and Cyp A occurred prior to cell death. CypA siRNA attenuated glutamate-induced cell death as detected by the Riedel T., Wiese S., Illes P. MTT-assay, impedance measurements (xCELLigence system), and by FACS analysis Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16-18, 04107 after Annexin V/ Propidium Iodide staining. Most intriguingly, CypA siRNA also Leipzig, Germany preserved the mitochondrial membrane potential as shown by FACS analysis after TMRE staining. Further, confocal microscopy showed that CypA silencing prevented Purinergic P2X receptors belong to the family of ligand-gated ion channels. They are mitomorphology alterations and blocked the release of mitochondrial AIF to the nucleus. non-selective cation channels, activated by extracellular ATP. One of the seven The inhibition of the AIF translocation to the nucleus was also shown by Western blot members of the P2X receptor family, the P2X3 receptor, is localized at the plasma analysis. membrane of sensory neurons and is involved in pain perception. Therefore, this In summary this study demonstrates that silencing of CypA prevents mitochondrial receptor is a possible target for new drugs in pain treatment. The development of such disruption and attenuates glutamate toxicity in vitro. Thus, CypA is a promising target for drugs can be supported by an exact knowledge of the receptor structure and function. mitoprotection as a basis for novel strategies of neuroprotection. There are many hints to the ATP binding site, but the interaction of the P2X receptor with its agonists and antagonists remains still unknown. In this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed ATP binding site of the homomeric human P2X3 receptor on the effect of nucleotide analogues. The mutant receptors were expressed in HEK293 327 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. Modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. Dose-response relationship of chlorogenic acids in humans Based on this fact, we were able to distinguish binding from gating, conductance, and 1 2 2 2 2 2 Richling E. , Williamson G. , Renouf M. , Steiling H. , Dionisi F. , Barron D. , desensitisation, using a Markov model that describes the complete channel behaviour 3 4 1 Scheppach W. , Melcher R. , Erk T. by a matrix of rate constants. The results were also checked for consistency with a 1University of Kaiserslautern Food Chemistry & Toxicology, Erwin-Schroedinger-Str. 52, structural hP2X3 model that we developed from the known zebra fish P2X4 crystal 67663 Kaiserslautern, Germany structure in the closed state. 2Nestle Research Center Nutrient Bioavailability, Vers-chez-les-Blanc PO Box 44, 1000 Lausanne 26, Switzerland 3Juliusspital Innere Medizin, Juliuspromenade 19, 97070 Würzburg, Germany 4Universitätsklinikum Würzburg Med. Klinik und Poliklinik II, Oberdürrbacher Str. 6, 330 97080 Würzburg, Germany
Up to now the question is unresolved how the ingested dose influences the absorption Voltage-dependent modulation of alpha adrenergic receptor signaling and metabolism of chlorogenic acids (CGA) from food. So far no studies have been 2A performed on the impact of the dose on CGA absorption, circulation and excretion. Rinne A., Birk A., Bünemann M. Recently we performed a dose-response study in a randomized, double-blinded, Philipps-Universität Marburg Institut für Pharmakologie und Klinische Pharmazie, Karl- crossover design with five ileostomy subjects. In three trials the volunteers consumed von-Frisch Str. 1, 35034 Marburg, Germany after a two day polyphenol free diet coffee with varying CGA content (HIGH 4525 µmol; MEDIUM 2219 µmol; LOW 1053 µmol). The CGA concentrations in plasma, ileal effluent G protein-coupled receptors (GPCRs) are proteins that regulate numerous signaling and urine were subsequently identified and quantified by HPLC-ESI-MS, HPLC-ESI- pathways by activation of intracellular G proteins. GPCRs are activated by extracellular MS/MS and HPLC-DAD. stimuli, such as light, hormones and neurotransmitters. Recent evidence suggests that The results showed that the consumption of higher CGA concentrations lead to a faster some GPCRs exhibit voltage-sensitivity leading to a modulation of their activity by the ileal excretion measured in the ileal effluents. This corresponded to the renal excretion membrane potential (VM). We used a FRET-based biosensor of the α2A adrenergic of 8.0 ± 4.9% (HIGH), 12.1 ± 6.7% (MEDIUM) and 14.6 ± 6.8% (LOW) of total CGA and receptor to analyze receptor activation at defined membrane potentials in HEK 293 cells metabolites. We found that CGAs with a caffeic acid moiety are predominantly sulphated by means of voltage-clamp recording. The biosensor was stimulated either with the and those with a ferulic acid moiety are predominantly conjugated via glucuronidation partial agonist clonidine or with the full agonist norepinephrine (NE) and receptor prior renal excretion. Furthermore, in the ileal effluents, sulphation of both structural activation was measured as decrease in the ratio of acceptor- /donor-fluorescence. units dominated. In plasma samples (after enzymatic deconjugation) the AUC values Receptor stimulation by NE was inhibited at depolarizing membrane potentials but were determined by the major CGA classes in coffee, the caffeoylquinic acids: 551.5 ± enhanced by hyperpolarization. Inhibition of NE activated receptors was strong at low 93.8 nM*h-1 (HIGH); 299.3 ± 79.6 nM*h-1 (MEDIUM) and 222.8 ± 91.3 nM*h-1 (LOW). No concentrations (500 nM: 60 % inhibition) but almost absent at saturating agonist major differences in the metabolic pattern were observed. Additionally, we were able to concentrations (100 µM: 9 % inhibition). Both agonist-induced and hyperpolarization- identify new metabolites of CGA in urine and ileal fluids. induced receptor activation exhibited a similar monoexponential time course and speed S76
of activation was primarily dependent on agonist concentration for both activation 333 modes. The latter indicates that depolarization lowers the apparent affinity of the NE receptor interaction and thus causes receptor deactivation by means of NE release. Application of clonidine (1 µM, VM=-90 mV) resulted in a FRET response that was Differential pro- and eukaryotic toxicity of silver released from nanocomposite inhibited by 40 % at +60 mV. In contrast to NE, strong receptor inhibition at +60 mV was surfaces increases the therapeutic window of silver in antibacterial treatments present even at super-saturating concentrations of clonidine (100 µM), suggesting that 1 2 3 2 2 voltage alters the equilibrium between active and inactive conformations of the receptor. Röhl C. , Hrkac T. , Podschun R. , Zaporojtchenko V. , Strunskus T. , Papavlassopoulos H.4, Garbe-Schönberg D.5, Faupel F.2 Voltage-dependence of the a2A adrenergic receptor also modulated downstream 1 receptor signaling: G protein activation or the recruitment of arrestins, which we Christina Albertina University Kiel Institute of Toxicology and Pharmacology for Natural Scientists, Brunswiker Straße 10, 24105 Kiel, Germany determined in FRET assays that directly detect Gαi protein activation or receptor-arrestin 2Christina Albertina University Kiel Institute for Materials Science - Multicomponent interactions, were both substantially inhibited at VM = +60 mV. Therefore we conclude Materials, Kaiserstraße 2, 24105 Kiel, Germany that negative membrane potentials promote active conformations of the a2A adrenergic 3 receptor, increase affinity of full agonists and enhance receptor signaling. Christina Albertina University Kiel Institute for Infection Medicine, Brunswiker Straße 4, 24105 Kiel, Germany 4Christian-Albrechts-Universität zu Kiel Institut für Toxikologie und Pharmakologie für Naturwissenschaftler, Brunswiker Straße 10, 24105 Kiel, Germany 5Christina Albertina University Kiel Department of Geology / ICP-MS Lab, Ludewig- 331 Meyn-Straße 10, 24118 Kiel, Germany
Silver has been used since ancient times as antimicrobial agent. Recently, silver gained Development of squamous skin carcinomas is supported by hyaluronan new attention due to its higher effectiveness in its nanoform. This led to new 1 1 2 2 1 Röck K. , Kellner M. B. , Hippe A. , Homey B. , Fischer J. W. developments of silver nanomaterials, e.g., for medical devices and consumer products. 1Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf Institut für Though, it is generally assumed that silver is less toxic for eukaryotes than for Pharmakologie und Klinische Pharmakologie, Moorenstraße 5, 40225 Düsseldorf, prokaryotes, concern is raised, if nanosilver at the same time might also increase Germany mammalian cytotoxicity. 2Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf Hautklinik, In our study we examined the toxicity of silver released from nanocomposite surfaces Moorenstraße 5, 40225 Düsseldorf, Germany with that of silver from AgNO3 solutions for adherent bacteria and mammalian cells. Therefore, we established an in vitro reference system which enabled us to compare the Squamous cell carcinoma (SCC) is the second most common skin cancer and strongly therapeutic window between prokaryotic toxicity and eukaryotic integrity in both associated with chronic UVB exposure of the skin. UVB induced changes of the exposure settings. We focussed especially on the comparability of the bacterial and extracellular matrix contribute to phenotypic responses of tumour and stroma cells. mammalian cell systems and the development of characterized Ag/TiO2 nanocomposite Hyaluronan (HA), as a main component of the dermal extracellular matrix, is synthesized coatings with well-defined silver filling factors and silver surface release, which could be by HA-synthase isozymes (HAS1-3) and is known to be involved in the regulation of varied over a wide concentration range. As reference cells the E. coli SAR 18 strain and several cellular mechanisms like proliferation, migration and survival. Aim of the present human dermal fibroblasts, which are of special relevance in the context of medical study was to evaluate the contribution of HA on tumourgenesis of UVB-induced SCCs. devices like implants or wound dressings, were chosen. Bactericidal effects were Human skin samples from healthy skin and SCCs were analysed via qRT-PCR. mRNA determined by direct growth visualization of the GFP-producing E. coli strain by levels of HAS1 and 2 showed no regulation, whereas HAS3, Hyaluronidases (HYAL) 1 epifluorescence microscopy. Mammalian cell growth and toxicity was determined by the and 2 were clearly upregulated. In vitro comparison of normal human keratinocytes and MTT assay, protein measurements and phase contrast microscopy. The Ag/TiO2 SCC cells (A431) showed the same expression patterns (HAS3 1.951 ± 0.386; HYAL1 samples were prepared by sputter co-deposition from two separate magnetron sources. 74.28 ± 22.1; HYAL2 4.229 ± 1.680 fold of keratinocytes). The amount of HA in the The silver surface concentration release was determined by XPS and the silver release supernatant of SCC cells was significantly increased in comparison to keratinocytes as by ICP-MS. In solution a concentration-dependent constant silver concentration could be measured by an HA-binding protein based assay. To evaluate the influence of HA on determined between 2 and at least 72 hours at the surface. While lowest bactericidal + 2 tumourgenesis in vivo, mice were UVB irradiated (80mJ/cm²) three times a week for 20 and cytotoxic concentrations of Ag from AgNO3 solutions with 0.64 and 0.95 mg/cm , weeks. During the experiment the mice received either chow with an inhibitor of HA respectively, differed only slightly, the therapeutic window increased significantly if Ag+ synthesis, 4-Methylumbelliferon (4-MU), or control chow. Mice treated with 4-MU was released from the nanocomposite surface. While the toxicity on the fibroblasts was developed significantly less tumours compared to non treated animals (18.25 ± 2.22 vs. unchanged the bactericidal potency increased at least one order of magnitude. 33.3 ± 4.1 tumours). In vitro 4-MU reduced the amount of HA in the supernatant of SCC Taken together, it can be concluded that local exposure factors i) can be modulated by cells (0.567 ± 0.1083 fold of control). Furthermore [H³-thymidine] incorporation revealed silver nanocomposites and ii) play an important role for the differential toxicity of surface reduced proliferation of SCC cells in response to 4-MU (0.421 ± 0.216 fold of control). silver on bacteria and mammalian cells. In conclusion the present data show that SCC cells extrude more HA, possibly related to increased levels of HAS3, in comparison to keratinocytes. Increased amounts of HA appear to be essential for the UVB induced tumourgenesis of SCCs in mice. This effect might be related to the pro-proliferative property of high molecular weight HA. 334 Furthermore biological active HA fragments derived from HA degradation by hyaluronidases (Hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and pro- inflammatory, thus possibly also promoting tumour growth and malignancy. C3bot binding to HT22 cells is dependent on posttranslational modification of the putative binding partner Rohrbeck A.1, Kolbe T.1, Schröder A.1, Hagemann S.1, Pich A.1, Ahnert-Hilger G.2, Just I.1 332 1Medizinische Hochschule Hannover Institut für Toxikologie, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 2Charite - Universitätsmedizin Institut für Integrative Neuroanatomie, Funktionelle Estradiol induced paracrine release of EGF from keratinocytes protects the Zellbiologie, Philippstr. 2, 10115 Berlin, Germany dermal hyaluronan/versican matrix during photoaging 1 1 1 2 3 2 2 Röck K. , Meusch M. , Fuchs N. , Tigges J. , Zipper P. , Fritsche E. , Krutmann J. , C3 exoenzyme (C3bot) a clostridial ADP-ribosyltransferase does not possess a cell- 3 3 1 Homey B. , Reifenberger J. , Fischer J. W. binding/-translocation domain. Nevertheless, C3 is able to efficiently enter intact cells, 1Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf Institut für including neuronal cells but the mechanism of uptake is not yet understood. Pharmakologie und Klinische Pharmakologie, Moorenstraße 5, 40225 Düsseldorf, In the present work, binding of C3bot to the hippocampus-derived HT22 cell line was Germany characterized by means of binding and blot overlay assays as well as mass 2IUF Leibniz-Institut für umwelt- medizinische Forschung GmbH, Auf'm Hennekamp 50, spectrometry analysis to identify binding partners of C3bot. The binding assays 40225 Düsseldorf, Germany established that C3bot bound in a concentration-dependent manner to HT22 cells. In the 3Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf Hautklinik, overlay assay we detected one clear band of 55 kDa. To elucidate whether glycosylation Moorenstraße 5, 40225 Düsseldorf, Germany is important for the C3bot-protein interaction, HT22 cells were incubated with glycosidase F resulting in a decreased binding of C3 to the 55 kDa band. To explore the Hyaluronan (HA) and versican are key components of the dermis and are responsive to involvement of phosphorylation in the binding of C3 to the putative binding protein, blot UVB induced remodeling. The aim of the present study was to investigate the molecular was pre-treated with CIP (Calf Intestinal Phosphatase) before overlay with C3bot. Pre- mechanisms of estrogen (E2) mediated effects on HA-rich ECM during actinic aging. 10 treatment greatly reduced the C3bot-protein interaction. Moreover, inhibition of de- weeks of UVB irradiation (3 x 1 MED (80 mJ/cm2), weekly) of hairless Skh-1 mice phosphorylation by vanadate before in intact cells showed an increased level of C3bot- caused a marked decline of dermal HA, which was aggravated by ovariectomy (OVX). protein interaction in the following overlay. Thus, interactions between C3bot and HT22 Subcutaneous substitution of estrogen (E2) by means of controlled release pellets cell proteins may require phosphorylation. abolished these effects confirming the stimulatory role of E2. The increase of dermal HA To further characterize the 55 kDa band as binding target of C3bot, the 55 kDa band correlated with induction of HA synthase HAS3 by E2. In addition the HA-binding was digested with trypsin and then subjected to LC-Orbitrap mass spectrometry proteoglycan versican was induced by UVB and further increased by E2. However in analysis. From this 55 kDa single gel band 141 proteins were identified. Further analysis cultured skin fibroblasts E2 reduced the expression of versican and had no effect on of the identified proteins will provide a possible interaction partner of C3bot. HAS3. Therefore, direct upregulation of HAS3 and versican in fiborblasts by E2 was In sum, protein overlay assays revealed that phosphorylation and glycosylation are excluded. However, E2 increased the expression of EGF in UVB irradiated skin in vivo critical for efficient C3bot-protein interaction. and in keratinocytes in vitro. EGF in turn upregulated the expression of HAS3 and versican in dermal fibroblasts. Furthermore the supernatants of Estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing EGF antibody or treatment of the fibroblasts with EGF receptor blocker erlotinib. Functionally, dermal HA and versican induction by E2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. Collectively these data suggest that E2 treatment increases the amount of dermal HA and versican via paracrine release of EGF which may be implicated in the pro-proliferative and anti-inflammatory effects of E2 during photoaging. S77
335 MAPK increased with ATP to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with UTP at 10 minutes of basal values, respectively. After 20 minutes, pre- drug values of MAPK phosphorylation were reached again. In summary, we noted an Solvent effects on enzyme kinetics in vitro ATP- and UTP-induced phosphorylation of ERK 1/2 and p38 MAPK in isolated neonatal rat cardiac myocytes. The involved receptor subtype(s) and the link between MAPK Rokitta D., Pfeiffer K., Gerwin H., Streich C., Fuhr U. phosphorylation and inotropic effect of ATP and UTP need to be elucidated. Uniklinik Köln Institut für Pharmakologie, Gleueler Str. 24, 50931 Köln, Germany
Kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. Such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are 338 regularly needed. To preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. Data on nature and extent of such solvent effects is sparse. HaMeeL: use of eLearning in teaching pharmacology and toxicology - the Halle In this study, we investigated the effects of methanol, ethanol, acetonitrile and experience 1 2 2 dimethylsulfoxide (1% to 4%) on the assessment of km, Vmax and Clint with regard to the Rulf K. , Gergs U. , Neumann J. 1-hydroxylation of midazolam via CYP3A4 and the CYP1A2 catalyzed metabolism of 1Martin-Luther-Universität Halle-Wittenberg, Medizinische Fakultät Institut für Klinische caffeine to paraxanthine in vitro. Epidemiologie, Magdeburger Straße 8, 06112 Halle (Saale), Germany 2 The presence of acetonitrile showed the highest Vmax value for paraxanthine formation Martin-Luther-Universität Halle-Wittenberg, Medizinische Fakultät Institut für but the lowest values for 1-hydroxymidazolam formation. The km value for midazolam Pharmakologie und Toxikologie, Magdeburger Straße 4, 06112 Halle (Saale), Germany showed no systematic effects of organic solvents, while for caffeine km was up to eight- fold lower for solvent free samples compared to solvent containing samples. Introduction: During the past three years, our faculty has started to integrate items of The present example suggests that the presence of organic solvents may considerably eLearning into the standard curriculum of a classical medical school: the “Hallesches influence enzyme kinetic parameters beyond a mere change in apparent activity. These Medizinisches eLearning - HaMeeL”. Our hypothesis was that these new eLearning effects are differing between enzyme-substrate systems and solvents. It remains to be tools would improve the willingness of students to spend more time into learning and this determined to which extent such effects compromise in vitro – in vivo extrapolations, and would lead to an improved outcome (in multiple choice tests). which solvents are most appropriate. Methods: Hence, we offered medical students (5th or 10th semester) additional learning environments. The courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. 336 Furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 - 12 previously used tests) and/or an online module on heart failure each week. We used the learning management system ILIAS software in combination with the content Atrial remodeling and arrhythmia induced by the transcription factor ER81 management system Stud.IP. All students were subjected to an introductory test (to assess knowledge prior to our Rommel C.1, Rösner S.1, Weiss S.1, Gilsbach R.1, Lother A.1, Kretz O.2, Hein L.1 1 teaching section and allowing us to exclude a conceivable bias due to previous Universität Freiburg Institut für Experimentelle und Klinische Pharmakologie und knowledge, involving basic items from prior teaching opportunities), a mid-term test and Toxikologie, Albertstraße 25, 79104 Freiburg, Germany 2 a final test to assess gain of knowledge. A maximum of 60 points could be obtained as a Universität Freiburg Anatomie und Zellbiologie, Albertstraße 17, 79104 Freiburg, sum of both tests. Germany Results: In the means 40% of students used the new eLearning tools (quizzes, heart failure module). However, there was no association between the use of self-assessment Introduction: The transcription factor ER81 (ETS related 81) belongs to the large family quizzes and examination results. The usage of the online quizzes increased in the of ETS-transcription factors that are involved in developmental processes and in the periods before the exams. However, usage of the heart failure module was pathogenesis of cancer. ER81 is activated by Gq- and Gs-coupled receptors leading to a accompanied by significantly increased scores in exams. Moreover, in a formalized phosphorylation of the transcription factor by MAP-kinases and protein kinase A, evaluation system, students positively commented on our eLearning efforts. respectively. Cardiac ER81 mRNA expression is increased in failing human hearts. Conclusions: While usage of our quizzes did not improve test marks, another more However mechanical unloading by a left ventricular assist device leads to normalization sophisticated clinically oriented eLearning module seemed to be improving test of ER81 expression. Thus, the aim of the present study was to investigate the cardiac outcomes marginally. function of ER81 in genetically modified mouse models. Methods and Results: We previously generated transgenic mice overexpressing ER81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αMHC) promoter by pronuclear injection and established independent transgenic lines. Electrocardiography (ECG) was assessed in mice at day 5 after birth (P5) and in adult 339 mice (3 months) during isoflurane anesthesia and by ECG telemetry in awake mice, respectively. ECG analysis revealed no differences between the genotypes at day 5 Thr188 after birth. However, we found a decreased heart rate, a replacement of regular P-waves Targeting of ERK phosphorylation attenuates cardiac hypertrophy but by an undulating baseline and frequent supraventricular extrasystoles in adult ER81αMHC preserves the anti-apoptotic effects of ERK1/2 transgenic mice. Next, isometric contractile force measurements on isolated left atria Ruppert C., Vidal M., Lohse M. J., Lorenz K. were carried out in organ baths. While WT left atria responded to increasing Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 concentrations of isoprenaline, NKH477 and calcium with an increase in contractility, the Würzburg, Germany maximal positive inotropic responses to these substances were severely blunted in ER81αMHC atria. We performed Western blots to identify potential aberrations of calcium Background and aims: The extracellular regulated kinases 1 and 2 (ERK1/2) play an handling and regulatory proteins. Phosphorylation of serine 16 of phospholamban (PLN) important role in cardiac hypertrophy and cell survival. ERK1/2 are phosphorylated at was reduced in ER81αMHC mice. In addition, protein phosphatase 1 (PP1) expression the so-called TEY motif, which in turn activates ERK1/2. Hypertrophic stimuli lead to an was significantly increased in ER81αMHC mice, which is consistent with the increased additional autophosphorylation threonine188 (Thr188). This autophosphorylation of dephosphorylation of phospholamban. Furthermore, we found a decreased expression ERK1/2 stimulates activation of nuclear ERK targets, which are known to induce of calsequestrin and Serca2a protein in ER81αMHC atria. Electron microscopy revealed hypertrophy. The aim of this study is to investigate whether specific targeting of the significant structural remodeling of ER81αMHC atria at 3 months of age. ERKThr188 phosphorylation affects both ERK functions – ERK mediated hypertrophy and Conclusion: Increased cardiac expression of the ETS-transcription factor ER81 leads to cardioprotective cell survival. structural and electrical remodeling of the atria. Thus, ER81 may play an important role Methods and results: For the analysis of cardiomyocyte hypertrophy in vitro, we in the pathogenesis of cardiac arrhythmias in chronic heart failure. stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. Cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (TAC). For analysis of cell survival, caspase activity and DNA fragmentation was determined upon hydrogen peroxide stimulation in vitro and 337 in response to TAC in vivo. To differentiate between inhibition of ERK1/2 activity and prevention of ERKThr188 phosphorylation, we either inhibited ERK activity with PD98059 or overexpressed a mutant of ERK2, which cannot be phosphorylated at Thr188 Signal transduction pathway of ATP and UTP in neonatal rat cardiac myocytes phosphorylation. While inhibition of overall ERK activity with PD98059 attenuated cell survival and Rothkirch D., Gergs U., Neumann J. hypertrophy in vitro, specific targeting of ERKThr188 phosphorylation by overexpression of Institute for Pharmacology and Toxicology Medical Faculty, Magdeburger Str. 4, 06097 the phosphorylation deficient mutant (ERK2T188A) attentuated phenylephrine induced Halle (Saale), Germany hypertrophy, but preserved the anti-apoptotic effects of ERK. Cardiac overexpression of ERK2T188A significantly reduced TAC-induced hypertrophy compared to wild-type ERK2 Extracellular ATP and UTP can be released from the heart during pathological overexpressing mice. In line with the in vitro experiments, ERKThr188 inhibition only conditions such as ischemia or hypoxia. In humans, ATP and UTP levels are increased prevented hypertrophy in the TAC model without promoting apoptosis. during myocardial infarction. ATP and UTP can act via P2-purinoceptors which are Conclusions: These results show that blockade of ERKThr188 phosphorylation further divided in P2X1-7 and P2Y1-14-receptors. As previously shown ATP and UTP can attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of ERK1/2. induce inotropic effects in cardiac preparations of mice and man. For rat articular Therefore, specific targeting of ERKThr188 phosphorylation might be a promising strategy chondrocytes and human intestinal cells it has been demonstrated that the MAPK for the treatment of pathological hypertrophy. cascade can be activated by ATP and UTP. Therefore, the cardiac effects of ATP and UTP on force of contraction probably occur via the MAPK pathway. To investigate the signal transduction pathway involved, we studied the effects of ATP and UTP on MAPK phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µM ATP as well as UTP transiently increased phosphorylation of ERK 1/2 and p38 MAPK with a maximum effect at 5 to 10 minutes after application of ATP and UTP in neonatal cardiac myocytes (n=3 preparations each). The maximum phosphorylation of p38 increased with ATP to 284% ± 68% (p<0.05) at 10 minutes and with UTP up to 204% ± 21% (p<0.05) at 5 minutes. The phosphorylation with ERK 1/2 S78
340 EA.hy926 cells and THP1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. ChIP and bandshift assays were performed to identify candidate transcription factors. Tissue distribution and function of PDE10 Results: In EA.hy926 cells, endogenous HIVEP1 expression was increased by proinflammatory cytokines TNFα and IL-1β. Simvastatin (1.2 and 2.4 µM) and Russwurm C., Jäger R., Koesling D., Russwurm M. atorvastatin (9 µM) - but not pravastatin or aspirin - both dose-dependently decreased Ruhr-Universität Bochum Pharmakologie und Toxikologie, Universitätsstrasse 150, basal and TNFα-stimulated HIVEP1 expression. The construct harbouring rs169713T 44780 Bochum, Germany exerted significantly higher transcriptional activity (TA) compared to rs169713C (P<0.001). For an intronic modulator, reporter gene assays demonstrated a regulatory Intracellular cAMP levels are determined by interplay of cAMP formation by adenylyl effect on HIVEP1 expression in EA.hy926 and THP1 cells. Cotransfection of SP1 and cyclases and cAMP degradation by phosphodiesterases (PDE). Eleven families of PDEs EGR1 led to an increase in TA, while WT1 exclusively upregulated TA of constructs are known. One of the most recently identified PDEs is PDE10, a PDE in principle comprising the intronic modulator. ChIP and bandshift assays combined with specific capable of hydrolysing cAMP as well as cGMP. PDE10 contains a tandem of so called antibody detection revealed binding of SP1 to the 5'-flanking region and the intronic GAF domains in its N-terminal regulatory domain that mediate activation by cAMP. modulator of HIVEP1. Because current knowledge about the tissue distribution of PDE10 was mostly based on Conclusion: Increased HIVEP1 expression during inflammatory conditions can be the analysis of mRNA distribution, we generated antisera against PDE10 to analyze repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. Basal tissue distribution of the protein level. Using these antibodies, we found a prominent HIVEP1 expression is regulated by SP1 combined in a transcription factor module with occurrence of the enzyme in testis and in brain, where it was confined to the striatum. EGR1 and WT1 under basal and/or inflammatory conditions. The rs169713 site Thus, PDE10 displays a comparably restricted tissue distribution which is in contrast to harbours potential activational capacity for HIVEP1 gene transcription and may that of many other PDEs. communicate with the SP1/EGR1/WT1 module. Low cAMP levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. Furthermore, studies using the nonspecific PDE10 inhibitor papaverine as well as specific PDE10 inhibitors suggest PDE10 as a target for the treatment of schizophrenia. Here we set out to analyze the contribution of PDE10 to cAMP degradation in striatum, to identify the physiological pathways PDE10 is involved 343 in and to clarify the functional impact of the proposed phosphorylation of the enzyme.
The specific KV7.2/3 channel opener ICA 27243 attenuates L-DOPA-induced dyskinesia in hemiparkinsonian rats 341 Sander S. E., Lambrecht C., Richter A. Freie Universität Berlin, Fachbereich Veterinärmedizin Institut für Pharmakologie und Toxikologie, Koserstr. 20, 14195 Berlin, Germany Identification of cGMP-dependent kinase I substrate complexes To date, the treatment of various movement disorders of the central nervous system is Salb K., Schlossmann J. still insufficient. In most cases this is due to the sparse knowledge of the Universität Regensburg Lehrstuhl Pharmakologie, Universitätsstrasse 31, 93053 pathophysiology. L-DOPA-induced dyskinesias (LID) represent a severe complication of Regensburg, Germany long-time pharmacotherapy in Parkinson’s disease that deserves novel therapeutics. An
increased activity of striatal projection neurons, which express KV7.2/3 channels, seems The cGMP-dependent kinases (cGKs) are components of the NO/cGMP/cGK-signalling to be involved in the pathophysiology of these spontaneous involuntary dystonic and pathway and have a great physiological importance in a multitude of tissues and organs choreatic movements. Previous studies demonstrated an antidyskinetic effect of the such as smooth muscles and platelets. Two isoforms of the cGKI and the cGKII are KV7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of known. cGKIα and cGKIβ differ only in their first ~ 100 amino acids which constitute the LID. In order to clarify if this effect was based on the modulation of KV7.2/3 channels, we leucine zipper and the autoinhibitory domains. The N-terminal leucine zipper domains examined the acute effects of the preferred KV7.2/3 channel opener ICA 27243 on LID in mediate homodimerization of the kinase and the interaction with diverse substrate this animal model. proteins. Since cGKIα and cGKIβ express different N-termini they interact with different Four weeks post 6-OHDA lesioning of the left forebrain bundle, dyskinesia was induced substrates. The cGKIβ isoform is assembled in a macrocomplex at the endoplasmic by chronic treatment with 10 mg/kg L-DOPA and 15 mg/kg benserazide for 20 days. reticulum (ER) with the intracellular calcium release channel Inositoltrisphosphate Three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score receptor I (InsP3R-I) and the Inositol-trisphosphate receptor associated cGMP kinase system from 0 to 4 over 180 min. For drug testing, ICA 27243 (5, 10 and 15 mg/kg) was substrate (IRAG). We investigated, whether IRAG also interacts with the InsP3R-II and administered intraperitoneal additionally to L-DOPA (or vehicle). Effects of drug action in the InsP3R-III in murine platelets and tissues. Additionally, we analyzed the interaction comparison to vehicle controls were detected by adding up the severity scores of each between the 52 amino acid peptide Phospholamban (PLB), which is also located at the 2+ observation time. Additionally, effects on parkinsonian symptoms were examined 20 min ER and regulates the ER calcium reuptake by the sarco/endoplasmic reticulum Ca - after drug administration using the block and the stepping test. ICA 24273 reduced the ATPase (SERCA), and the two cGKI isoforms. severity of dyskinesia significantly at all doses while no negative impact on the We performed cGMP-agarose experiments with murine WT and IRAG-KO platelets to antiparkinsonian effect of L-DOPA was observed. Whereas the antidyskinetic effect was examine the IRAG-InsP3R interactions. The InsP3R-II isoform was neither bound to restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats cGMP-agarose nor detected in the anti-IRAG immunoprecipitate. On the other hand, treated with 10 mg ICA 27243. A higher dose of 15 mg did not further enhance the InsP3R-III from WT but not from IRAG-KO platelets was bound to cGMP-Agarose. antidyskinetic effect. Hence, InsP R-III interacts directly with IRAG but not with cGKIβ in murine platelets. 3 The results of our study suggest that the antidyskinetic effect of the KV7 channel opener However, in colon smooth muscle lysate, InsP R-III not only interacted with the IRAG 3 retigabine was based on its action on striatal KV7.2/3 channels. In line with the results of protein but was also detected in the anti-cGKIα-immunoprecipitate. previous studies with retigabine, this action does not seem to interfere with the Phospholamban from WT and IRAG-KO platelets was also bound to cGMP-agarose. antiparkinsonian effect of L-DOPA. Subsequent immunoprecipitation experiments with the respective antibodies against the two cGKI isoforms revealed that PLB interacted both with cGKIβ and cGKIα. These This study was supported by the Micheal J. Fox Foundation. results were supported by analysis of colon smooth muscle tissue from WT and IRAG- KO mice. In conclusion, IRAG interacts with InsP3R-I and InsP3R-III but not with InsP3R-II in murine platelets and colon smooth muscle tissue. Moreover, Phospholamban is an interacting partner of both the cGKIα and the cGKIβ isoform. 344
Statin-induced myopathy in an engineered skeletal muscle model is not due to increased iNOS activity 342 Sanders S. - J., Tiburcy M., Zimmermann W. Universitätsmedizin Göttingen Pharmakologie, Robert-Koch-Str. 40, 37075 Göttingen, The human immunodeficiency virus type 1 enhancer binding protein 1 (HIVEP1) is Germany regulated by proinflammatory stimuli and statins Background: Skeletal muscle toxicity is the major side effect of HMG-CoA-reductase Salomon A.1,2, Schmitz B.2, Herrmann M.3, Rötrige A.1, Brand E.3, Morange P. E.4, 5 5 5 1,2 inhibitors (statins) and can be simulated in engineered skeletal muscle. Statins are Cambien F. , Tiret L. , Trègouët D. - A. , Brand S. - M. known to exert “pleiotropic” effects, e.g. reducing endothelial dysfunction by inducing NO 1Leibniz-Institute for Arteriosclerosis Research, Domagkstr. 3, 48149 Münster, Germany 2 synthases and NO production. The role of NO synthases in skeletal muscle under statin Medical Faculty of the Westfalian Wilhelms-University of Münster Department of treatment is largely unknown. Interestingly, some skeletal muscle pathologies (e.g. Molecular Genetics of Cardiovascular Disease, Domagkstraße 3, 48149 Münster, Duchenne muscular dystrophy) may be exacerbated by increased iNOS activity. Here Germany 3 we tested whether or not statin- induced skeletal muscle toxicity would be associated University Hospital Münster Internal Medicine D, Domagkstraße 5, 48149 Münster, with enhanced NO synthesis. Germany Methods and Results: We generated engineered skeletal muscle (ESM) from rat 4Université de la Méditerranée INSERM UMR S 626, 13385 Marseille Cedex 5, France 5 skeletal muscle cells, matrigel and collagen. ESMs displayed typical skeletal muscle Faculté de Médecine Pitié-Salpêtrière INSERM U525, 91 blvd de l’Hôpital, 75634 Paris properties (differentiated muscle fibres, tetanic contractions). Under baseline conditions cedex 13, France ESM expressed eNOS most abundantly, followed by iNOS and nNOS (n=4-5). Myotoxic Cerivastatin (0.01, 0.1, 1 µM for 5 days) caused a concentration-dependent decrease of Objective: HIVEP1 binds NF-ĸB and other proinflammatory consensus sequences, and contractile force (p<0,05, n=17-20) paralleled by an increase in iNOS transcript is suggested to be involved in inflammatory processes. We recently identified two (mean±SEM: 0.01 µM 4±0.9-fold, n=3 p<0.05; 0.1 µM 9.3±2.8-fold, n=3 p<0.05) and tagging SNPs, one positioned 90 kb upstream (rs169713) and another in exon 4 protein (0.01 µM 5.1±2-fold, n=4 p<0.05; 0.1 µM 7.8±0.8-fold, n=3 p<0.05). Mevalonic (rs2228220) of the HIVEP1 gene, to be replicatively associated with venous thrombosis acid fully prevented the iNOS increase suggesting that the induction is HMG-CoA in GWAs and follow-up studies (AJHG, 2010; PLoS ONE, 2011). reductase-dependent. To test whether iNOS may contribute to the decrease in Methods: Total RNA isolation was performed after treatment of vascular endothelial contractile force we co-treated ESM with 1400W, a specific iNOS inhibitor. We applied 5 cells (EA.hy926) with proinflammatory cytokines or statins (24h). Serial HIVEP1 µM of 1400W, a concentration found to potently reduce lipopolysaccharide (LPS)- promoter deletion constructs were cloned into the pGL3-Basic vector, a potential induced NO-production in cultured myotubes. However, we did not observe a rescue enhancer fragment, harbouring rs169713C/T, into the pGL3-Promoter vector. In S79
effect (n=9-15). Also, L-NAME (10 mM), an unspecific NOS inhibitor, did not improve 347 contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). To further investigate the role of NO for muscle function we treated the ESMs with increasing concentrations of the NO-donor SNP. Only high concentrations of SNP (10 Improved glucose tolerance, less chronic adipose tissue inflammation and µM) caused a reduction of contractile force. Combined treatment with cerivastatin and reduced adipose tissue mass in mice with adipocyte-specific loss of TAK1 0.1 µM SNP showed a tendency towards improved force development in ESM. Conclusions: Statins increase iNOS activity in our skeletal muscle model (ESM). Sassmann A., Offermanns S., Wettschureck N. However, this does not seem to functionally contribute to myopathy in ESM. Increased Max-Planck-Institut für Herz- und Lungenforschung Pharmakologie, Ludwigstr. 43, production of NO may in fact be a protective measure. ESM may help to dissect 61231 Bad Nauheim, Germany clinically relevant functional changes in statin myotoxicity. TGF-β activated kinase 1 (TAK1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like LPS, interleukin-1 or TNFa to IKK, p38 and JNK activation. Chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance 345 in states of obesity. To investigate the role of TAK1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific Cre mouse line AdipoqCreERT2 with animals carrying floxed alleles of the TAK1 gene. AdipoqCreERT2; TAK1fl/fl animals and Characterization of primary skin fibroblasts of patients with 3M syndrome and Cre negative control littermates are viable and fertile and do not show any mutations in the CUL7 gene developmental defects. After tamoxifen induction and high fat diet feeding adipocyte- Meyer K., Hieber M., Engelhardt S., Sarikas A. specific TAK1 knockout mice show improved glucose tolerance and lower fasting insulin Technische Universität München Institut für Pharmakologie und Toxikologie, levels compared to control animals. In line with this, serum levels of the adipose tissue- Biedersteiner Str. 29, 80802 München, Germany specific hormone resistin are reduced in adipocyte-specific TAK1 knockout mice. These findings are accompanied by a lower state of chronic inflammation of adipose tissue as Introduction: indicated by a dramatic reduction of adipose tissue macrophage number and lower 3M syndrome is an autosomal-recessive disorder characterized by pre- and postnatal serum levels of TNFα and interleukin-6. Stimuli like TNFα, interleukins and TGF-β growth retardation (< - 4 SD), facial dysmorphism and skeletal anomalies. The majority released from macrophages and adipocytes are known to promote obesity-related of patients harbor missense mutations of the CUL7 (76%) or OBSL1 (16%) gene, adipose tissue inflammation. When stimulated with these substances TAK1 deficient respectively. CUL7 constitutes an E3 ubiquitin ligase that is involved in the regulation of adipocytes show reduced activation of JNK and p38 which both play an important role in the insulin-like growth factor 1 (IGF-1) signaling pathway via ubiquitin mediated the development of insulin resistance. Interestingly, we observe a lean phenotype in degradation of insulin receptor substrate 1 (IRS-1). adipocyte-specific TAK1 knockout mice when fed a high fat diet which reflects a Objective: reduction of white adipose tissue mass. Currently we are investigating the molecular To investigate the role of CUL7 mediated IRS-1 degradation in the pathogenesis of 3M mechanisms underlying the reduced adiposity and lower state of chronic inflammation in syndrome. adipose tissue. Methods and Results: Primary skin fibroblasts of seven 3M syndrome patients (six with CUL7 mutations, one with a OBSL1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (FACS), cell morphology and cellular senescence 348 (histochemistry), IRS-1 protein concentrations and activation of the IGF-1 signaling pathway (Western Blot). The proliferation rate of 3M patient fibroblasts was significantly increased when compared to control cells. In contrast, IRS-1 protein levels and Growth regulation in small cell lung cancer via Gq/11- and G12/13-dependent activation of the PI3K/Akt and ERK MAPK pathway were only increased in a subset of signaling 3M cells that carried CUL7 mutations, but not in cells from a patient with the OBSL1 Schäfer E. A. M.1, Büch T. R. H.1, Hauswald M.2, Stohr S.1, Gudermann T.1, Aigner A.2 mutation. No significant differences in cell cycle profile, cell morphology or cellular 1 senescence were observed in 3M patient fibroblasts when compared to control cells. To Ludwig-Maximilians Universität Walther-Straub-Institut für Pharmakologie und Toxikologie, Goethestr. 33, 80336 München, Germany determine the pathogenetic contribution of increased IRS-1 levels to the observed 2 phenotype, human IMR90 fibroblasts were stably transfected with retroviral vectors Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16-18, 04107 encoding IRS-1. Despite 20-fold overexpression of IRS-1 compared to empty vector Leipzig, Germany controls, no significant effect of IGF-1 stimulation on proliferation rate or PI3K/Akt and Erk MAPK signaling was observed. Growth of small cell lung cancer (SCLC) cells is regulated via the autocrine stimulation Summary and Conclusion: of G protein coupled receptors (GPCRs), i. e., neuropeptide and muscarinic acetyl Skin fibroblasts of 3M patients with CUL7 mutations displayed an increased proliferation choline (ACh) receptors. The activation of Gq/11 and calcium-dependent GPCR pathways rate and enhanced activation of the IGF-1 signaling pathways. Despite accumulation of results in the stimulation of ERK signaling which is necessary for the mitogenic effects of IRS-1 in fibroblasts from a subset of 3M patients with CUL7 mutations, no neuropeptides or ACh on SCLC cells. In contrast, the role of calcium-independent pathomechanistic role for IRS-1 could be demonstrated. Collectively, our data indicate GPCR signaling and its interplay with Gq/11-regulated pathways in SCLC cells are less that a dysregulated IGF-1 signaling may contribute to the pathogenesis of 3M syndrome, well defined. The aim of our studies was to characterize the molecular make-up and the yet in an IRS-1 independent manner. interaction of these pathways, and to delineate the phenotypic effects of calcium- dependent and -independent signaling cascades in SCLC cells. Using a panel of SCLC cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. This calcium increase was sufficient to activate the 346 tyrosine kinase Pyk2 and subsequently the ERK1/2 cascade. The role of Pyk2 for the growth of SCLC cells was further supported by the fact that inhibition of Pyk2 using a siRNA approach or a novel specific inhibitor, PF431396, exerted pronounced cytotoxic Pharmacases.de - a student-centered eLearning project of clinical pharmacology effects on SCLC cells, whereas non-SCLC cells were less sensitive. Interestingly, the Zollner B., Berg C., Gros N., Muß N., Oestreicher D., Engelhardt S., Sarikas A. inhibition of G12/13 signaling by siRNA-mediated G(alpha)12 or G(alpha)13 knockdown Technische Universität München Institut für Pharmakologie und Toxikologie, also markedly reduced the growth of SCLC in vitro or in subcutaneous tumor xenografts, Biedersteiner Str. 29, 80802 München, Germany and increased the sensitivity of SCLC cells towards certain cytostatics. To further define the role of calcium-dependent signaling via Pyk2 versus the role of calcium-independent Introduction: signaling via G12/13, we tested the effect of Pyk2 inhibition in cells with impaired G12/13 Pharmacases.de is a novel e-learning website of clinical pharmacology that presents signaling. Notably, Pyk2 and G12/13 double inhibition led to an even increased clinically relevant aspects of pharmacology and toxicology in an interactive and multi- proliferation. Thus, we propose that dysbalanced G protein signaling favoring either medial manner. Pyk2 activation or G12/13-dependent cascades inhibits the growth of SCLC cells, whereas Aims and Objective: the parallel inhibition of both pathways restores again the balance and the growth The aim of the project Pharmacases.de was to develop an innovative concept for capacity in this tumor entity. creating high quality eLearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. Methods and Results: 349 A peer-teaching concept was developed in which final year medical students with the elective pharmacology (PJ Wahlfach Pharmakologie) independently researched and wrote eLearning lessions ("pharmacases"). Subject-specific expertise was acquired by Dendritic cells play an important role in the initial immune response against consulting elective students of other disciplines. At present (11/2011), this "peer pulmonary toxins in lung network" consists of elective students of nine cooperating institutions (pathology, Schäfer M.1, Pohl C.1, Moisch M.1, Steinritz D.2, Kehe K.2, Kirkpatrick C. J.1 microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, 1 pediatrics) at the Technische Universität München. The average time for the generation Johannes Gutenberg University Mainz Institute of Pathology, Langenbeckstrasse 1, 55101 Mainz, Germany of one eLearning lession by the peer network was 10 days. To date, the website 2 consists of 49 pharmacases that are available to all students online Bundeswehr Institute of Pharmacology and Toxicology, München, Germany (http://www.pharmacases.de). The website also contains a discussion forum and evaluation form for direct feedback. On average, Pharmacases.de has 1000 visitors per Dendritic cells (DCs) are essential for the initial immune response and for the defence month with the following evaluation results: "excellent": 76%, "good": 15% and against inhalated pulmonary toxins and carcinogens in lung. To differentiate DCs, the "satisfactory": 9% (n=33). cell line THP-1 were used for 7 days and stimulated with various cytokines (IL-4, GM- Summary and Conclusion: CSF, TNF-a, Ionomycin). The DCs were characterized by flow cytometry with different The didactic concept of Pharmacases.de enabled the efficient generation of high quality typical dendritic cell markers (for example CD11c, CD209, CD83) and by eLearning content in a student-centered and interdisciplinary manner. The peer-teaching immunfluorescence compared to monocytes. approach supports the collaborative skills of final year medical students and facilitates The bronchial tract contains up to 800 DCs per mm² and therefore we established a the transfer of theoretical pharmacological knowledge into clinical practice. triple culture model to mimic the situation in vivo. The triple culture consists out of primary human epithelial cells from small bronchi (HBEC) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and DCs which were S80
added after the differentiation phase of the bronchial cells. During the cultivation time the 352 HBEC formed an epithelial layer expressing both tight and adherens junctions. They also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 Hz and the transepithelial resistance values were stable between 600 to 800 Ω·cm². A human in vitro alveolar-capillary triple-culture model with an inflammatory Pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the triple- component: Development of a pulmonary cell culture system culture model is the basis for investigations of the toxic effects at cellular level. Lung- Scheele N.1,2, Schmidt A.1, Pohl C.2, Kirkpatrick C. J.2, Thiermann H.1, Steinritz D.1 toxic substances such as organophosphates are usually absorbed through inhalation. 1 Organophosphates are dangerous nerve agents for the human organism. At high Institut für Pharmakologie und Toxikolgie der Bundeswehr, Neuherbergstrasse 11, 80937 München, Germany concentrations organophosphates damage in the coculture without DCs the cell-cell 2 contacts of the epithelial layer. In the triple culture DCs firstly respond to inhaled Johannes-Gutenberg-Universität Mainz Institut für Pathologie, Langenbeckstrasse 1, organophosphates and seem to compensate effects on the other cells. 55101 Mainz, Germany
In summary, it is very important to understand the pathogenic mechanisms of lung injury Inhalation of toxicants such as sulphur mustard (SM), an alkylating chemical warfare in relation to the role of dendritic cells in lung. They could play an essential role in agent, cause pulmonary complications like respiratory failure, pulmonary edema and therapy against damage of organophosphates in the lung. secondary pneumonia. In order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. In this model the human lung adenocarcinoma epithelial cell line (H441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (Iso-Has) represents the endothelial site. 350 Acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. LDH) and electrochemical indicators (e.g. transepithelial resistance). SM-mediated pulmonary injury is Co-purification of ARF GTPase-activating protein GIT1 and Cavb3 characterized by the increased secretion of proinflammatory mediators (e.g. IL-6). A Schalkowsky P., Wissenbach U., Fecher-Trost C., Flockerzi V. shortcoming of this model is the missing inflammatory component in the lung. Universität des Saarlandes Institut für Experimentelle und Klinische Pharmakologie und Aim of the present project is the addition of macrophages to the established co-culture Toxikologie, Kirrbergerstraße, 66421 Homburg, Germany model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. High-voltage activated Ca channels are assembled from pore-forming α1 subunits and The effect of SM on this triple-culture model is characterized with special regard to the two interaction of epithelial cells and macrophages. The human acute monocytic leukemia distinct types of auxiliary subunits, Cavβ1-β4 and, maybe, α2δ1-δ4. By a Cavβ3-specific cell line (THP-1) was stimulated to allow differentiation into macrophages. Validation of antibody based affinity chromatography the Cavβ3 protein was highly enriched from rat the cellular differentiation was checked by specific clusters of differentiation (e.g. brain microsomal membranes. Proteins associated with Cavβ3 were identified by mass CD206) using flow cytometric analysis. After successful differentiation into spectrometry (LC-ESI-MS/MS) and include α1-subunits, α2δ-subunits and β-subunits. In macrophages, these inflammatory cells were added to the co-culture model before and addition to these expected interacting proteins additional proteins were co-purified with after exposure with SM, respectively. the Cavβ3 protein, including the G protein-coupled receptor kinase-interactor 1 (GIT1). The cytotoxicity of SM on the triple-culture model was evaluated by XTT assays and The 770aa GIT1 is a ubiquitously expressed multidomain protein which may serve as a TER measurements. Furthermore, immunohistochemical staining of tight junction scaffold to bring together molecules to form signaling modules controlling, for example, proteins (e.g. ZO-1) and of adherens junction proteins (e.g. E-cadherin) was conducted vesicle trafficking, cytoskeletal organization and cell migration. In rat brain lysates the to enhance the knowledge of the function of the intercellular junction in injured and GIT1 and Cavβ3 proteins were co-immunoprecipitated by the antibodies for Cavβ3 and rejuvenated regions as well as the interaction of epithelial cells and macrophages. GIT, respectively. We cloned the GIT1 cDNA from mouse brain and co-expressed it with the Cavβ3 subunit in HEK cells. Like in brain lysates the GIT protein was retained by Cavβ3 precipitated by the antibody for Cavβ3 and Cavβ3 was retained by the GIT1- protein precipitated by the antibody for GIT1. Both proteins, Cavβ3 and GIT1 are 353 endogenously co-expressed in mouse embryonic fibroblasts (MEF). We could not observe potassium-induced voltage-activated Ca influx in these acutely prepared cells. Accordingly, MEFs can be used as a model system to study the impact of Cavβ3-GIT1 Para-phenylenediamine is a substrate and inhibitor of N-acetyltransferase 1 interaction in the absence of functional Cav channels. In addition, using MEFs from activity in THP-1 cells Cavβ3-deficient mice enables us to control the impact of Cavβ3 on GIT1 function. Vice versa down-regulation of GIT1 by specific siRNAs might allow to control the impact of Scheitza S., Dierolf D., Blömeke B. GIT1 on Cavβ3 function. As read-outs we use cell migration assays and monitor Universität Trier Ökotoxikologie, Universitätsring 15, 54286 Trier, Germany receptor-dependent and receptor-independent calcium signaling in these cells. N-Acetyltransferase 1 (NAT1) catalyzes the N/O acetylation of various aromatic amines. For the contact allergen para-phenylenediamine (PPD) we showed that concentrations above 50 µM are accompanied with inhibition of NAT1 activity in human keratinocytes [1]. In the following we investigated the impact of PPD on NAT1 activity in antigen- 351 presenting cells using dendritic cell-like cells, namely the monocytic THP-1 cells. Measured NAT1 activity of THP-1 was comparable to those found in primary keratinocytes. A 24h treatment of THP-1 cells with physiologically relevant Effects of sphingosine-1-phosphate and FTY720 on epidermal hyperproliferation concentrations of PPD (10-200 µM) led to a 47% reduction of NAT1 activity. Comparable and inflammation in an imiquimod induced mouse model of psoriasis results were found for mono-acetylated PPD (MAPPD) whereas di-acetylated PPD 1 1 2 1 Schaper K. , Kietzmann M. , Kleuser B. , Bäumer W. demonstrated no inhibition. Time-dependent studies found a significant decrease in 1Institut für Pharmakologie, Toxikologie und Pharmazie Stiftung Tierärztliche enzyme activity already 8h after application of PPD or MAPPD while NAT1 mRNA levels Hochschule Hannover, Bünteweg 17, 30559 Hannover, Germany were not modified. These results are indicative for a substrate-dependent inhibition. 2Institut für Ernährungstoxikologie Universität Potsdam, Arthur-Scheunert-Allee 114-116, Further investigations concentrated on the restoration of NAT1 activity after treatment 14458 Nuthetal, Germany with PPD or MAPPD. Here we found that N-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. Independent of the enzymatic The sphingolipid sphingosine 1-phosphate (S1P) is a mediator that modulates various activity, certain compounds are known to oxidise the catalytic cysteine or form adducts physiological functions of skin cells. S1P has distinct direct effects on keratinocytes as it with the NAT1 protein. Therefore we studied whether PPD and/or oxidised PPD diminishes proliferation and induces differentiation which is a classical goal of psoriasis including the trimer Bandrowski´s Base interact additionally with recombinant NAT1 therapy. Furthermore, S1P modulates the function of various immune cells, mainly to an protein itself in the absence of acetyl-coenzyme A. We found that all compounds but anti-inflammatory direction. Thus, the strategy of targeting immune cells with locally MAPPD bind to NAT1 protein after 2h. The greatest inhibition was found for oxidised acting S1P was explored in an experimental animal model of psoriasis vulgaris, the PPD (up to 50%). Due to the greater inhibition by oxidized PPD we propose that recently established imiquimod induced psoriasis mouse model and in the mouse tail oxidation products interact with the protein whereas PPD itself modulates NAT1 enzyme test. activity in a substrate-dependent mode of action. Overall we demonstrated that PPD can Topical administration of imiquimod onto back and ear skin led to a distinct inflammatory inhibit NAT1 in two different ways. response characterized by epidermal hyperproliferation, scaling and redness which was The work was partially financed by Federal Office of Public Health (FOPH), Switzerland scored with a modified PASI (psoriasis area and severity index). The positive control and Stiftung zur Förderung begabter Studierender und des wissenschaftlichen diflorasone diacetate and S1P, but not FTY720 reduced the epidermal hyperproliferation Nachwuchses by topical administration onto ear skin, indicating a mode of action for S1P via the S1P2 receptor, which is not activated by FTY720. There was also a moderate reduction of References inflammatory cell influx and edema formation in ear skin by S1P treatment, which was [1] J. Bonifas, S. Scheitza, J. Clemens, B. Blomeke J Pharmacol Exp Ther. 2010, 334, even more pronounced by treatment with diflorasone diacetate. The PASI determined on 318-326. back skin was, however, only significantly reduced by diflorasone diacetate. The discrepancy between outcome on ear and back skin remains elusive. In the mouse tail assay, the influence of S1P in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. Whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, S1P lacked such an effect, indicating a different mode of action in epidermal differentiation. Taken together, these results imply that topical administration of S1P might be a new option for the treatment of mild to moderate psoriasis lesions.
Study funded by DFG (BA 2071/3-1; KL 988/7-1)
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354 cortical interstitium. In contrast to cGKIα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. Here, we focused on the function of cGKI in the renal interstitium emphasizing a Functional analysis of the GLA gene promoter region and the potential impact on functional differentiation of both isoforms. The interstitium exists mainly of fibroblasts Fabry's disease playing a prominent role in the interstitial fibrosis. Accordingly, cGKI could also be involved in this pathophysiological process. Therefore, we studied whether cGKI Schelleckes M.1, Lenders M.1, Guske K.1, Schmitz B.1,2, Brand S. - M.2, Brand E.1 1 influences renal fibrosis which was induced by unilateral ureter obstruction (UUO). University Hospital Münster Internal Medicine D, Albert-Schweitzer-Campus 1, At first we analysed the role of the NO/cGMP signaling by application of cGMP Gebäude A1, 48149 Münster, Germany 2 increasing YC1 or ISDN. Thereby we detected antifibrotic effects of these substances. Medical Faculty of the Westfalian Wilhelms-University Muenster Department of Subsequently we tested whether these effects are mediated by cGKI by using mutant Molecular Genetics of Cardiovascular Disease, Albert-Schweitzer-Campus 1, Gebäude mice. On the one hand we examined αSM-rescue mice (expressing cGKIα only in D3, 48149 Münster, Germany smooth muscle under the control of the SM22 promotor with a cGKI-KO background) and cGKI-KO mice (expressing no cGKI). On the other hand we used tgtg mice Objective: Fabry's disease is a rare progressive multisystem disorder resulting from expressing more cGKIα in smooth muscle than wt mice (transgenic cGKIα under the deficiency of the lysosomal enzyme alpha-galactosidase A (GLA, EC 3.2.1.22). We control of the SM22 promotor). hypothesize that genetic GLA variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of Fabry's disease phenotypes. This study focuses on the characterization of the GLA promoter, identification of functional genetic variants and impact of transcription factor EB (TFEB), a regulator of lysosomal genes. 357
Methods: We screened 4011 bp of the 5'-flanking region of GLA in 60 patients with Fabry's disease and 60 controls for genetic variants. Serial promoter deletion constructs Cytotoxic and genotoxic effects of a panel of nanoparticles in three different for reporter gene assays were designed and identified genetic variants were introduced human epithelial cell lines. by site-directed mutagenesis. Constructs were transiently transfected into immortalized Thongkam W., Gerloff K., van Berlo D., Albrecht C., Schins R. human kidney epithelial (IHKE) cells and human vascular endothelial cells (EA.hy926) to IUF – Leibniz Research Institute for Environmental Medicine Particle Research, Auf'm determine transcriptional promoter activity (TA). Hennekamo 50, 40225 Düsseldorf, Germany
Results: Sequencing of patients’ DNA revealed five genetic variants in the 5'flanking Due to the steeply increased use of nanomaterials for commercial and industrial region of GLA, significantly more frequent in Fabry's patients compared to control group applications, toxicological assessment of their potential harmful effects is urgently (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles needed. Moreover, the continuous development of novel materials requires the p<0.0027). We identified two regions, a proximal one between -110 and -425 and a implementation of hazard-predicting models to prevent potential health effects resulting distal region between 1106 and -1421 with significant TA, in both cell lines. Co- from human exposure. In the present study, we studied the toxic potential of a set of transfection with TFEB activated TA of both regions significantly up to 5.3-fold (p<0.001). nanoparticles (NP) with varying physicochemical properties in human A549 lung In IHKE cells, insertion of the minor T allele (rs2071225) significantly enhanced basal TA epithelial cells, HepG2 liver epithelial cells and HK-2 proximal tubule epithelial cells. The of the proximal promoter region (p=0.0006), while insertion decreased basal TA used nanomaterials incorporated five TiO2 samples, two ZnO samples (i.e. uncoated and (p<0.0001) of the distal promoter portion. The combined insertion of the minor C alleles coated), two multi-walled carbon nanotube (MW-CNT) samples and a nanoparticulate (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly Ag sample. Cells were treated with NP at doses ranging from 0.3 to 80 µg/cm2 for increased basal TA of the distal promoter region (p=0.0037). cytotoxicity and from 06 to 40 µg/cm2 for genotoxicity. DNA damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the Conclusion: Our results indicate that three genetic variants, overrepresented in Fabry's WST-1 assay. Marked contrasts in cytotoxic and DNA damaging properties were patients, are located within transcriptionally active regions, possibly altering TF binding observed among the different materials. The overall strongest responses were observed sites and therefore, affecting GLA expression. Future analysis will assess the impact of with the uncoated ZnO-NP sample and with Ag-NP, although effects were found to GLA promoter variants and GLA regulation by TFEB with respect to Fabry's phenotypes. depend on the cell type. Notably, the DNA damaging effect of Ag-NP could at least partly be attributed to its dispersant. Present results form part of a growing data set which are generated in the framework of the EU FP7 project ENPRA (FP7-NMP) to establish dose-response relationships and a mathematical model to predict the hazard 355 of nanoparticles.
Ceramide synthase 6 plays a critical role in the development of experimental autoimmune encephalomyelitis 358 Schiffmann S.1, Ferreiros N.1, Birod K.1, Eberle M.1, Pfeilschifter W.2, Ziemann U.2, Scholich K.1, Grösch S.1, Geisslinger G.1 1Uniklinikum Frankfurt Inst. für Klnische Pharmakologie, Theodor-Stern-Kai 7, 60590 Increased spontaneous HPRT mutant frequency in V79 cells expressing human Frankfurt am Main, Germany cytochrome P450 1B1 2Uniklinikum Frankfurt Department Neurologie, Theodor-Stern-Kai 7, 60590 Frankfurt Schlechtweg A., Esch H., Martínez Jaramillo D., Lehmann L. am Main, Germany University of Wuerzburg/Institute of Pharmacy and Food Chemistry Section of Food Chemistry, Am Hubland, 97074 Wuerzburg, Germany Multiple sclerosis (MS) and its animal counterpart experimental autoimmune encephalomyelitis (EAE) have a major inflammatory component that drives and The hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay in Chinese orchestrates both diseases. Ceramides (Cer) are known as mediators of inflammatory hamster V79 lung fibroblasts (V79 cells) represents a widely-used mammalian test processes, but until now their role in MS was not elucidated. We measured the ceramide system to detect gene mutations. Since V79 cells do not express any cytochrome-P450- levels in the cerebrospinal fluid of MS patients and control patients using LC-MS/MS. dependent monooxygenase (CYP) isozymes, usually an activating system has to be Interestingly, the C16:0-Cer levels were 1.9 fold increased in MS patients. This translates added. Therefore, V79 cells expressing human (h) CYP isozymes have been into the finding that C16:0-Cer levels were also significantly elevated in the lumbar spinal commercialized. To test these V79 cells for their use in the HPRT test, V79 h1A1 and cord of EAE mice. The raised C16:0-Cer levels in the lumbar spinal cord were caused by h1B1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanine- a transiently increased expression of ceramide synthase (CerS) 6 in macrophages. Nitric resistant clones per 106 clonable cells (SMF), (ii) the stability of which over 4 weeks (w), oxide (NO) and tumor necrosis factor alpha (TNF-α) secreted by interferon gamma (INF- and (iii) the mutational spectrum (MS) of cDNA from mutant clones. MS of cDNA was γ ) induced macrophages play an essential role in the development of MS. determined by isolation of total RNA, reverse transcription/amplification of the coding Astonishingly, RNAi experiments reveal that CerS6 and its product C16:0-Cer are region by polymerase chain reaction and Sanger sequencing of the amplification mediators of INF-γ induced NO/TNF-α release in RAW macrophages. Moreover, product. Activity of CYP isozymes was verified by ethoxyresorufin-O-deethylase (EROD) treatment of EAE mice with L-cycloserine prevented the increase of C16:0-Cer and of assay. (i)/(ii) Whereas the SMF of V79 cells (w2:13±4; w4:2±1) and V79 h1A1 (w2:17±4; iNOS/TNF-α expression and caused a remission of the disease. In summary, CerS6 w4:3±0) only varied within the range of historical controls, SMF of V79 h1B1 increased plays a critical role in the initial phase of MS, most likely by regulating the NO and TNF-α continuously over time (w2: 36±6; w4: 68±13). (iii) Although the SMF of V79 and V79 synthesis. This let us speculate, that a substance designed to inhibit CerS6 and h1A1 were similar, the mutational spectrum of V79 cells was characterized by as many therefore to limit the inflammatory effects of C16:0-Cer may represent a new drug in MS transversions as transitions and deletions of exon 4 or exon 4+5, whereas the therapy. mutational spectrum of V79 h1A1 was characterized exclusively by transversions and deletion of exon 7+8. Surprisingly, with 57 out of 59 cDNAs derived from V79 h1B1 mutant clones, no amplification product was detected. First results indicate that there is at least one gene mutation in the untranslated region before and behind the coding 356 region precluding amplification with the original primers. (ii)To reduce the SMF of V79 h1B1, cells with wildtype HPRT activity were cloned and one clone with an EROD activity which did not differ significantly from the original cell population was further Role of cGMP-dependent protein kinase I for kidney fibrosis characterized. Initially, SMF of the clone varied between 0.7±0.6 and 1.2±0.0. Yet its SMF was unstable reaching up to 104±6. In conclusion, the mutational spectrum differed Schinner E.1, Hofmann F.2, Schlossmann J.1 1 between the V79 cell lines. Furthermore, h1B1 expression seemed to enhance SMF in Uni Regensburg Pharmakologie/Toxikologie, Universitätsstraße 31, 93053 V79 cells. Even though a temporary reduction of the SMF by cloning was possible, SMF Regensburg, Germany 2 of V79 h1B1 cells was unstable. TU München Pharmakologie/Toxikologie, Biedersteinerstraße 29, 80802 München, Germany cGMP is synthesized via nitric oxide- or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. Hence, the integration of cGMP signaling via cGMP-dependent protein kinases (cGK) might play a critical role for renal physiology. Both isozymes were detected in arterioles, mesangium and within the
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Antiaggregatory effects of a new identified galactolipid and a phytosterol from TRPC3 expression governs Orai1-mediated control of NFAT signalling in mast Allilum ursinum cells Schlegel F.1, Sabha D.2, Hiyasat B.1, Grötzinger K.2, Hennig L.3, Mohr F. - W.1, Rauwald Schleifer H.1, Doleschal B.1, Poteser M.1, Katrin T.1, Frischauf I.2, Romanin C.2, H. - W.2, Dhein S.1 Groschner K.1 1Herzzentrum Leipzig FuL/Chirurgie, Strümpellstr. 39, 04289 Leipzig, Germany 1Karl-Franzens-Universität IPW, Pharmakologie und Toxikologie, Universitätsplatz 2, 2Universität Leipzig Institut für pharmakologische Biologie, Brüderstr. 34, 04103 leipzig, 8010 Graz, Austria Germany 2Johannes Kepler Universität Institut für Biophysik, Altenberger Straße 69, 4040 Linz, 3Universität Leipzig Organische Chemie, Johannisallee 29, 04103 Leipzig, Germany Austria
We wanted to investigate the possible antithrombotic effects and elucidate the chemical Orai and STIM proteins have been identified as central components of the highly Ca2+ identity of the active principles involved in inhibitory effects against ADP-induced selective, store-operated current in immune cells (ICRAC). The molecular basis of aggregation of human platelets by wild garlic, Allium ursinum L. selective Orai-mediated activation of the calcineurin/NFAT pathway and the crosstalk Method: Bioassay-guided isolation procedure was used followed by spectrometric with other channel and scaffold molecules of the TRPC family are still incompletely identification of pure active compounds. For the bioassay, blood was taken from healthy understood. human volunteers and platelet rich plasma (PRP) was prepared for turbidimetric platelet Using patch clamp recordings complemented by fluorescence and TIRF microscopy we aggregation tests. PRP, stimulated with 20µM ADP, was treated with extracts of different investigated interactions between Orai1 and TRPC3 in plasma membrane microdomains polarities, fractions and isolated single compounds from Allium ursinum. The extracts of RBL-2H3 mast cells. were investigated by thin layer chromatography, HPLC, mass spectroscopy, ESI-MS Orai1-mediated CRAC currents, activated by passive store depletion, were found and 1d/2d 1H/13C-NMR spectroscopic techniques. For references the ADT-Antagonist significantly reduced by over-expression of TRPC3. This negative impact of TRPC3 on MeS-AMP was used. ICRAC was independent of channel function as the TRPC3 pore dead mutant (E630K) Result: Fresh Allium ursinum leaves were extracted with ethanol, which was the potent inhibited ICRAC to a similar extent as wild type TRPC3. Importantly, despite a reduction in form that effectively inhibited ADP-induced aggregation of human platelets. This ICRAC, NFAT translocation in TRPC3 overexpressing RBL cells remained unchanged, or ethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase was even slightly promoted. Store depletion-induced NFAT translocation in RBL cells containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only was as well unaffected by TRPC3E630K but substantially reduced by TRPC3 mutants with weak activity on platelet aggregation, the ethyl acetate and especially the chloroform either i) eliminated FKBP12/calcineurin binding (P704Q) or ii) deficiency in PKC partitions showed highest aggregation inhibiting potency. Thus, in our bioassay effects phosphorylation (S712A). Moreover, inhibition of PKC phosphorylation by of alliins/allicins could be neglected. The chloroform phase, possessing the strongest (GFX109203X; 3 µM) strongly suppressed NFAT signaling. activity, was separated into 28 fractions by gradient elution open CC on silica gel. The We suggest TRPC3 as a scaffold that links Orai-mediated Ca2+-entry to most active fractions 11-17 were separated again yielding 10 subfractions. This afforded NFAT/calcineurin signaling within plasma membrane microdomains. 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-D- glucopyranoside, the structures of which were determined by ESI-MS and 1d/2d 1H/13C-NMR spectroscopic techniques. Furthermore, the diminutive amounts of volatile oil of A.ursinum leaves obtained by steam distillation according to Ph.Eur. could be 362 evaluated as a third aggregation inhibiting principle. Conclusions: At the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory Neurally-induced bronchoconstriction in human and guinea pig precision-cut lung action on ADP-induced aggregation in human blood platelets. As a major constituent, slices the galactolipid 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet Schlepütz M.1, Rieg A. D.1, Bernau M.1, Spillner J. W.2, Perez-Bouza A.3, Autschbach found in Allium spec., appears as a new, highly useful marker substance for A.ursinum 2 1 1 drugs, or their pharmaceutical preparations. R. , Uhlig S. , Martin C. 1 RWTH Aachen University Institute of Pharmacology and Toxicology, Wendlingweg 2, 52074 Aachen, Germany 2RWTH Aachen University Department of Cardiac and Thorax Surgery, Pauwelsstr. 30, 52074 Aachen, Germany 360 3RWTH Aachen University Institute of Pathology, Pauwelsstr. 30, 52074 Aachen, Germany
Opportunities and potential pitfalls when using Computer Assisted Sperm Introduction: Precision-cut lung slices (PCLS) are well suited to study peripheral airway Analysis (CASA) responses in different species. Airway tone is under close control of the autonomic 1 1 2 1 Schleh C. , Takawale P. , Plassmann S. , Leoni A. - L. nervous system and dysregulation may contribute to airway hyperresponsiveness as 1BSL Bioservice Scientific Laboratories GmbH In Vivo Pharmacology and Toxicology, observed in human lung diseases such as asthma. Hence, the aim of the present study Behringstr. 6/8, 82152 Planegg/Munich, Germany was to characterize neurally induced bronchoconstriction (BC) in guinea pigs (GP) and 2PreClinical Safety (PCS) Consultants Ltd, Gartenstrasse 7, 4132 Muttenz, Switzerland to compare the results with those in human PCLS. Methods: PCLS were prepared from GP or human lung tissue. Nerve endings in PCLS Introduction were activated by electric field stimulation (EFS) or capsaicin addition. Cholinergic nerve In recent years, public attention focused more and more on risk factors which may responses were proven by atropine. Capsaicin was used to show excitatory non- impair sperm quality and thereby human reproduction. In this context, for example adrenergic non-cholinergic (eNANC) responses. Ruthenium red or SKF96365 were used pesticides, alcohol, cigarettes, and even mobile phones are discussed. A variety of to confirm transient receptor potential (TRP) channel contributions upon eNANC parameters exists including sperm counts as well as sperm motility, which are activation. considered to be two of the most important parameters to evaluate sperm quality in Results: GP and human PCLS were both sensitive to EFS and airways contracted to animal models with the final aim to assess human risk. In recent years Computer 39±26% of the initial airway area (%-IAA) and 63±21%-IAA, respectively. In frequency Assisted Sperm Analysis (CASA) devices mostly replaced the formerly used manual response curves half maximal response was found at 7.0±1.2 Hz for guinea pig PCLS counting and manual motility assessment. However, although CASA offers multiple and 8.1±0.8 Hz for human PCLS. EFS-induced BC was inhibited by atropine in both opportunities and can allow for an objective and more detailed evaluation, several pitfalls species. Capsaicin contracted GP to 18±15%-IAA. 60% of human PCLS were exist which can alter the results profoundly and consequently compromise the quality of responsive to capsaicin and airways contracted to 72±10%-IAA, respectively. In GP the data and ultimately the validity of a study. Ruthenium red and SKF96365 blocked capsaicin- as well as EFS-induced BC. Method Conclusion: GP and human PCLS contain atropine sensitive cholinergic and capsaicin The aim of the present study was to establish and validate the CASA device TOX IVOS sensitive eNANC nerve endings. Since GP PCLS were sensitive to TRP channel Sperm Analyzer from Hamilton Thorne and thereby to gain detailed knowledge about the inhibitors, the involvement of those channels can be characterized with respect to lung practical advantages but also intricacies which may alter the obtained results. In this diseases. In conclusion, GP PCLS resemble the human distal lung innervation and regard healthy adult male rats (10-12 weeks old) were used. Ultrasonic sound resistant represent a useful model to study neural airway pharmacology. sperm heads were isolated from the testis and in addition, sperms were isolated from the Cauda epididymis. Testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. Results 363 Different instrument settings modulate both – sperm motility and testicular sperm counts. In this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. In addition, the modification of the isolation The Erk1/2-pathway is involved in PKC-induced Nox4 up-regulation procedure can lead to variable results especially for sperm motility. Conclusion Schlufter F., Xia N., Förstermann U., Li H. Isolation procedures as well as instrument settings can alter the results. Consequently, Universitätsmedizin Mainz Institut für Pharmakologie, Obere Zahlbacher Straße 67, in an experimental setting, potential adverse effects can be confounded with 55131 Mainz, Germany methodologically mediated apparent findings exerted via inappropriate use of the device – depending on the respective conditions in the test laboratory. This study demonstrates NADPH oxidases (Nox) are major producers of reactive oxygen species in the vascular the relevance of standardization of testing conditions adopted for computer assisted wall and Nox4 is the most abundant Nox isoform in human endothelial cells. We have sperm analysis and the need for a robust validation prior to use in experimental settings. previously shown that treatment of human EA.hy 926 endothelial cells with phorbol 12- myristate 13-acetate (PMA) for 48 h leads to an up-regulation of Nox4 expression. This effect of PMA is mediated by protein kinase Cα, because it is preventable by the PKC inhibitor Gö 6983 and by PKCα-siRNA. The present study is aimed to investigate the signal transduction cascade downstream of PKCα. PMA-induced Nox4 up-regulation can be attenuated by PD 98,059 (an Erk1/2 inhibitor), but not by SP 600125 (a JNK inhibitor), indicating in the involvement of Erk1/2. Consistently, PMA treatment leads to a sustained activation of Erk1/2, and siRNA- mediated knockdown of Erk1/2 markedly reduces the PMA-induced Nox4 up-regulation. S83
H89, an inhibitor of the mitogen- and stress-activated protein kinases (MSKs) has no 366 effect on the PMA-stimulated Nox4 expression, indicating that MSKs are not the target molecules of Erk1/2 in this scenario. On the contrary, knockdown of the transcription factor Elk-1 by siRNA significantly reduces the PMA-induced Nox4 up-regulation. In Are High Plasma Sodium Concentrations Linked To Endothelial Cell Stiffness And conclusion, Erk1/2 and Elk-1 are involved in the PKCα-induced Nox4 up-regulation. Amiloride Response?
Schmitz B.1, Lenders M.2, Drüppel V.3, Kasprzak B.4, Kusche-Vihrog K.3, Oberleithner H.3, Brand S. - M.1, Brand E.2 1Medical Faculty of the Westphalian Wilhelms-University Münster Department of 364 Molecular Genetics of Cardiovascular Disease, Albert Schweitzer Campus 1, 48149 Münster, Germany 2University Hospital Münster Internal Medicine D, Nephrology, Hypertension and Determination of spontaneous mutation frequencies in normal human mammary Rheumatology, Albert-Schweitzer-Campus 1, 48149 Münster, Germany gland tissue using the Random Mutation Capture technique 3Westphalian Wilhelms-University Münster Institute of Physiology II, Schlossplatz 2, Schmalbach K., Lehmann L. 48149 Münster, Germany University of Wuerzburg Section of Food Chemistry, Am Hubland, 97074 Wuerzburg, 4University Hospital Münster Department of Vascular and Endovascular Surgery, Albert- Germany Schweitzer-Campus 1, 48149 Münster, Germany
Annually, over 57,000 women develop breast cancer in Germany. The accumulation of Objective: Hypertension and arterial stiffness is influenced by environmental and genetic mutations in mammary gland tissue during lifetime may be reasonable for genetic factors. High plasma sodium concentration leads to mechanical stiffening of developing breast cancer. In particular mutations in tumor suppressor genes, e.g. p53, endothelial cells resulting in endothelial dysfunction and elevated blood pressure. Here seem to play an important role in developing cancer. Up to now, lack of a method we investigated whether endothelial cell stiffness of ex vivo preparations of human sensitive enough to determine the expected very low spontaneous mutation frequency arteries is linked to plasma sodium concentrations and functional genetic variants of the (SMF) in normal mammary gland tissue precluded the investigation of the role of mineralocorticoid receptor (NR3C2), rs2070951 modulating blood pressure, renin, and spontaneous mutations acquired in the p53 gene in epidemiological studies. The only aldosterone levels, and rs5534, which alters a miRNA binding site. test with the potential to determine low SMFs was the Random Mutation Capture (RMC) Design and methods: Twenty patients were enrolled after a vein stripping procedure assay, a genotype selective method which detects mutants that render the mutational and collateral arterial blood vessels were prepared for atomic force microscopy (AFM). sequence non-cleavable by the TaqI restriction enzyme after accumulation of the target Plasma sodium concentration was routinely determined and DNA for genotyping was sequence. Therefore, the suitability of the RMC assay to determine SMF in p53 gene in extracted from EDTA blood samples. Sodium levels >140 mmol/L were defined as ’high’. normal human mammary gland tissue was evaluated. Thus, the RMC assay was After application of 5 µM amiloride, a specific blocker of the endothelial sodium channel optimized concerning (i) DNA isolation, (ii) PCR conditions, and (iii) amount of mammary (ENaC) changes in endothelial cell stiffness, were defined as ‘weak’ (≤10%), or ‘strong’ gland tissue. (i) Genomic DNA from normal human mammary gland tissue, obtained (>10%). Statistical analyzes were performed by ANOVA. from healthy women who underwent mamma reduction surgery for cosmetic reasons, Results: In ex vivo artery preparations of patients with high sodium levels (n=12), was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). The target sequence in intron 6 of p53 gene was captured by hybridization with a Both NR3C2 variants were associated with a change >10% in endothelial stiffness after complementary uracil-containing DNA-probe synthesized via polymerase chain reaction amiloride treatment. The rs2070951 C allele was significantly associated with a strong (PCR), followed by magnetic separation from the remaining genomic DNA. The copy amiloride response (p=0.024), while the rs5534 A allele only showed a trend towards number of the target sequence was quantified by competitive PCR. The number of stronger amiloride effects (p=0.06). mutants was detected after cleavage of the target DNA with TaqI by means of PCR with Conclusion: Our findings indicate that high plasma sodium concentration results in an a primer set flanking the restriction site. (iii) With 2 g of normal mammary gland tissue a increased endothelial amiloride response and thus influencing mechanical stiffness, SMF of 2.2±1.4x10-7 per base pair was determined indicating the RMC assay suitable for modulated by functional NR3C2 variants. Our novel approach linking patients’ sodium SMF determination. In conclusion, the SMF in the p53 gene in normal human mammary levels and genetic status to endothelial stiffness by AFM will be further evaluated in gland tissue was determined for the first time, enabling the future investigation of factors larger clinical settings. influencing the SMF during breast cancer development.
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Protein Expression Changes in BaP-Exposed Human Bladder Cancer Cells from Anti-inflammatory role of the cAMP effectors Epac and PKA: implications in Spliceosome Activation Towards Redistribution of the Cytoskeleton After Long- chronic obstructive pulmonary disease Term Exposure to Subacute Concentration Schmidt M., Oldenburger A., Maarsingh H., Meurs H. Schmitz-Spanke S., Pink M., Jeske E., Stempelmann K., Rehn S., Verma N., University of Groningen Department of Molecular Pharmacology, Antonius Deusinglaan Rettenmeier A. W. 1, 9713 AV Groningen, Netherlands Universitätsklinikum Essen Institut für Hygiene und Arbeitsmedizin, Hufelandstr. 55, 45122 Essen, Germany Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway Introduction smooth muscle (ASM) cells, may contribute to the development of chronic obstructive Polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (BaP)) have pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed been assumed to play a role in the development of bladder cancer. The objective of the at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little present study was to unravel cellular and in particular cytoskeletal response to BaP. To is known on its exact mechanism of action. We report here that next to the β2-agonist follow the sequential steps of chemical carcinogenesis the differential proteomic profile fenoterol, direct and specific activation of either exchange protein directly activated by was analyzed at early and late time points. The study was carried out in a superficial cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced human bladder cancer cell line (RT4) exposed to 0.5 µM BaP, a subacute concentration IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBα- based on results of proliferation (BrdU) and DNA damage (TUNEL) tests. degradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) Methods: phosphorylation, which was selectively reduced by PKA activation. CSE decreased Cells of a human bladder cancer cell line (RT4) were exposed to 0.5 µM BaP for 24 h Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 (n=5), 4 wk (n=7) and 8 wk (n=3). Proteins of whole cell lysate were separated by two- expression was also reduced in lung tissue from COPD patients. In conclusion, Epac dimensional electrophoresis (Fig. 1). Differentially expressed proteins were identified by and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF- matrix-assisted laser desorption/ionization-time of flight analysis. Cortactin, actin and κB and ERK, respectively, pointing at these cAMP effectors as potential targets for anti- tubulin were immunohistochemical stained. Changes in migration and colony forming inflammatory therapy in COPD. However, cigarette smoke exposure may reduce anti- ability were assessed by scratch wound-healing assay and soft-agar colony formation. inflammatory effects of cAMP elevating agents via down-regulation of Epac1. Results: References By using several databases (UniProt, Reactome, Panther) the identified proteins were Roscioni, S.S., Maarsingh, H., Elzinga, C.R.S., Schuur, J., Menzen, M., Halayko, A.J., categorized into different functional classes such as mRNA processing, translation, Meurs, H., & Schmidt, M. (2010) Epac as a novel effector of airway smooth muscle protein metabolic process, or several areas associated with the organization of the relaxation. J.Cell.Mol.Med., 15, 1551-1563. cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are Roscioni, S.S., Dekkers, B.G.J., Prins, A.G., Meurs, H., Schmidt, M. & Maarsingh, H. involved in the processing of pre-mRNA (20 %) and protein metabolism (25 %). This (2011) cAMP inhibits airway smooth muscle phenotype modulation. Brit. J. Pharmacol. pattern changed after 8 wk of treatment. Then, 48 % of the proteins affected the 162, 193-209. cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to Roscioni, S.S., Prins, A.G., Dekkers, B.G.J., Elzinga, C.R.S., Halayko, A.J., Meurs, H. & pre-mRNA processing. In the immunhistochemical staining, the treated cells appeared Schmidt, M. (2011) Functional roles of Epac and PKA in human airway smooth muscle after 8 wk of exposure larger and rounder predominantly due to the modifications of the phenotype plasticity. Brit. J. Pharmacol., 164, 958-969. actin cytoskeleton. Merged images of actin and cortactin revealed that both proteins co- localized in invadopodiae. After 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. In conclusion, BaP exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-to- mesenchymal transition. Two coordinators of a cell-type-specific splicing program, Epithelial Splicing Regulatory Proteins 1 and 2, are currently being validate by PCR.
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significantly reduced time on the rotating rod compared to C57Bl/6 wildtype controls, which may indicate an impaired cerebellar function. We conclude that DARC in fact modulates brain function. Surprisingly, this appears to be happening under homeostatic conditions, although DARC binds for the most part to inflammatory chemokines. It remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. It may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by DARC.
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Transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms Schnepf R.1, Zolk O.1, Muschler M.2, Fromm M. F.1, Wendler O.3, Traxdorf M.3, Iro H.3, Zenk J.3 1Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich- Alexander Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany 2Klinik für Psychiatrie, Sozialpsychiatrie und Psychotherapie, Medizinische Hochschule Hannover Center for Addiction Research, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 3Hals-Nasen-Ohren-Klinik, Kopf-und Halschirurgie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraße 1, 91054 Erlangen, Germany
fused master gel : Background: Membrane transporters may affect the disposition and thereby treatment Representative 2-DE gel of RT4 cells exposed to 0.5 µM BaP for 8 wk. Protein spots efficiency of anticancer drugs in human head-neck squamous cell carcinoma (HNSCC). which were differentially expressed compared to control and identified were marked. The gene expression profile of transporters in HNSCC, however, is unknown and was evaluated in this study. Moreover, we evaluated mechanisms by which transporters are regulated in HNSCC. We focused on the role of the nuclear pregnane X receptors (PXR, NR1I2) and epigenetic mechanisms.
Methods and results: Real-time RT-PCR revealed a significantly increased mRNA expression of SLCO1A2 and SLCO1B3 and a significantly decreased expression of 368 transporters such as SLCO2B1, SLCO2A1 and ABCC3 in human HNSCC tissue samples compared to adjacent normal mucosa. Moreover, an association between SLCO2B1 mRNA levels in tumor tissues and five-year survival of HNSCC patients was Cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated observed (χ2=6.59; P=0.010; n=34). Bisulfite sequencing revealed that promoter CpG protein kinase pathway islands of ABCC3 and SLCO2A1 were not methylated and thus these genes were not 1 1 2 1 Schmuhl E. , Ramer R. , Peters K. , Hinz B. epigenetically silenced in HNSCC tissues. In the HNSCC-derived UMSCC-1 and SCC- 1Institut für Toxikologie und Pharmakologie, Universität Rostock, Schillingallee 70, 15 cell lines, transcript expression of transporters (e.g., ABCC3, SLCO2A1; P<0.001) 18057 Rostock, Germany and PXR (NR1I2; P<0.001) was markedly induced by the DNA methyltransferase 2Abteilung für Zellbiologie, Universität Rostock, Schillingallee 69, 18057 Rostock, inhibitor decitabine. Cotreatment with the prototypical PXR activator rifampicin Germany significantly reversed decitabine-induced ABCC3 and SLCO2A1 expression.
Mesenchymal stem cells (MSCs) are known to be involved in various regenerative Conclusions: Transporter expression profiles significantly differed between HNSCC and processes such as cardiac, ocular, skin and bone tissue healing. However, little is normal mucosa and expression levels of SLCO2B1 may serve as a marker for known about the pharmacotherapeutical options aiming at tissue healing steps such as prognosis. Modulation of the epigenome with the anticancer drug decitabine the mobilization and homing of MSCs. Here, we show that cannabidiol (CBD), a non- substantially affects transporter expression in UMSCC-1 and SCC-15 cells, suggesting psychoactive cannabinoid, stimulates the migration of human adipose-derived MSCs in epigenetic regulation mechanisms. Moreover, interactions between epigenetic and both Boyden chamber and in vitro scratch wound assays. In Boyden chambers CBD nuclear receptor-mediated mechanisms in transporter regulation occur. (0.01 – 3 µM) was shown to promote cell migration in a time- and concentration This work was in part supported by the Johannes und Frieda Marohn Foundation. dependent manner. This promigratory action was inhibited by AM-630 (CB2 receptor antagonist) and by O-1602 (G protein-coupled receptor [GPR] 55 agonist). Moreover, CBD activated the mitogen-activated protein kinase (MAPK) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Blockade 371 of ERK activation by PD98059 prevented CBD-stimulated MSC migration, whereas inhibition of p38 MAPK by SB203580 was inactive in this respect. Furthermore, AM-630 and O-1602 were found to attenuate CBD-induced ERK activation. An ERK-dependent The role of HCN2 in neuropathic and inflammatory pain promigratory action was likewise demonstrated for the phytocannabinoid ∆9- Schnorr S.1, Eberhardt M.2, Reeh P.2, Ludwig A.1, Herrmann S.1 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue R(+)- 1 methanandamide. We conclude that CBD promotes MSC migration via receptor- Friedrich-Alexander-Universität Erlangen-Nürnberg Institut für Experimentelle und dependent ERK activation, possibly contributing to tissue healing. Klinische Pharmakologie und Toxikologie and, Fahrstraße 17, 91054 Erlangen, Germany 2Friedrich-Alexander-Universität Erlangen-Nürnberg Institut für Physiologie und Pathophysiologie, Universitätsstraße 17, 91054 Erlangen, Germany 369 The pacemaker current Ih is carried by hyperpolarization-activated cyclic nucleotide- gated cation channels (HCN1-4) and contributes to cellular excitability in the heart and the nervous system. HCN1 and HCN2 are the two most abundant HCN subunits in The Duffy Antigen Receptor for Chemokines modulates balance, locomotion and peripheral sensory neurons with HCN2 being the prevalent isoform in nociceptive small anxiety-like behavior in mice under homeostatic conditions sized C-fibre dorsal root ganglion (DRG) neurons. We examined the role of HCN2 for 1 1 2 3 1 Schneider E. , Gao J. - L. , Holmes G. , Peiper S. C. , Murphy P. M. peripheral sensitization and spontaneous neuronal activity in neuropathic and 1NIAID/NIH Molecular Signaling Section, 10 Center Drive, Bethesda, Maryland 20892- inflammatory pain. We generated a conditional deletion of HCN2 by using a nociceptor 1886, United States specific Cre-transgene driven by the Nav1.8 promoter. The nociceptor-specific knockout 2NIAID/NIH Inflammation Biology Section, 10 Center Drive, Bethesda, Maryland 20892- of HCN2 in DRG neurons (nospHCN2KO) was confirmed by quantitative RT-PCR and 1886, United States western blot. Immunohistochemical staining revealed that the deletion of HCN2 was 3Jefferson Medical College Pathology, Anatomy & Cell Biology, 1020 Locust Street JAH mainly restricted to small sized DRG neurons. The conditional loss of HCN2 resulted in 279, Philadelphia, Pennsylvania 19107, United States a significant reduction of Ih positive small diameter DRG neurons pointing to a central role of this isoform to the HCN current in nociceptive neurons. Behavioral studies The Duffy Antigen Receptor for Chemokines (DARC) binds promiscuously many showed that the lack of HCN2 did not influence basal pain responses but led to a inflammatory chemokines without showing intracellular signal transduction. It is mainly significant reduction in mechanosensation in both neuropathic and inflammatory pain expressed on endothelial cells of postcapillary venules and on red blood cells, where it models. However, thermosensation of the mutants was only decreased in neuropathic acts as a transendothelial transporter of chemokines and as a chemokine sink, pain conditions. In wild-type animals, intraperitoneal, intraplantar and even intrathecal respectively. Surprisingly, as shown for human and mouse brain, DARC is also injection of the HCN channel blocker ZD7288 nearly eliminated tactile allodynia caused expressed at high density in the cerebellum. However, nothing is known about the by inflammation in contrast to thermal hyperalgesia which remained unaffected. In function of DARC in this location. contrast, pain thresholds in nospHCN2KO mice did not significantly increase after We addressed this question by subjecting C57Bl/6 wildtype and DARC-deficient mice to pharmacological block of Ih. Additionally, experiments revealed that the inflammatory a series of behavior experiments including Morris water maze-, elevated plus maze-, condition induced an upregulation of HCN2 protein in the spinal dorsal horn compared to rotarod- and actometer tests. While the results from the water maze experiments are saline injected mice. Our results suggest that HCN2 might be a new target in the ambiguous, elevated plus maze trials show a strong aversion of DARC-/- mice to walk to treatment of neuropathic and inflammatory pain. the end of the open arm, which is consistent with anxiety-like behavior. Moreover, DARC-/- mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). Finally, DARC-/- mice spend a S85
372 properties of M2 receptors, the low affinity fraction is not associated with any other G protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. Moreover, high affinity agonist binding appears A life-cell imaging-based screening for toxin effects on neurotransmitter receptor solely in membrane homogenates but not in experiments conducted with living cells, function probably due to their high intracellular concentration of guanylnucleotides. Taken together the chemical knock-out of G proteins revealed that the high affinity Schöbel N., Hausherr V., Sisnaiske J., Hengstler J. G., van Thriel C. binding of agonists in membrane homogenates is associated with the interaction of the Leibniz Research Center for Working Environment and Human Factors NB Tox, muscarinic M2 receptor with Gi proteins. The low affinity binding cannot be related to Ardeystr. 67, 44139 Dortmund, Germany another G protein, although the muscarinic M2 receptor exhibits promiscuous G protein signalling properties. Interestingly data obtained with living cells do not reveal any high The proper functioning of the central, as well as the peripheral nervous systems is of affinity binding of agonists. outstanding importance to the survival and well-being of humans. Yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and References occupational toxins. Some of these toxins preferentially target neural cells. These [1] Whalen JW et al. (2011) Trends in Molecular Medicine 17 (3): 126-139 neurotoxins can exert their devastating effects by very different modes of action. [2] De Lean A et al. (1980) Journal of Biological Chemistry 255 (15): 7108-7117 Neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth Support of the DFG is gratefully acknowledged (HO 1368/12-1 MO 821/2-1). and elongation. These processes can be identified by specialized in vitro tests. Furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. As another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. Countless natural and synthetic substances have 375 been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. However, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure Stress-inducecd hyperalgesia is mimicked by oral administration of are still poorly studied with respect to their effects on neurotransmitter receptors. Thus, corticosterone the need for a better understanding of the susceptibility of neurotransmitter systems for Schramm E., Koch E. toxic effects exerted by these substances is of outstanding importance for the protection Dr. Willmar Schwabe Pharmaceuticals Preclinical Research, Willmar-Schwabe-Straße 4, of human health. 76227 Karlsruhe, Germany Here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. Prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (HPA)- Different neuronal cells were tested for their sensitivities for classical neurotransmitters axis and may affect the sensitivity of pain perception. However, it is not yet known using life-cell imaging experiments. In more detail, we examined the proportion of whether the alterations of HPA-axis and increased pain sensitivity are related. To create responding cells and determined the EC50 values for the most prominent a long lasting stressful situation, male Wistar rats were exposed to a restraint-stress for neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one 1 h daily over a period of two weeks. The effect of stress on the HPA-axis was hand, namely SH-SY5Y and LUMES cells, and primary mouse neurons on the other determined by adrenal morphology and stress hormone levels, the influence on hand. With these data at hand, we are now able to identify and characterize the effects mechanical pain sensitivity was evaluated by the Randall-Selitto paw pressure test. On of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. day 15 the animals exhibited a significant mechanical hyperalgesia. They also showed The use of an in vitro imaging-based physiological test system is at the interface increased ACTH and corticosterone plasma levels and an enlarged zona fasciculata of between non-functional in vitro approaches and in vivo toxicity tests, thus, giving the adrenal gland, indicating a dysregulation of the HPA-axis. mechanistic insight into neurotoxic processes without requiring animal experiments. For testing the correlation of HPA-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in Wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. These animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the 373 adrenal glands and reduced ACTH levels. The results show that chronic stress leads to a dysfunction of the HPA-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of Apomorphine acts on TRPA1 channels corticosterone. Thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. Scholze A., Schaefer M., Hill K. Universität Leipzig - Universitätsmedizin Rudolf Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr. 16-18, 04107 Leipzig, Germany
Apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist 376 and is clinically used to treat “off-states” in patients suffering from Parkinson´s disease. Adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. The Alternaria mycotoxins AOH and AME induce CYP1A1 and apoptosis in murine We wanted to test whether an activation of TRP (transient receptor potential) channels hepatoma cells dependent on the aryl hydrocarbon receptor 1 1 2 3 1 in sensory neurones contributes to the perception of pain after apomorphine injection. Schreck I. , Deigendesch U. , Burkhardt B. , Marko D. , Weiss C. While the warm/heat receptors TRPV1, TRPV2, TRPV3, and TRPV4 and the cold 1Karlsruhe Institute of Technology, Campus North Institute of Toxicology and Genetics, receptor TRPM8 were insensitive towards apomorphine treatment, TRPA1 could Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany concentration-dependently be modulated by apomorphine. Low micromolar 2Karlsruhe Institute of Technology, Campus South Institute of Applied Biosciences, apomorphine concentrations potently activated heterologously expressed TRPA1 Chair of Food Chemistry, Adenauerring 20a, 76131 Karlsruhe, Germany channels in a stably transfected cell line (HEK293-TRPA1), as well as natively 3University of Vienna Department of Food Chemistry and Toxicology, Währinger Str. 38, expressed TRPA1 in cultured dorsal root ganglion neurones. On the other hand, when 1090 Vienna, Austria using higher concentrations of apomorphine, we observed inhibition of TRPA1 activity. Previous studies have shown that subcutaneously administered apomorphine produces Mycotoxins are secondary metabolites of fungi including the genus Alternaria (black a biphasic dose response relationship in rats, inducing hyperalgesia at low doses mold). Alternaria fungi are known to infest different types of foodstuffs and produce whereas high doses of the substance cause antinociception. From our studies we diverse toxins amongst them the mycotoxins alternariol (AOH) and alternariol methyl conclude that such in vivo effects of apomorphine are presumably mediated by ether (AME) which are potential carcinogens. As planar compounds, AOH and AME are activation/inhibition of TRPA1 expressed in sensory neurones preferentially metabolized by cytochrome P450 (CYP) 1A1 and 1A2. The most prominent regulator of CYP1A1 is the dimeric transcription factor complex AhR/ARNT, which is activated by planar ligands. Therefore we studied the activation of AhR/ARNT by AOH and AME and monitored CYP1A1 induction in murine hepatoma cells (Hepa- 374 1c1c7). Indeed, AOH and AME enhanced the levels of CYP1A1 in Hepa-1c1c7 cells but not in cells with inactivated AhR (Hepa-1c1c12) or ARNT (Hepa-1c1c4). Furthermore, we studied the cytotoxicity of AOH and AME. By using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and In depth-analysis of biphasic agonist competition curves at muscarinic M2 receptors micronuclei formation. Both AOH and AME reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after AOH treatment or Schrage R.1, Klöckner J.2, Holzgrabe U.2, Mohr K.1 1 micronuclei formation. The observed effects where, except for the induction of Pharmazeutisches Institut Pharmakologie und Toxikologie, Gerhard-Domagk-Str. 3, apoptosis, independent of AhR/ARNT. 53121 Bonn, Germany 2 In summary, AOH and AME activate the AhR/ARNT pathway to induce CYP1A1 Institut für Pharmazie und Lebensmittelchemie Pharmazeutische und medizinische expression and apoptosis. However, the predominant cytotoxic effect of AOH and AME Chemie, Am Hubland, 97074 Würzburg, Germany in hepatoma cells is a profound reduction in cell numbers, which is independent of the AhR/ARNT pathway. Agonist binding to a G Protein-coupled receptor (GPCR) induces a conformational change of the receptor protein, which results in the activation of receptor-associated References heterotrimeric G proteins [1]. In radioligand binding studies, conducted to investigate Schreck I, Deigendesch U, Burkhardt B, Marko D, Weiss C. The Alternaria mycotoxins ligand binding to specific GPCRs, receptors are usually probed with radioantagonists. As alternariol and alternariol methyl ether induce cytochrome P450 1A1 and apoptosis in in other GPCRs [2], agonists of the muscarinic M2 receptor exhibit biphasic kinetics and murine hepatoma cells dependent on the aryl hydrocarbon receptor. Arch Toxicol. 2011 biphasic competition curves with radioantagonists, indicating a more complex situation Nov 26. [Epub ahead of print] probably caused by G protein interactions. Here, we present a detailed study of the binding of agonists to muscarinic M2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knock- out of G proteins. In addition to membrane homogenates living cells were employed. We demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with Gi proteins. However, despite promiscuous signalling S86
377 inactivation of CREM display impaired cardiac contraction and relaxation, decreased expression of SERCA and down-regulation of β1-adrenoceptors. To elucidate the underlying functional mechanisms on the cellular level we here investigated cellular 2+ PLASMA NITRITE IN PATIENTS WITH RENAL IMPAIRMENT: A PILOT STUDY electrophysiology and Ca -cycling in ventricular cardiomyocytes from CREM KO mice (KO). Schröder D.1, Passauer J.1, Fauler J.2 1 TU Dresden Nephrology, Department of Medicine III, Fetscherstraße 74, 01307 Methods and Results: Dresden, Germany 2 Adult ventricular cardiomyocytes were isolated from KO and wildtype (WT) mice (age TU Dresden Institute of Clinical Pharmacology, Fiedlerstraße 27, 01307 Dresden, 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. Germany Action potentials (APs) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp). AP duration parameters were not different between Background: Nitric oxide is an important mediator of vascular homeostasis. Plasma groups (in ms; mean±SEM; APD50: KO, 7.5±0.8; WT, 6.1±0.7. APD90: KO, 44.7±3.6; nitrite is now regarded as a storage compound of nitric oxide (NO) which significantly WT, 40.6±4.0; KO n=51/13, WT n=42/13 [cells/mice]). contributes to vasodilatation and organ protection during ischemia. At present little is Intracellular calcium transients and sarcomere shortening were measured under basal known about the role of nitrite in patients with renal impairment. conditions and acute isoprenaline stimulation (10-6 M) after loading ventricular Question: Are plasma nitrite concentrations in patients with renal impairment different cardiomyocytes with Indo-1/AM. Basal transient amplitudes were not significantly from those in healthy volunteers? different between groups despite a tendency to an amplitude reduction in KO myocytes Methods: In a pilot study we determined baseline plasma nitrite levels in patients with (RU; KO, 0.112±0.004; WT, 0.131±0.006; KO n=153/10, WT n=150/10). Basal calcium renal insufficiency (RI, n=19), in patients on dialysis (D, n=30) and in healthy control transient decay was delayed in KO myocytes (τ of decay in s; KO, 0.75±0.02*; WT, subjects (C, n=11) by chemiluminescence (NOA280, Sievers instruments, USA). In 0.68±0.02; *p<0.05 vs WT). Absolute and relative sarcomere shortening was reduced in addition, we measured changes in nitrite concentrations during 8 minutes of complete KO myocytes and the time constant of relaxation increased. Under acute exposition to forearm ischemia in D (n=5) and C (n=5). Finally we determined changes in nitrite isoprenaline Ca2+transient amplitude increased less in KO than in WT (KO, 0.33±0.01*; concentrations during a four-hour dialysis session in D (n=20). WT, 0.38±0.01; *p<0.05 vs WT) which was also reflected by a less pronounced Results: Nitrite plasma concentrations were 182±92 in D, 162±95 in RI and 67±28 sarcomere shortening in KO. However Ca2+ transient decay and cell relaxation seemed nmol/L in C showing an inverse correlation to GFR (MDRD, r=0.35;p<0.005). During to improve under isoprenaline and was even slightly faster in KO vs WT. hemodialysis nitrite concentrations decreased to 82±25% (1h), 84±43 (2h) and 63±21 (4h) of the pre-dialysis baseline concentrations. During forearm ischemia we observed a Conclusions: significantly more pronounced decrease in nitrite concentrations to 80±10% (2min), Inactivation of CREM seems to have no consequences for AP duration and possibly 73±12 (6min) and 59±11 (8min) of baseline in D compared to C (91±12 ; 84±15 and associated ion channels but leads to impairment of Ca2+ cycling and sarcomere 82±13% ; p<0,05 by ANOVA). shortening under basal conditions well explaining the previous findings in vivo. Our Conclusion: 1) Renal impairment is associated with increased plasma nitrite results show that CREM is essential for a regular excitation-contraction coupling in the concentrations compared with healthy volunteers. 2) The high NO2 plasma mouse heart. concentrations observed in D can not be explained by the dialysis procedure itself. 3) (supported by the IZKF Münster) There are significant differences in nitrite utilization during ischemia between dialysis patients and control subjects. Further studies have to clarify the role of nitrite in patients with renal impairment and have to unravel the underlying mechanisms. 380
New mechanistic insights in NO/cGMP actions in the vasculature 378 Schulte K., Koesling D., Mergia E. Medizinische Fakultät Ruhr-Universität Pharmakologie und Toxikologie, Universitätsstr. Toxicological facts from text: How text mining can make the difference in life 150, 44781 Bochum, Germany science research 1 2 3 4 3 5 Hypertension, a major risk factor for cardiovascular diseases, is associated with vascular Schroeder M. , Alvers M. R. , Grune B. , Landsiedel R. , Luch A. , Sauer U. G. , changes resulting in increased vascular contractility und vascular peripheral resistance. Spielmann H.6, Wächter T.1,2 1 A prominent factor in the development and maintenance of hypertension is the renin- Biotechniologisches Zentrum (BIOTEC), TU Dresden Lehrstuhl für Bioinformatik, angiotensin-aldostrerone system. Angiotensin II (Ang II) is the main mediator of this Dresden, Germany 2+ 2 system and a powerful vasoconstrictor. Ang II increases the intracellular Ca Transinsight GmbH Semantic Technologies, Dresden, Germany concentration thereby activating myosin light chain (MLC) kinase, which enhances MLC 3Bundesinstitut für Risikobewertung (BfR), Berlin, Germany 4 phosphorylation and subsequent vascular contraction. Opposite to angII-induced BASF Product Safety Experimental Toxicology and Ecology, Ludwigshafen, Germany vascular contraction, NO/cGMP pathway promotes vascular relaxation by decreasing 5Scientific Consultancy - Animal Welfare, Neubiberg, Germany 2+ 6 Ca concentration and lowering MLC phosphorylation. Responsible for MLC FU Berlin Institut für Pharmazie, Berlin, Germany dephosphorylation is the MLC phosphatase (MLCP), whose activity is regulated by different phosphorylations. Phosphorylation of MLCP via RhoA-activated Rho-kinase Special purpose databases are the first place for researchers in the life sciences to enhances phosphatase activity while phosphorylation via the cGMP-dependent protein obtain expert curated data. Naturally, such resources are limited in terms of timeliness kinase has been proposed to decrease enzymatic activity. and comprehensiveness. The literature database PubMed alone lists more than To investigate the interplay of AngII with the NO/cGMP pathway, we treated wild-type 20,000,000 scientific abstracts, and 700,000 are newly added every year. The protein and KO mice lacking the cGMP forming NO receptor, NO-GC1, with AngiotensinII (1.44 sequence database UniProtKB stores over 10,500,000 sequences, a hundred times mg / kg BW / d) for two weeks. In addition to various cardiovascular parameters, more than ten years ago. Turning these data into meaningful information and making it physiological changes in vascular reactivity of aortic rings of AngII-treated WT and NO- accessible to both humans and computers have become an essential part of biological GC1 KO mice were assessed in organ bath experiments and correlated with discovery and biomedical research. biochemical parameters as the phosphorylation state of MLC, MLCP and Rho-kinase Text mining techniques have proven useful to extract the missing links in areas such as activities examined by immunoblot analysis. Analysis of cGMP levels revealed that AngII drug-target and drug-disease prediction, the extraction of mutation-phenotype treatment decreased cGMP in WT mice to levels comparable to those of the KO mice associations, or the prediction of protein-protein and protein-ligand interactions. By which were unaltered by the treatment. systematically extracting information from available literature, reports or patents, text Our study will provide further mechanistic insights in the molecular interactions between mining techniques can help to refine existing categorical knowledge stored in databases, constrictor and dilator stimuli in the vasculature. and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. In the area of regulatory toxicology we developed Go3R, the first knowledge-based search engine for alternative methods to animal experiments. The system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, 381 but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. The up-to-date taxonomic- structured “table of contents” provided by Go3R allows for search in the literature listed Investigation on the Genotoxicity of Different Sizes of Gold Nanoparticles in PubMed or the Toxicology Data Network (TOXNET) in a fast and comprehensive Administered to the Lungs of Rats 1 1 2 1 1 1 manner. The semantically enriched platform supports the user during the query Schulz M. , Lan M. - H. , Brill S. , Strauss V. , Treumann S. , Gröters S. , van 3 1 formulation, allows for bibliographic analysis, and reveals existing relations to Ravenzwaay B. , Landsiedel R. replacement, reduction, and refinement of animal experiments. 1BASF SE Experimental Toxicology and Ecology, 67063 Ludwigshafen am Rhein, Germany 2BASF SE Occupational Medicine, 67063 Ludwigshafen am Rhein, Germany 3BASF SE Product Safety, GV/TB, 67063 Ludwigshafen, Germany 379 Nanomaterials are already used today and offer even greater use and benefits in the future. The progress of nanotechnology must be accompanied by investigations of their Impaired cardiac excitation-contraction-coupling in mice with complete potential harmful effects. For airborne nanomaterials, lung toxicity is a major concern inactivation of the CREM gene and obviously the particle size is discussed as a critical property directing adverse effects. While standard toxicological test methods are generally capable of detecting the Schulte J. S., Tekook M., Schmitz W., Müller F. U. toxic effects, the choice of relevant methods for nanomaterials is still discussed. We Westfälische Wilhelms-Universität Institut für Pharmakologie und Toxikologie, have investigated two genotoxic endpoints - alkaline Comet assay in lung tissue and Domagkstraße 12, 48149 Münster, Germany micronucleation in polychromatic erythrocytes of the bone marrow - in a combined study 72 hours after a single instillation of 18 µg Gold nanoparticles (NP) into the trachea of Rationale: male adult Wistar rats. The administration of three test materials differing only in their The structurally related transcription factors cAMP response element binding protein primary particle size (2-, 20- and 200-nm) did not lead to relevant DNA damage in the (CREB) and cAMP response element modulator (CREM) mediate a regulation of gene mentioned tests. The measurement of clinical pathology parameters in bronchoalveolar transcription in response to cAMP and are expressed in the heart. Mice with complete S87
lavage fluid (BALF) and blood indicated neither relevant local reactions in the animals’ 384 lungs nor adverse systemic effects. Minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. In conclusion, under the conditions of this study the different sized Gold NP tested were non-genotoxic and Human Pregnane X Receptor genotype of the donor but not of the recipient is a showed no systemic and local adverse effects at the given dose. risk factor for delayed graft function after renal transplantation
Schwab M.1,2, Schaeffeler E.1, Kruck S.3, Gauer S.4, Nies A.1, Winter S.1, Bedke J.3, Geiger H.4, Hoefeld H.4, Kleemann J.4, Asbe-Vollkopf A.4, Engel J.4, Burk O.1, Hauser I.4 1Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, 382 70376 Stuttgart, Germany 2University Hospital Tuebingen Dept. Clinical Pharmacology, Otfried-Müller-Str. 45, 72076 Tuebingen, Germany Platelet dense granule secretion mediates platelet-dependent enhancement of 3University Hospital Tuebingen Department of Urology, Hoppe-Seyler-Str. 3, 72076 tumor cell transmigration and formation of metastases Tübingen, Germany Schumacher D., Strilic B., Wettschureck N., Offermanns S. 4University Hospital Johann Wolfgang Goethe-University Deptartment of Nephrology, MPI für Herz- und Lungenforschung Offermanns, Ludwigstr. 43, 61231 Bad Nauheim, Med. Clinic II, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany Germany Delayed graft function (DGF) is an important immediate complication in renal Tumor cell metastasis to distant organs is the primary cause of mortality in cancer transplantation significantly contributing to decreased long-term allograft survival. In patients. Tumor cells leave the primary tumor, intravasate, survive in the circulation and addition to donor- and recipient-related risk factors altered renal excretion of xenobiotics extravasate through the endothelial cell layer to grow in the target organ. It has long by membrane transporters may influence DGF as well. Using recipients’ and donors’ been known that blood platelets play an important role in tumor cell survival and DNA, we assessed the impact of genetic variants on DGF for the transporter proteins, P- dissemination, but the mechanism by which platelets promote metastasis remained glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2), and the nuclear unclear. Given that platelets are found closely associated with tumor cells shortly after pregnane X receptor (PXR/NR1I2), regulating the transcription of drug metabolizing vascular arrest, we explored whether platelets can facilitate the transmigration of tumor enzymes and membrane transporters. In our local cohort of transplant patients (n=178) cells through the endothelium and thereby promote extravasation of tumor cells into the DGF occurred in 27.5 %. Logistic regression analysis using four different genetic models organ parenchyma. The ability of various mouse and human tumor cells like Lewis-Lung (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor’s carcinoma cells (LLC1), B16F10 melanoma cells or human neuroblastoma cells (SH- PXR rs2276707 8055TT genotype was significantly associated with DGF (recessive SY5Y) to transmigrate through an endothelial cell layer was strongly enhanced by model: OR, 9.0; 95%CI, 1.75-46.3; p=0.004 unadjusted), even after correction for seeding tumor cells together with mouse or human platelets onto the endothelial cell multiple testing (p=0.035 Holm-adjusted). When we performed multivariate analysis layer. This indicates that platelets facilitate tumor cell transmigration in vitro. We found including genetic and 16 clinical co-variates (i.e. age, gender, HLA mismatches, panel- that platelet granule secretion is involved in this process as supernatant from platelets reactive antibodies, immunosuppression using CNI, T cell-depleting agents, anti-IL-2 incubated with tumor cells but not from resting platelets was sufficient to enhance tumor receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors cell transmigration. Additionally, no platelet-mediated increase of tumor cell or graft loss) again DGF was significantly associated only with the PXR rs2276707 TT transmigration was observed in dense granule secretion-defective platelets of Munc13-4 genotype of the donor (recessive model: OR, 16.08; 95% CI, 2.0-129.46; p=0.0046 deficient mice. Thus, dense granule secretion is required for platelet-dependent tumor unadjusted) which held true after correction for multiple testing (p=0.04). For ABCC2 cell extravasation in vitro. While the growth and weight of primary tumors after variants only the donor rs17222723 3563T>A genotype correlated with DGF by subcutaneous injection of LLC1 and B16 cells was indistinguishable between wild-type univariate (OR, 4.65; 95%CI, 1.56-13.90; p=0.005 unadjusted) as well as by multivariate mice and animals lacking Munc13-4, the number of metastases in the lung was strongly analysis (OR, 5.5; 95%CI, 1.37-22.11; p=0.01; Table 4) but not after correction for reduced in Munc13-4-deficient animals. The strong decrease in formation of metastases multiple testing (p=0.12). For variants in the ABCB1 gene no significant associations in Munc13-4 deficient mice was also observed after i.v. injection of LLC1 and B16F10 with DGF were detected for both the donor’s and the recipient’s genotype. In summary, cells. Thus, platelet dense granule secretion plays a critical role in tumor cell metastasis our findings suggest for the first time that PXR may be a risk gene for the development by enhancing tumor cell transmigration through the endothelial cell layer. of DGF independently from previously identified risk factors.
Supported by the Robert-Bosch Foundation, Stuttgart, Germany, the BMBF grant 03IS2061C (Berlin, Germany) and by the Ferdinand Eisenberger grant of the German 383 Society of Urology (ID KrS1/FE-10).
Formation of DNA adducts in mouse tissues by the Brassica ingredient 1- methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3- 385 indolylmethyl alcohol Schumacher F.1, Engst W.2, Monien B. H.1, Florian S.1, Frank H.3, Seidel A.3, Glatt H.1 1 Formation, morphology and structural requirements for formation of microtubule Deutsches Institut für Ernährungsforschung (DIfE) Potsdam-Rehbrücke protrusions by Clostridium difficile toxin CDT Ernährungstoxikologie, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany 2Deutsches Institut für Ernährungsforschung (DIfE) Potsdam-Rehbrücke Analytik, Schwan C., Kruppke A. S., Nölke T., Aktories K. Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany Institut für Experimentelle und Klinische Pharmakologie und Toxikologie I, Albertstr. 25, 3Biochemisches Institut für Umweltcarcinogene, Prof. Dr. Gernot Grimmer-Stiftung, 79104 Freiburg, Germany Lurup 4, 22927 Grosshansdorf, Germany Clostridium difficile is an anaerobe, gram-positive pathogen. It causes antibiotic- Glucosinolates are secondary metabolites present at substantial levels in cruciferous associated diarrhoea and pseudomembranous colitis by production of the Rho GTPase- vegetables, such as broccoli and cabbage. After injury of plant tissue they are activated glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile by the enzyme myrosinase to form various electrophilic degradation products like strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium isothiocyanates. We previously showed that 1-methoxy-3-indolylmethyl (1-MIM) difficile transferase). CDT is taken up via receptor-mediated endocytosis after binding to glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and the Lipolysis Stimulated Lipoprotein Receptor (Papatheodorou et al., PNAS 2011). In the mammalian cells after activation by myrosinase. The induction of mutations could be cytosol, CDT ADP-ribosylates actin at Arg 177, thereby actin polymerization is blocked, correlated with the formation of DNA adducts [1]. resulting in disruption of the F-actin network. We have identified and synthesized the major DNA adducts N2-(1-MIM)-dG and N6-(1- CDT and other binary actin-ADP-ribosylating toxins induce redistribution of microtubules in MIM)-dA. Moreover, we developed a highly sensitive UPLC-ESI-MS/MS method for the cell interior and formation of long (>150 µm) microtubule-based protrusions at the selective MRM quantification of these adducts using stable-isotopic labeled adducts as surface of intestinal epithelial cells which increase bacterial adherence (Schwan et al., PLoS internal standards. Pathog 2009). The clostridial actin-ADP-ribosyltransferases influence the dynamicity of While the plant enzyme myrosinase is probably almost completely inactivated after microtubules and their capture at the cell cortex by indirectly affecting different microtubule cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good regulating proteins like CLASP2 and ACF7. Besides the influence of CDT on microtubule heat and pH stability. Enzymes of individual intestinal bacteria are able to cleave the regulatory proteins, the formation of protrusions depends on plasma membrane glycosidic bond of the glucosinolates, which leads to the formation of reactive composition. Depletion of cholesterol, the breakdown of sphingomyelin or inhibition of metabolites within the gut lumen. We were able to detect significant levels of N2-(1- sphingolipid-synthesis reduce the formation of microtubule-based protrusions. Surprisingly, MIM)-dG and N6-(1-MIM)-dA in a dose-dependent manner in the large intestine of mice most of the CDT-induced processes contain membrane-tubules derived from the treated orally with isolated 1-MIM glucosinolate. The peak levels of N2-(1-MIM)-dG and endoplasmatic reticulum (ER). The remodeling of the ER is microtubule dependent and is N6-(1-MIM)-dA in the murine large intestine were reached 8 h after a single mainly mediated by Stim1 that usually functions as a calcium sensor in the ER and administration of 600 µmol 1-MIM glucosinolate/ kg body weight. The oral application of activates the store operated Orai1 calcium ion channels in the plasma membrane. The data the relatively stable metabolite 1-MIM alcohol to mice led to the formation of identical suggest that toxin-induced changes of the microtubule system including alterations of the DNA adducts. This benzylic alcohol can be activated by sulfotransferases to an ER, may affect trafficking and ER-dependent signalling. electrophilic sulfo conjugate. In contrast to the intact glucosinolate the orally administered 1-MIM alcohol generated significant levels of N2-(1-MIM)-dG and N6-(1- MIM)-dA not only in the large intestine but also in other tissues, such as the liver, of mice. 386
References [1] H. Glatt, C. Baasanjav-Gerber, F. Schumacher, B. H. Monien, M. Schreiner, H. Neuroprotection by bilobalide: what is the mode of action? Frank, A. Seidel, W. Engst, Chem.-Biol. Interact., 192 (2011) Schwarzkopf T. M., Klein J. J.W. Goethe Universität Frankfurt Pharmakologisches Institut für Naturwissenschaftler, Max von Laue Str. 9, 60438 Frankfurt, Germany
Bilobalide is a neuroprotective constituent of Ginkgo biloba with an unknown mechanism of action. In the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. Microdialysis probes were S88
implanted into the striatum of CD-1 mice, and dialysates were obtained while a Slices of spinal cord and muscle tissue were dissected from mice embryos (E 14-15), monofilament was inserted for 60 min via the common carotid artery (CCA) to block fixed to coverslips and incubated in roller tubes for about 2-4 weeks. Spontaneous perfusion through the middle cerebral artery (MCA). While glucose levels dropped muscle activity was recorded by video microscopic techniques and was quantified immediately upon middle cerebral artery occlusion (MCAO), glutamate concentrations in offline. the microdialysates – as measured with a CMA 600 analyzer – rose extensively during Muscle force production in mice diaphragm preparations was elicited by indirect field ischemia to more than 2000% of baseline level. Both glucose and glutamate levels stimulation technique in a 12 chamber organ bath and quantified as time–force diagrams recovered rapidly when MCAO was terminated after 60 min. When bilobalide (10 µM) (AUC) that were expressed as relative changes of the muscle force compared to the was perfused into the striatum through the microdialysis probe during MCAO, glucose control data. levels dropped but the neurotoxic rise of glutamate was significantly attenuated and Application of the nerve agent VX (0.75 µM) resulted in a strong reduction of muscle reached only 500% of baseline level (p<0.01). activity in the co-culture and of muscle force production in the diaphragm muscle. After In the following experiments, we investigated the activity of mitochondria in ischemic obidoxime (10 µM) was added spontaneous muscle activity in the co culture recovered brain. Ischemia was induced by MCAO, and ischemic as well as “healthy” tissue from from 0.45 ± 0.24 Hz to 1.67 ± 0.24 Hz (control 1.79 + 0.22 Hz). Muscle force remained the opposite hemisphere was obtained. Mitochondria were isolated and mitochondrial stable over the next days. The VX-blocked muscle force of diaphragm was restored to respiration was monitored using the Oroboros® oxygraph. Significant deficits of 69.9 ± 21.3 % by obidoxime compared to control. Muscle force production after indirect respiration were observed after ischemia. In the healthy hemisphere, the respiratory stimulation was stable for 6 hours only. states (leak I+II, complex I+II+IV, oxidative phosphorylation (OXPHOS) and electron Our results suggest that the organotypic nerve-muscle co-cultures may be an transport system (ETS) capacity) showed a decrease of oxygen consumption to 60-70% appropriate tool for the screening of new therapeutic approaches in restoration of of sham-operated mice. In the ischemic hemisphere, several values were lower at 40% blocked neuromuscular transmission. Moreover, the model offers an additional of sham-operated mice (leak I+II, complex II+IV,OXPHOS and ETS) whereas complex I advantage as long-term experiments may be performed and pre- and postsynaptic showed a remarkably low respiratory capacity of 16% of baseline. Direct addition of effects may be assessed directly. Additionally, the number of experimental animals bilobalide (10 µM) to post-ischemic mitochondria caused a 2-fold increase of complex I could be reduced. activity in vitro. Pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before MCAO caused a significant, 3-fold improvement of complex I respiration when measured ex vivo. These data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex I function during ischemia. This action 389 likely explains its neuroprotextive activity in vivo.
Function of the cAMP dependent transcription factor CREM in PDGF induced proliferation of vascular smooth muscle cells 387 Seidl M. D.1, Steingräber A. K.2, Klugstedt C. T.1, Fischer J. W.3, Schmitz W.1, Müller F. U.1 1Westfälische Wilhelms-Universität Institut für Pharmakologie und Toxikologie, Antioxidant Enzyme Expression in Macrophages Exposed to a Dental Resin Domagkstr 12, 48149 Münster, Germany Monomer 2Klinik und Poliklinik für Hautkrankheiten, Universitätsklinikum Münster Kutane Krifka S., Petzel C., Hiller K. - A., Schmalz G., Schweikl H. Vaskuläre Biologie, Von-Esmarch-Str. 58, 48149 Münster, Germany Universitätsklinikum Regensburg Poliklinik für Zahnerhaltung und Parodontologie, 3Universitätsklinikum Düsseldorf Institut für Pharmakologie und Klinische Franz-Josef-Strauss Allee 11, 93042 Regensburg, Germany Pharmakologie, Universitätsstr. 1, 40225 Düsseldorf, Germany
Unreacted resin monomers such as 2-hydroxyethyl methacrylate (HEMA) are The modulation of gene expression by the transcription factor CREM (cAMP responsive- environmental stressors released from dental composites after incomplete element modulator) represents a fundamental mechanism of gene control in response to polymerization. The production of reactive oxygen species (ROS) is a major response of elevation of intracellular cAMP levels. In vascular smooth muscle cells (VSMCs) CREM cells to monomer exposure. Moreover, adverse effects of monomers including delayed is involved in the regulation of cell proliferation and apoptosis supporting its relevance cell differentiation or mineralization processes, DNA damage or apoptosis are for vascular proliferative diseases. Mice with a global inactivation of CREM (CKO) associated with increased ROS production. The intracellular redox homeostasis is showed a significant increase in neointima formation after ligation of the carotid artery controlled by the major non-enzymatic antioxidant glutathione (GSH), and antioxidant and an increase of atherosclerotic plaque formation after high fat diet on an ApoE enzymes. Here, we hypothesized that cells exposed to HEMA responded by a knockout background compared to wildtype controls (WT). On the cellular level a CREM differential expression of antioxidant enzymes such as superoxide dismutase (SOD-1), deficiency was associated with a 1.3 fold increased proliferation rate of primary VSMCs catalase (CAT) or glutathione peroxidase (GPx1/2). RAW246.7 mouse macrophages after stimulation with the platelet-derived growth factor (PDGF; n=10 from 6 isolations). were exposed to HEMA (0-8mM) for 24h, and protein expression was analyzed by Microarray analysis and subsequent realtime-RT-PCR validation revealed that the Western blotting. To study the influence of intracellular GSH on enzyme expression, alpha-type platelet-derived growth factor receptor (Pdgfra) the regulator of G-protein GSH synthesis was reduced by the inhibitor buthionine sulfoximine (50µM BSO), or signaling 5 (Rgs5) and peptidylprolyl isomerase A (Ppia) were 1.5-2.5 fold upregulated enhanced by 2-oxothiazolidine-4-carboxylate (5mM OTC) and N-acetyl cysteine (10mM in PDGF treated CKO VSMCs compared to WT VSMCs (n=6). Transcripts of Rgs5 (2.6 NAC). Expression of SOD-1 found in untreated cultures was decreased in the presence fold, Std. Err.: 0.80-7.58) and Ppia (1.8 fold, Std. Err.: 1.01-3.30) were also upregulated of HEMA and even further reduced by BSO. In contrast, NAC counteracted HEMA- in the carotid artery of CKO mice in comparison to WT mice (n=10-12). induced inhibition of SOD-1 expression. CAT expression was not detected in untreated We conclude that CREM deficiency is associated with transcriptional changes of Rgs5, cells, however, the enhanced expression of CAT in cells exposed to HEMA indicated the Pdgfra, Ppia, which might explain the increased proliferation rate in CKO VSMCs and decomposition of abundant levels of hydrogen peroxide. The minor influence of BSO or the increased responsiveness to pathophysiological conditions. (Supported by the IZKF OTC showed that expression of CAT was independent of GSH levels while a decrease Münster). of HEMA-induced CAT expression in the presence of NAC indicated reduced oxidative stress. GPx1/2 was expressed in untreated cultures, and its down-regulation by BSO indicated that this enzyme was primarily responsible for H2O2 decomposition. The inhibitory effect of HEMA on GPx1/2 expression was enhanced by BSO but 390 counteracted by OTC or NAC. These findings indicate that H2O2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like HEMA. Abundant H2O2 production leads to the activation of CAT expression and a The role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3- feed-back inhibition of SOD-1 expression. The HEMA-induced reduction of GPx1/2 kinase γ by Gβγ and H-Ras expression is most likely a consequence of reduced GSH levels because of the Shymanets A.1, Ahmadian M. R.2, Krause E.3, Wetzker R.4, Harteneck C.1, Nürnberg formation of glutathione disulfide (GSSG) or by GSH-HEMA adducts. 1 Supported by the Deutsche Forschungsgemeinschaft (Schw 431/13-1) B. 1 Institut für Pharmakologie und Toxikologie Abteilung Pharmakologie und Experimentelle Therapie, Wilhelmstraße 56, 72074 Tübingen, Germany 2Institut für Biochemie und Molekularbiologie II, Universitätsstraße 1, 40225 Düsseldorf, Germany 388 3Leibniz-Institut für Molekulare Pharmakologie, Robert-Roessle-Str 10, 13125 Berlin, Germany 4Institut für Molekulare Zellbiologie, Hans-Knöll-Str 2, 07745 Jena, Germany An in vitro co-culture model of nerve- and muscle tissue for the study of new treatment options for organophosphorus compound poisoning Phosphoinositide 3-kinase γ (PI3Kγ) controls a plethora of cellular responses. PI3Kγ, a 1 2 1 1 Seeger T. , Antkowiak B. , Worek F. , Thiermann H. heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is 1Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 regulated by Gβγ and Ras. Earlier we speculated that p101 binds to Gβγ to translocate Munich, Germany cytosolic PI3Kγ to the plasma membrane, enabling direct activation of p110γ (Brock et 2Department of Anaesthesiology University of Tuebingen Experimental Anaesthesiology al., J. Cell Biol. 2003). However, the p87 subunit does not function as Gβγ adapter Section, Schaffhausenstrasse 113, 72072 Tübingen, Germany (Kurig et al., PNAS 2009). Since the impact of each non-catalytic subunit in regulating p110γ by Gβγ and Ras still remains elusive, we studied their role in detail. The life-threatening effects of certain organophosphorus compounds such as soman or Gβ1γ2 variants harbouring mutations in positions involved in interaction with Gα subunit fenamiphos cannot be antagonized adequately by the treatment with atropine and were purified from Sf9 cells and tested for their ability to activate PI3Kγ. We observed oximes. Alternative approaches are necessary. Since the adequate restoration of that p101, but not p87, was able to rescue the stimulatory activity of Gβ1γ2 mutants disturbed muscle function is considered to be life-saving, a model is needed for incapable to activate p110γ (Shymanets et al., Biochem. J. doi:10.1042/BJ20111664). screening of potentially therapeutic substances. An established model for the To further study the functional impact of the non-catalytic subunits on PI3Kγ regulation, development of such new therapies is the diaphragm preparation. However, this model we have designed phospholipid vesicles containing similar amounts of recombinant requires a large number of animals and experimental available time frame is limited to PI3Kγ variants. Although p87/p110γ exhibited stronger sensitivity to Gβ1γ2 than p110γ, some hours. Here, the organotypic nerve-muscle co-culture may be an appropriate the activity of vesicles-associated p101/p110γ was significantly higher as compared to alternative, because a large number of specimens with low numbers of animals and a vesicles-associated p87/p110γ or p110γ in the absence and in the presence of Gβ1γ2. long period of investigation over several days is possible. In the present study, the To study an effect of Ras proteins on PI3Kγ variants, recombinantly expressed H-Ras restoration of VX paralysed muscle function with obidoxime was investigated by using was purified from Sf9 cells. The posttranslational processing and lipidation of the protein both models. was verified by mass spectrometry analysis. The impact of H-Ras on regulation of S89
p87/p110γ and p101/p110γ differed, which may explain integration of PI3Kγ variants in shortening of the snout, we studied the effect of PMT on bone cells. Here we studied the different signalling pathways. Taken together, p87 and p101 subunits implement discrete effect of the toxin on osteoblast differentiation in ST-2 cells and in primary osteoblasts functions in respect to (i) membrane recruitment of PI3Kγ and (ii) regulation of enzymatic from rat calvaria. ST-2 cells are stromal derived cells, which can be differentiated into activity by Gβγ and H-Ras. osteoblasts or adipocytes. The toxin inhibits the differentiation of ST-2 cells into osteoblasts studied by determination of specific osteoblast markers. Additionally, PMT represses the induction of transcription factors essential for osteoblast differentiation. Moreover, the principal pathways activated by PMT to induce these effects were 391 investigated.
Preparation of consolidated exposure scenarios for mixtures under REACh Sica M., Dorn S., Mostert V. 394 DR. KNOELL CONSULT GmbH, Marie-Curie-Str. 8, 51377 Leverkusen, Germany
Under REACh, formulators of mixtures need to include substance-related information Ventilation induces differing pro-inflammatory responses in two different mouse into extended safety data sheets (eSDS), if mixtures are classified as dangerous strains according to the Dangerous Preparation Directive (Directive 1999/45/EC). One way to Siegl S., Uhlig U., Uhlig S. add information on substances into eSDS of mixtures is to generate exposure scenarios University Hospital Aachen Institut für Pharmacologie und Toxikologie, Wendlingweg 2, (ESs) for mixtures. 52074 Aachen, Germany In order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: The critical Introduction component approach (CCA) relies on DNELs and PNECs for all substances, their Ventilator-induced lung injury (VILI) is a serious problem in intensive care medicine. Its concentrations in the mixtures as well as substance and use-specific availability mechanisms are only incompletely understood, although it is widely accepted that parameters (ECHA, 2008). In contrast, the DPD+ method is based on the legislation for ventilation-induced inflammation (biotrauma) makes an important contribution. The classification of preparations (Directive 1999/45/EC). The DPD+ method defines a lead isolated perfused mouse lung (IPL) is a valuable tool to investigate the mechanisms of substance for each route of human exposure and for the aquatic environment (CEFIC, VILI. Several studies have shown considerable differences between various mouse 2010). However, each of these methods has certain limitations. strains with respect to lung mechanics and inflammatory responses. Therefore, we The aim of the present work is to improve the preparation of consolidated ESs for a hypothesized that the pulmonary responses to mechanical ventilation differ between number of mixtures and provide information about their safe use. To this end, we first C57Bl/6 and BALB/c mice. In addition, this study introduces the novel half lung adopted information on risk management measures (RMMs) and operational conditions technique that allows to obtain lung tissue from the same mouse at two different time (OCs) of the lead substances using the DPD+ methodology. points. At the same time, we considered the specific conditions of use of the mixtures (e.g. Methods spraying, brush painting). We then conducted risk assessments by deriving DNELs for Isolated perfused mouse lungs from C57Bl/6 or BALB/c were subjected to high the mixtures and using exposure modelling tools recommended under REACh (e.g., (25cmH2O) or low pressure (8cmH2O) ventilation for 240 minutes. After 180 minutes the ECETOC TRA, RISKOFDERM, ART). We compared the outcome of these assessments left lung was removed and used for Western Blot analysis. The right lung was ventilated with results obtained from the application of the DPD+ methodology. for another 60 minutes. By the end of experiment the right lung was removed and qRT- The work presents the results of application of DPD+ approach and the CCA and PCR performed. During the whole experiment perfusate sample were taken from the indicates possible improvements for the risk assessment of mixtures. venous catheter and used for protein quantification by ELISA. Results References It was possible to remove half of the lung and to further ventilate the other half without ECHA, 2008. Guidance for downstream users. acute changes in lung mechanics. In both strains high pressure ventilation elicited a CEFIC, 2010. REACh Practical Guide on Exposure Assessment and Communication in significantly higher cytokine release than low pressure ventilation. C57Bl/6 mice showed the Supply Chains. Part III: Mixtures under REACH. higher TNF, IL-1β and amphiregulin levels after high pressure ventilation, whereas BALB/c exhibited increased production of CXC chemokines (Cxcl-1, Cxcl-2) and IL-6. Kinase activities (JNK, Akt, ERK1/2, p38 MAP kinase) were increased in high pressure ventilated animals, but were strain independent. 392 Conclusion: The novel half lung technique builds on the well established IPL method. It permits to separately analyze the left and the right lung at different time points during continual Seasonal influences in the prescription rate of drugs for cardiovascular and ventilation. This method reduces animal numbers by 50% and allows statistical within respiratory disease (ATC codes C and R). subject analysis. Using this method, the present study showed that inflammatory response to mechanical ventilation differ between C57Bl/6 and BALB/c. These findings Siegert J., Brecht B., Böhme A., Tuttas K., Schindler C., Kirch W. show that the biotrauma response in mice depends on the strain that is studied. TU- Dresden Institut fuer Klinische Pharmakologie, Fiedlerstrasse 27, 01307 Dresden, Germany
To check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (ATC codes C and R) a 395 survey covering all prescriptions of a specimen German general regional health insurance (AOK plus, data for Saxony, largest health insurance service, approx. 50 % of all Saxonian citizens are inscribed to this service) for the years 2005 to 2007 was Effects of Functionalized Polystyrene Nanoparticles on Human Macrophages analysed on a monthly basis. Haas K., Loos C., Lunov O., Hafner S., Syrovets T., Simmet T. The number of prescriptions for cardiovascular drugs changed approximately +/- 20 % Ulm University Institute of Pharmacology of Natural Products & Clinical Pharmacology, around the mean for the different month without a clear seasonal pattern. For respiratory Helmholtzstr. 20, 89081 Ulm, Germany drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 – 70 % above average prescription figure Macrophages play an important role as an integral part of the first line of immune during spring time (February to May) and a 25 to 40 % trough in late summer/autumn defense. Two different macrophage populations have been described. M1 macrophages (July to October). produce proinflammatory cytokines and are involved in inflammatory processes. By The data have to be analysed for further cofounders (e.g. influenza prevalence, contrast, M2 macrophages release anti-inflammatory cytokines and extracellular matrix environmental conditions etc.) to fully understand the fluctuations observed. The components. They can enhance wound repair and angiogenesis, but they can also prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced promote tumor progression. Recently, industrial nanoparticles have raised concerns by multiple factors aside seasonal influences. because of their putative toxic effects. On the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. Thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. Macrophages as phagocytosing 393 cells scavenge nanoparticles circulating in the bloodstream. Therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. Monocytes were isolated from buffy coats and differentiated to Pasteurella multocida toxin prevents osteoblast differentiation by activation of macrophages with macrophage colony-stimulating factor. Carboxy- (PS-COOH) and heterotrimeric G proteins amino- (PS-NH2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity Siegert P., Aktories K., Orth J. index and their zeta potential were determined by dynamic light scattering. The Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Abt. I, Albertstr. macrophages were cultured in the presence or absence of different concentrations of 25, 79104 Freiburg i. Br., Germany PS-COOH and PS-NH2 nanoparticles for up to 6 days. Analysis of cell viability revealed that PS-NH2 but not PS-COOH concentration- and time-dependently reduced the Pasteurella multocida toxin (PMT) is a major virulence factor of Pasteurella multocida macrophage viability. By annexin V/propidium iodide double staining we could show that causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. PS-NH2 trigger apoptosis in macrophages. We further polarized macrophages to either The toxin modulates various signaling pathways by acting on the heterotrimeric G M1 or M2 using IFN-γ and LPS or IL-4, respectively. These macrophage populations proteins Gαq, Gα12/13 and Gαi. PMT activates Gq to increase inositol phosphate were characterized by their expression of extracellular markers by flow cytometry and production via phospholipase Cβ and alteration of gene expression via the JAK/STAT their production of cytokines by ELISA. The effects of functionalized polysterene pathway. The toxin also activates RhoA via Gαq and Gα12/13 family proteins. We showed nanoparticles on the cytokine production and surface marker expression of M1 and M2 that the underlying mechanism of the activation of heterotrimeric G proteins is a macrophages were analyzed. Our data indicate that surface functionalization is a critical deamidation of an essential glutamine residue leading to a constitutive activation of the parameter in the nanoparticle-induced toxicity in human macrophages. G protein. This work was supported by the DFG SPP1313. Because PMT is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or S90
396 analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. Using flow cytometry, Western immunoblotting and fluorescent microscopy, we show that endothelial cells of different Differential Uptake of Functionalized Polystyrene Nanoparticles by Human origin express the plasmin receptor complex composed of annexin A2 and S100A10. Macrophages and Monocytic Cells Addition of plasmin to human umbilical vein endothelial cells (HUVEC) induced time- and concentration-dependent cytotoxic effects in the cells. In addition, within 30 min Loos C., Lunov O., Syrovets T., Simmet T. plasmin triggered a rapid and prolonged expression of free radical oxygen species Ulm University Institute of Pharmacology of Natural Products & Clinical Pharmacology, (ROS) in endothelial cells as analyzed by microscopy and fluorometry using the ROS- Helmholtzstr. 20, 89081 Ulm, Germany sensitive dye carboxy-H2DCFDA. The ROS production in endothelial cells was accompanied by cell detachment. Fluorometric and Western blot analysis of caspase 3 Nanoparticles are currently used for various medical applications including imaging, activation in the cells treated with plasmin showed that plasmin induced apoptotic cell diagnosis and drug delivery. Due to particle size and surface area, their fundamental death in endothelial cells, which was evident already several hours after exposure to properties differ significantly from those of corresponding bulk materials. Nanoparticles plasmin. Thus, plasmin might induce production of ROS in endothelial cells, their circulating in the blood are mainly sequestrated by the reticuloendothelial system that detachment and apoptosis, events which might be relevant for the development of consists predominantly of phagocytic macrophages. Macrophages express a variety of atherosclerosis. cellular receptors for sensing and internalizing particular material like viruses, This work was supported by the DFG. microorganisms, and foreign particulate matter including nanoparticles. Therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. Introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. 399 Tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. Here we show that carboxy- (PS-COOH) and amino- Molecular Interactions of NF-κB Proteins with the Sesquiterpene Lactone functionalized (PS-NH2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, THP-1 monocytic leukemia cells, and by PMA- Helenalin differentiated THP-1 cells via different mechanisms. In buffer, macrophages and THP-1 Syrovets T., Büchele B., Lunov O., Simmet T. rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles Ulm University Institute of Pharmacology of Natural Products & Clinical Pharmacology, were taken up to a higher extent. The uptake of both nanoparticles was drastically Helmholtzstr. 20, 89081 Ulm, Germany reduced in media containing serum. Using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between CD64 Sesquiterpene lactones (STL) comprise a large group of secondary plant metabolites receptors and the particles determines the macrophage uptake of particles by that constitute the active principle of a number of traditional anti-inflammatory phagocytosis, whereas particle internalization by THP-1 cells occurred via dynamin II- phytomedicines. Specifically helenalin and parthenolide have recently gained dependent endocytosis. By contrast, PMA-differentiated THP-1 cells took up the considerable attention as lead compounds or putative therapeutics for the treatment of particles via macropinocytosis. In line with the in vitro data, more intravenously applied inflammation and possibly cancer. Both compounds have been shown to interfere with PS-COOH particles accumulated in liver tissue, whereas PS-NH2 were preferentially the signal transduction through inhibition of the nuclear factor κB (NF-κB). Whereas the targeted to tumor tissue. These data show that the amount of particle internalization, the inhibitory effects of the STL on NF-κB are undisputed, their molecular mechanism of uptake mechanisms, and kinetics differ significantly among primary cells and model action remains a matter of debate. Surface plasmon resonance (SPR) analysis allows tumor cells, whether differentiated or not, and that they are further critically dependent label-free measurement of molecular interactions. Yet, analysis of the interaction of on the particle opsonisation by serum proteins. immobilized recombinant proteins with small molecular ligands remains a technically This work was supported by the DFG SPP1313. challenging task. In the present study we used SPR technology to investigate the molecular interaction of the STL helenalin with putative intracellular target proteins such as the NF-κB protein p65/RelA, the catalytic subunits of the IKK complex, namely IKKα and IKKβ, and the intracellular antioxidant glutathione (GSH). At physiological pH 7.4, 397 helenalin interacts with RelA (KD = 4.8 µM), yet it failed to bind either IKKα or IKKβ. Hence, when DNA with NF-κB binding consensus sequence was immobilized on sensor chips, the binding of RelA was inhibited by helenalin with an IC50 of 5.0 µM. Moreover, Functionalized Polystyrene Nanoparticles Activate the NLRP3 Inflammasome in we provided several lines of evidence that STL may modify RelA on cysteine 38 by a Human Macrophages Michael-type addition. This interaction was confirmed by molecular docking that identified the best matching interaction between RelA and helenalin with predicted Lunov O., Syrovets T., Loos C., Simmet T. hydrogen bonding interactions between helenalin and residues Arg35, Lys37, Gly44 and Ulm University Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ile118 of RelA. Consistent with our hypothesis that helenalin interacts with sulfhydryl Helmholtzstr. 20, 89081 Ulm, Germany groups at pH 8.0, helenalin was also able to interact with reduced, but not oxidized,
glutathione with a KD of 24 µM, though no significant interaction was observed at pH 7.4. Specifically designed and functionalized nanoparticles hold great promise for a variety of Thus, we showed that the sesquiterpene lactone helenalin interacts with the NF-κB biomedical applications. To ensure their safe application, such particles require a protein RelA but not with IKKα or IKKβ. Moreover, at physiological pH, helenalin does rigorous analysis of their effects on cell functions. Here we demonstrate that amino- not interact with glutathione to any significant extent. Direct interaction of helenalin with functionalized polystyrene nanoparticles (PS-NH2) of ~100 nm in diameter in contrast to RelA leading to inhibition of RelA-DNA binding and transactivation might present the carboxy- (PS-COOH) and nonfunctionalized (PS) particles induce an NLRP3 molecular mechanisms underlying the anti-inflammatory effects of STLs. inflammasome activation and the subsequent release of IL-1β in human macrophages. Amino-functionalized PS nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. This resulted in mitochondrial damage and formation of reactive oxygen species. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing 400 a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein resulting in a conformational change of the pyrin Modeling Receptor-Mediated Uptake of Polymer-Functionalized Iron Oxide domain of the NLRP3 protein as predicted by molecular modeling. TXNIP interaction Nanoparticles by Macrophages 1 2 1 1 with NLRP3 led to assembly of the NLRP3 inflammasome complex, to caspase-1 Lunov O. , Zablotskii V. , Syrovets T. , Simmet T. activation, and release of IL-1β. Using an in vitro knockdown approach, we showed that 1Ulm University Institute of Pharmacology of Natural Products & Clinical Pharmacology, PS-NH2 induced activation exclusively of NLRP3, whereas other inflammasomes Helmholtzstr. 20, 89081 Ulm, Germany remained unaffected. Treatment of macrophages with N-acetyl-L-cysteine, a scavenger 2ASCR, Institute of Physics, 18221 Prague, Czech Republic of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of IL-1β caused by PS-NH2 nanoparticles. These data reveal a Although nanosized materials are quickly taken up by macrophages, our understanding novel mechanism of the NLRP3 activation induced by amino-functionalized of the involved processes is still rather limited. Therefore, we analyzed the uptake of nanoparticles and provide a strategy as to how such an effect can be functionally diagnostically used carboxydextran-coated iron oxide nanoparticles of two different antagonized by supplementation with a radical scavenger. sizes, superparamagnetic iron oxide nanoparticles of 60 nm (SPIO) and ultrasmall This work was supported by the DFG SPP1313. superparamagnetic iron oxide nanoparticles of 20 nm (USPIO), by human macrophages. By pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor A-dependent endocytosis. Further, we created a mathematical 398 model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of Effects of Plasmin on Endothelial Cells internalized particles and their extracellular concentration. The model also provided information on the individual and collective wrapping time of the nanoparticles and Maheswaran V., Schmidt Z., Koshova O., Lunov O., Syrovets T., Simmet T. described the relation between biophysical parameters such as cytoskeletal forces, Ulm University Institute of Pharmacology of Natural Products & Clinical Pharmacology, membrane elasticity, and the uptake time. Finally, we gained information on the minimal Helmholtzstr. 20, 89081 Ulm, Germany linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. The calculated The semi-permeable barrier of the endothelial cell lining of the blood vessels has parameters were further confirmed experimentally using spinning disc confocal important synthetic and metabolic functions including transport of cells and microscopy. Thus, the new model provides important insights into the biophysical biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. processes involved in endocytosis of nanoparticles by human macrophages. Plasmin is a serine protease, which is generated from its zymogen plasminogen under This work was supported by the DFG SPP1313. physiological and pathological conditions. Small amounts of plasmin are produced in the context of contact activation during inflammation. Consistently, increased generation of plasmin has been reported during atherosclerosis. We have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. Therefore, we S91
401 counteracting effects of brain-derived neurotrophic factor (BDNF) and calpeptin in these systems. To create the hexagonal array we used a Poly(dimethylsulfoxide) bilayer stencil Selective Poisoning of Ctnnb1-Mutated Mouse Liver Tumors by Single Application comprising through holes for adhesion spots and interconnecting tracks. Plasma of Acetaminophen stencilling a PEG-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. Singh Y., Schwarz M., Braeuning A. Results: In both systems, the developing and mature network, we found not only a Institut für experimentelle und klinische Pharmakologie und Toxikologie Abteilung concentration dependant effect of ACR and BDNF but also a time dependant effect with Toxikologie, Wilhelmstraße 56, 72074 Tübingen, Germany a limited capability of the developing system to regenerate, even in the presence of ACR. The co-treatment of the cells showed that inhibition of calpains by calpeptin might Deregulation of the β-catenin signaling pathway plays an important role in the reduce the effect of intracellular elevated Ca2+, a known neurotoxic mechanism of ACR. development of hepatocellular tumors. Activating mutations in Ctnnb1 (encoding β- Moreover, the neurothrophin BDNF acts via TrkB receptors on pathways stimulating Catenin) are frequently observed in murine and human liver tumors (e.g. human neurite outgrowth and thereby counteracting the adverse effect of ACR. hepatoblastomas). Activation of β-Catenin signaling induces an overexpression of Conclusion: With the NFA we provide a rapid and simple way to analyze neurite several cytochrome P450 (CYP) enzymes, including CYP2E1. outgrowth and connection formation in real time. By spatially standardizing the array we Cytotoxicity of acetaminophen (AAP) is based on its CYP2E1-catalyzed metabolism to provide assay coordinates to streamline the analysis process and bring it towards high the electrophilic compound N-acetyl-p-benzo-quinone imine, which forms covalent throughput testing. Furthermore preliminary data showed that modification of the surface adducts with cellular macromolecules if depletion of glutathione occurs. Treatment with with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse AAP should therefore lead to a selective damage of CYP2E1-overexpressing Ctnnb1- neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via mutated hepatoma cells. integrins. Mice were injected with a single dose of the liver carcinogen N-Nitrosodiethylamine (DEN) and subsequently treated with the tumor promoter phenobarbital to select for Ctnnb1-mutated tumors. Administration of a single dose of AAP (300 mg/kg of body weight) followed the tumor promotion protocol. Two days after treatment immunohistological analysis of the livers showed about 90% 404 necrotic tissue in the larger tumors which were positive for glutamine synthetase (GS), a marker for Ctnnb1-mutated tumor cells. By contrast, GS-negative tumors remained unaffected. At later time points we observed regeneration processes with infiltration of Transcriptional regulation of Nox4 by histone deacetylases 1,2 3 4 1 1 1 1 the necrotic tissue by inflammatory cells followed by fibrotic cells. Proliferation of normal Siuda D. , Zechner U. , Prawitt D. , Xia N. , Kleinert H. , Förstermann U. , Li H. hepatocytes surrounding the damaged areas could also be observed. However, 1Universitätsmedizin der Johannes Gutenberg-Universität Institut für Pharmakologie, repopulation of parts of the former tumor areas by remaining GS-positive tumor cells Obere Zahlbacher Str. 67, 55131 Mainz, Germany was also detected. 2Universitätsmedizin der Johannes Gutenberg-Universität Centrum für Thrombose und These results suggest that treatment with AAP might serve as a future therapeutic Hämostase (CTH), Langenbeckstraße 1, 55131 Mainz, Germany possibility to selectively poison CYP2E1-overexpressing hepatoma. 3Universitätsmedizin der Johannes Gutenberg-Universität Institut für Humangenetik, Langenbeckstraße 1, 55131 Mainz, Germany 4Iniversitätsmedizin der Johannes Gutenberg-Universität Molekulargenetisches Labor der Kinderklinik, Langenbeckstraße 1, 55131 Mainz, Germany 402 Nox4 is a member of the NADPH oxidase family, which represents a major source of reactive oxygen species (ROS) in the vascular wall. Nox4-mediated ROS production Release of 5,6-Epoxyeicosatrienoic acid (5,6-EET) upon neuronal activity induces mainly depends on the expression levels of the enzyme. The present study is aimed to TRPA1-dependent mechanical pain hypersensitivity investigate the regulation mechanisms of Nox4 transcription by histone deacetylase 1 2 1 1 3 1 1 (HDAC). In cultured human EA.hy 926 endothelial cells, treatment with the pan-HDAC Sisignano M. , Park C. - K. , Angioni C. , Zhang D. , Grant A. , Lu R. , Schmidtko A. , inhibitors (scriptaid, trichostatin A, TSA, and suberoylanilide hydroxamic acid, SAHA) Ji R. - R.2, Woolf C.4, Geisslinger G.1, Scholich K.1, Brenneis C.1,4 1 leads to a drastic decrease in Nox4 mRNA expression. A similar down-regulation of Klinikum der Goethe-Universität Pharmazentrum Frankfurt/ZAFES, Institut für Klinische Nox4 mRNA expression can be achieved with siRNA-mediated knockdown of HDAC3. Pharmakologie, Theodor Stern Kai 7, 60590 Frankfurt, Germany 2 HDAC inhibition in endothelial cells is associated with enhanced histone acetylation in Brigham and Women's Hospital, Harvard Medical School Department of the human Nox4 promoter region, with no significant changes in DNA methylation. Anesthesiology, Sensory Plasticity Laboratory, Pain Research Center, Boston, Consistently, scriptaid-treated cells show increased chromatin accessibility in Nox4 Massachussets United States promoter. In addition, we provide evidence that c-jun plays an important role in 3King's College London Wolfson Centre for Age Related Disease, London, Great Britain 4 controlling Nox4 transcription. Knockdown of c-jun with siRNA leads to a marked down- Harvard Medical School, Children's Hospital F. M. Kirby Neurobiology Center, regulation of Nox4 mRNA expression. In response to scriptaid treatment, the binding to Department of Neurobiology, Boston, Massachussets United States c-jun to the Nox4 promoter region is reduced despite the open chromatin structure. In parallel, the binding of polymerase IIa to the Nox4 promoter is significantly inhibited as Epoxyeicosatrienoic acids (EETs) are CYP-epoxygenase (CYP450) derived metabolites well, which may explain the reduction in Nox4 transcription. In conclusion, HDAC of arachidonic acid (AA) which act as endogenous signaling molecules in multiple inhibition decreases Nox4 transcription in human endothelial cells by preventing the biological systems. We investigated the specific contribution of 5,6-EET to Transient- binding of transcription factor(s) and polymerase(s) to the Nox4 promoter. This is very Receptor potential-(TRP)-channel activation in nociceptor neurons, and its consequence likely because of a hyperacetylation-mediated steric inhibition. for nociceptive processing. We found that during capsaicin-induced nociception 5,6- EET-levels increased in the DRG and it is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux [10 nM] in cultured DRG-neurons which was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices 5,6-EET dose-dependently enhanced the frequency, but not the amplitude of 405 spontaneous excitatory postsynaptic currents (sEPSC) in lamina II neurons that also respond to mustard oil (AITC), indicating a presynaptic mechanism. Furthermore, 5,6- EET-induced enhancement of sEPSC frequency was abolished in TRPA1 null mice, Cyclopentenone prostaglandins induce oxidative DNA-damage in hamster lung suggesting that 5,6-EET pre-synaptically facilitates spinal cord synaptic transmission via fibroblast V79 cells 1,2 3 2 TRPA1. Finally, intrathecal injection of 5,6-EET caused mechanical hyperagesia in wild Solecki G. M. , Schrenk D. , Esselen M. type but not TRPA1 null mice. We conclude that 5,6-EET is synthesized upon acute 1Universität Wien Institut für Lebensmittelchemie und Toxikologie, Währinger Straße 40, activation of nociceptors and leads to mechanical hypersensitivity via TRPA1 at central 1090 Wien, Austria afferent terminals in the spinal cord. 2Technische Universität Kaiserslautern Fachrichtung Lebensmittelchemie und Toxikologie, Erwin-Schrödinger-Straße Gebäude 56, 67663 Kaiserslautern, Germany 3Technische Universität Kaiserslautern Fachrichtung Lebensmittelchemie und Toxikologie, Erwin-Schrödinger-Straße Gebäude 52, 67663 Kaiserslautern, Germany 403 Introduction Prostaglandins (PG) are hormones which are formed during inflammatory processes Micropatterned neuronal cells – a new tool for neurotoxicity testing and drug from arachidonic acid by cyclooxygenases and prostaglandin synthases [4]. In the discovery in vitro subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2,3]. These come along with mercapto groups of Sisnaiske J.1, Hardelauf H.2, Frimat J. - P.3, Waide S.2, Schöbel N.1, Hausherr V.1, 2 2 1 2 1 amino acids in a Michael addition reaction associated with activation of cellular enzyme Baumann J. , Dinh N. - D. , Hengstler J. G. , West J. , van Thriel C. cascades [1] that potentially contribute to their possessed antiinflammatory, 1IfADo - Leibniz-Institut für Arbeitsforschung, Ardeystr. 67, 44139 Dortmund, Germany 2 antineoplastic and antiviral effects [5]. However little is known so far about possible Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 adverse health effects.We addressed the question whether selected cyclopentenone Dortmund, Germany 3 prostaglandins (cyPG) exhibit potential mutagenic and genotoxic properties in the University of Twente, Enschede, Netherlands hamster lung fibroblast cell line V79.
Introduction: Neurite outgrowth and plasticity of neuronal networks are essential Methods processes e.g. during brain development and learning. Thus, morphological readouts of Induction of DNA damage was investigated by single cell gel electrophoresis assay neuronal connectivity are thought to be important endpoints to assess neurotoxic effects (SCGE). The impact of cyPG on cellular redox status was detected by total glutathione of environmental chemicals as well as when discovering new drugs. (tGSH) assay. The induction of micronuclei and apoptosis was determined by staining To analyze neurite outgrowth and connectivity level rapidly and easily in vitro we with 4’,6-diamidino-2-phenylindole (DAPI). Furthermore the hypo-xanthine-guanine developed the Network Formation Assay (NFA) (PCT/EP2010/002811). This platform phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. Results and Summary Methods: To demonstrate the feasibility of the NFA we performed experiments in which 12,14 15-deoxy-∆ -prostaglandin J2 (15dPGJ2), followed by prostaglandin A2 (PGA2), we disrupted mature neurite networks or inhibited generating networks of human SH- showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic SY5Y cells with different concentrations of acrylamide (ACR). We also observed the S92
effects. Furthermore, the 15dPGJ2 and PGA2 –induced significant decrease in the tGSH 408 level in V79 may contribute to the observed increase in oxidative DNA-damage. However, none of the tested cyPG exhibited mutagenic properties in the hprt assay. In conclusion, a potential in vitro genotoxicity of cyPG has been observed which may be Is formaldehyde a good example for a “genotoxic carcinogen” with a threshold involved in carcinogenesis associated with chronic inflammation. mode of action?
References Speit G. [1] Díez-Dacal B and Pérez-Sala D (2010).Sci World J; 13: 655-675. Institut für Humangenetik, Universität Ulm, 89069 Ulm, Germany [2] Fitzpatrick FA and Wynalda MA (1983). J Biol Chem; 258: 11713-11718. [3] Lee JB, Crowshaw K, Takman BH, Attrep KA (1967). Biochem J; 105: 1251-1260. Formaldehyde (FA) induces toxic and genotoxic effects in directly exposed cells (site of [4] Smith WL, Marnett LJ, DeWitt DL (1991). Pharmacol Ther; 49:153-179. contact). Several studies in which FA was administered to rats by inhalation showed [5] Straus DS and Glass CK (2001). Med Res Rev; 21: 185-210. evidence of tumor induction in nasal epithelium. There is also some epidemiological evidence that FA causes nasopharyngeal cancer in humans. Although FA is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. There is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. However, a decisive role of 406 mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. The basic assumption is that mutagens have a non-threshold mode of action. A threshold mode of action for a chemical is likely when a substance with a Assessment of the Sensitizing and Irritative Potential of Preservatives by Loose-fit known mutagenic potential does not induce mutations at low concentrations due to a Coculture-based Sensitization Assay (LCSA) specific type of reaction with the genetic material and / or physiological protective Sonnenburg A. mechanisms. Because FA is a directly acting DNA-reactive substance, a threshold mode Charité Universitätsmedizin Berlin Institut für Klinische Pharmakologie und Toxikologie, of action may only be considered because of physiological protective mechanisms. After Luisenstr. 7, 10117 Berlin, Germany inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. Furthermore, FA is efficiently inactivated by enzymatic pathways. If FA Parabens and methylisothiazolinone are used as preservatives in personal care reaches and damages the nuclear DNA, DNA repair mechanisms act as efficient post- products. Sensitization to parabens and methylisothiazolinone is relatively rare lesion protection mechanisms. In vivo inhalation studies with rats indicated that FA considering their wide use in cosmetics, but only few quantitative or clinical data exist. induces primary DNA damage in the nasal epithelium but increased mutation Therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, frequencies were not measured. Data are now available to show the relative distribution phenyl- and benzylparaben, and methylisothiazolinone in the loose-fit coculture-based of endogenous versus exogenous DNA adducts in different locations of the nose and sensitization assay (LCSA) developed by our working group. The coculture of primary other organs. Considering the FA concentrations present in every living cell and the human keratinocytes and allogenic dendritic cell-related cells (DC-rc) in this assay background levels of endogenous DNA adducts, appropriate risk assessment and the emulates the in vivo situation of the human skin. Sensitization potential of the test identification of practical thresholds for FA-induced genotoxicity become feasible. substances was determined by flow cytometric analysis of the DC-rc maturation marker CD86. Determination of the concentration required to cause a half-maximal increase in CD86-expression (EC50) allowed a quantitative evaluation. The irritative potential of the substances was assessed by 7-AAD (7-amino-actinomycin D)-staining. The 409 concentration required to devitalize 50 % of the examined cells compared to a zero control was termed EC50%. Parabens exhibited weak (methyl-, ethyl-, propyl- and isopropylparaben) or strong (butyl-, isobutyl-, pentyl- and benzylparaben) sensitizing Fraud and misconduct in clinical trials potential, phenylparaben was found to be a moderate sensitizer, with EC -values 50 Steffen C. ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). Due to a formerly Federal Institute for Drugs and Medical Devices Clinical Trials Unit, Kurt-Georg- pronounced cytotoxicity (EC50% = 70.94 µmol/l), we could not estimate an EC50-value for methylisothiazolinone. Sensitization potential of parabens correlated with side chain Kiesinger-Allee 3, 53175 Bonn, Germany length. Parabens showed no (methyl- and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl- and benzylparaben), only pentylparaben Plagiarism in scientific publications has been the subject of public ("Guttenplag") and was rated to be irritative. Apart from phenyl- and benzylparaben, irritative potential also scientific debate. Plagiarism violates, however, "only" the intellectual property of its correlated with side chain length but did not correlate strictly with the sensitization authors. In clinical trials, misconduct may also endager the health of patients. potential. Overall, we were able to demonstrate and compare the sensitizing potential of The suppression or falsification of data in clinical trials may mislead patients and doctors parabens in this in vitro test. It was weak for methyl- and propylparaben, the most to use worthless treatments. Clinicians may be provoked to repeat these trials, thereby commonly used parabens. Furthermore, we showed an irritative potential for most of the wasting time and money. perservatives. Thus the LCSA is a useful in vitro test to compare the sensitizing potential In clinical trials, misconduct includes everything from suppression or their repeated of xenobiotics. publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. The peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. They will have to rely on these data, the calculations and 407 statistics that are presented to them. Some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. Other kind of fraud, as the suppression of patient data in Phosphorylation of the neurodegeneration-related Septin 4 by protein kinase clinical trials (the deletion of "non-responders", according to Cinderella: The good ones DYRK1A at serine 107 affects protein stability go into the pot, the bad ones go into your crop) can only be detected by the National 1 2 1 Soppa U. , Tejedor F. J. , Becker W. Competent Authorities performing an inspection according to Good Clinical Practice. 1Institute of Pharmacology and Toxicology, Medical Faculty of RWTH Aachen University, Even if the results of such an inspection become public as in the case of Ukrain Wendlingweg 2, 52074 Aachen, Germany (Gansauge et al. 2002), there is no institution that will further analyze and publish the 2Instituto de Neurosciencias, Consejo Superior de Investigaciones Cientificas (CSIC) y misconduct or initiate the retraction of the incriminated paper. A German agency similar Universidad Miguel Hernandez, 03550 Alicante, Spain to the US Office of Scientific Integrity could foster Good Clinical Practice in Germany. Although it is sometimes very difficult to decide whether improper results arise from Septins are GTP-binding proteins forming heterooligomeric complexes and filaments by fraud or error, a bias for the source of funding is obvious. Trials funded by for-profit interactions of the 13 family members. These complexes have important functions by organizations were significantly more likely to recommend the experimental drug as building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular treatment of choice (Als-Nielsen et al. 2003). Medicinal products with disputed efficacy distribution as well as their regulation remain largely unclear. Septin 4 (SEPT4) was such as orally applied enzymes for systemic action, bacterial preparations for irritable found in neurodegeneration related protein aggregates and is associated with migration bowel syndrome and food supplements are to be reviewed with special attention. of cortical neurons. Here we first describe a potential mechanism for regulation of Further examples from the author’s experience will be presented. SEPT4 by phosphorylation via Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). DYRK1A is overexpressed in Down syndrome and supposed to be References involved in neurodevelopment and neurodegeneration. By site directed mutagenesis of Gansauge F, Ramadani M, Pressmar J, Gansauge S, Muehling B, Stecker K, Cammerer Flag tagged mouse SEPT4 and overexpression in HeLa cells we identified serine 107 as G, Leder G, Beger HG (2002) NSC-631570 (Ukrain) in the palliative treatment of the major phosphorylation site of DYRK1A and generated a phosphospecific antibody. pancreatic cancer. Results of a phase II trial. Langenbecks Arch Surg. 386(8):570-4 Transient coexpression of SEPT4 and DYRK1A in HeLa cells increased phosphorylation Als-Nielsen B, Chen W, Gluud C, Kjaergard LL (2003) Association of funding and of serine 107 by 50% in relation to basal phosphorylation. In contrast, cotransfection of conclusions in randomized drug trials: a reflection of treatment effect or adverse events? the kinase deficient DYRK1A mutants K188R and D287N did not increase serine 107 JAMA 290(7):921-8. phosphorylation. Moreover we could show, that inhibition of kinase activity by the DYRK1A inhibitor harmine reduced phosphorylation of exogenous SEPT4 at serine 107 about 25% in HeLa cells. Furthermore, down regulation of DYRK1A by RNA interference lead to decreased phosphorylated serine 107. These results indicate that endogenous DYRK1A contributes to SEPT4 phosphorylation in HeLa cells. Finally we analyzed protein stability of wild type SEPT4 compared to the phosphorylation resistant S107A mutant in HeLa cells by inhibition of translation with cycloheximide. We found that in living cells the SEPT4 S107A mutant is more stable than wild type SEPT4. In summary, our results suggest phosphorylation at serine 107 by DYRK1A as a novel mechanism to regulate SEPT4 stability and indicate a possible link of these proteins in cellular processes.
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2+ + The Ca -activated K channel (KCa) of intermediate conductance (SK4) plays a TRPC1 suppresses the calcium permeability thereby affecting neuronal migration. major role for cell cycle progression and proliferation of breast cancer cells Storch U.1, Forst A. - L.1, Philipp M.2, Gudermann T.1, Mederos Y Schnitzler M.1 Steinle M., Ripplinger A., Ruth P., Lukowski R. 1Ludwig-Maximilians-Universität München Walther-Straub-Institut für Pharmakologie und Uni Tübingen Pharmakologie, Toxikologie und Klinische Pharmazie, Auf der Toxikologie, Goethestr. 33, 80336 München, Germany Morgenstelle 8, 72076 Tübingen, Germany 2Philipps-Universität Marburg Pharmakologisches Institut, Karl-von-Frisch-Str. 1, 35043 Marburg, Germany Background: KCa are expressed in multiple human tumors. In non excitable tumor cells + 2+ the opening of KCa causes a K efflux which in turn increases the driving force for Ca The classical transient receptor potential (TRPC) channel subfamily is regarded as non- 2+ entry needed for cell cycle progression. Since elevated [Ca ]i favors further KCa selective, calcium permeable cation channels involved in a wide range of physiological 2+ opening, KCa may establish a feed-forward regulation of Ca influx. In the present study events that require calcium signaling. Until now, the specific roles of TRPC channels in 2+ we analyzed whether KCa channels of SK4 and BK type play a role in sustaining Ca neuronal function are still elusive. Given that TRPC1 is able to form receptor-operated oscillations at G1- to S-phase transition of primary mouse tumor cells and in human heterotetrameric channel complexes with other TRPC channel subunits, we investigated breast cancer cells, aiming towards a better understanding how KCa modulate tumor cell the role of TRPC1 for receptor-operated calcium influx in the heterologous expression proliferation. system as well as in neurons. For this electrophysiological whole-cell measurements, 2+ Methods: KCa expression was quantified by qPCR in human breast cancer biopsies, fluorimetric calcium measurements, Mn quenching and qPCR analysis were applied. transgenic MMTV/c-neu+ mouse mammary tumors and primary tumor cells derived Furthermore, the effect of TRPC1 knock-down on neuronal migration was monitored thereof. The identity of the tumor cells was verified by gliolan staining. Proliferation of the performing scratch assays, videomicroscopy and G-actin/F-actin assays. Employing primary mammary tumor cells in the presence or absence of BK and SK4 modulators these techniques, we found that recombinant TRPC1 was not able to function as a was tested using a real time cell monitoring system. The cellular DNA content as a homomeric channel. Instead, TRPC1 subunits formed functional receptor-operated measure for cell ploidity was determined by propidium iodide staining and flow heteromeric channel complexes with TRPC3, 4, 5, 6, and 7. Heteromers containing 2+ cytometry. Changes in [Ca ]i oscillations and peak amplitude were determined using TRPC1 subunits showed significantly decreased calcium permeation in heterologous Ca2+ indicator FURA-2AM. cell systems. Mutation of amino acids in the putative pore forming region of TRPC1 Results: SK4, but not BK, expression is detectable in human and mouse breast cancer further reduced calcium permeability. In GnRH neurons endogenously expressing biopsies and in primary tumor cells derived from the MMTV/c-neu+ mouse model. SK4 TRPC1, 2, 5, and 6, downregulation of TRPC1 by shRNA resulted in increased basal inhibition by TRAM-34 dose-dependently (0,1 to 10 µM) inhibits the growth of primary cytosolic calcium concentrations and elevated calcium permeability. TRPC1 was not mammary tumor cells probably by a G1 cell cycle arrest. Ca2+ oscillations in proliferating involved in store-operated cation influx in GnRH neurons. Moreover, TRPC1 suppressed MMTV/c-neu+ tumor cells were ablated upon pharmacologic inhibition of SK4 channels. the migration of GnRH neurons without affecting cell proliferation. These findings Conclusion: Regulation of Ca2+-dependent cell cycle progression is dependent on SK4 suggest a novel regulatory mechanism relying on the expression of TRPC1 and the activity. Blocking SK4 disrupts a feed-forward loop that coordinates Ca2+ influx via TRP subsequent formation of heteromeric TRPC channel complexes with reduced calcium or CRAC channels in tumor cells. The consequences of SK4 inhibition in mammary permeability, thereby fine-tuning neuronal migration. tumors in vivo will be discussed.
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Flavonoide aus Zitrusfrüchten als potente TRPM3-Blocker - Citrus fruit flavonoids Synthesis of a triphenylphosphonium substituted derivative of as potent TRPM3 blockers 5-hydroxymethyl-5-methylpyrroline N-oxide Straub I.1, Mohr F.2, Stab J.2, Konrad M.2, Oberwinkler J.2,3, Schaefer M.1 1 2 3 3 Stolze K. , Rohr-Udilova N. , Patel A. , Rosenau T. 1Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr. 1Veterinärmedizinische Universität Wien Institut für Pharmakologie und Toxikologie, 16-18, 04107 Leipzig, Germany Veterinärplatz 1, A1210 Wien, Austria 2Universität des Saarlandes Institut für experminentelle und klinische Pharmakologie 2Medizinische Universität Wien Gastroenterologie und Hepatologie, Klinik für Interne und Toxikologie, Universitätsklinikum Geb. 46, 66421 Homburg, Germany Medizin III, A1090 Wien, Austria 3Philipps-Universität Marburg Institut für Physiologie und Pathophysiologie, 3Universität für Bodenkultur Dept. Chemie, Muthgasse 18, A1190 Wien, Austria Deutschhausstr. 1-2, 35037 Marburg, Germany
ESR combined with spin trapping is a well-known analytical approach to detect free The transient receptor potential melastatin-3 (TRPM3) is a calcium permeable non- radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of selective cation channel that can be activated by the neurosteroid pregnenolonesulfate carbon-centered free radicals, which are involved in oxidative stress. Our aim was to (PregS) or heat. TRPM3 is expressed in various tissues, including insulin-secreting β- modify the established spin trap 5,5-dimethyl-pyrroline N-oxide (DMPO) with a functional cells and a subset of sensory neurons from dorsal root (DRG) and trigeminal ganglia. side chain, which can be used further as anchor for moieties enabling the spin trap to The ability of PregS to evoke TRPM3-like currents in pancreatic β-cells and to induce penetrate mitochondrial membranes, such as the positively charged insulin secretion indicated its involvement in blood glucose regulation. However, TRPM3- triphenylphosphonium substituent. /- mice show so far no metabolic deficits but further investigations are recommended to Several synthetic routes were tested to introduce a 4-carboxybutyl- evaluate its function in insulin secretion. Further studies showed that TRPM3 is a triphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline N- nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. We oxide (HMMPO). While the activation of the carboxy group via the corresponding performed a calcium-based screening of a compound library (Spectrum Collection) that chloride was not successful, the use of a mixed anhydride with acetic acid appeared to identified several natural compounds as TRPM3 blockers. The most potent blockers be a promising way, although the reaction is considerably slower. Preliminary spin were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon trapping experiments have been performed with model systems generating superoxide, from ononis spinosa. The IC50 values of the substances are in the low micromoles hydroxyl-, and carbon-centered radicals. ranges. Electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the TRPM3 blockers. Furthermore, we could show that these blockers are effective on endogenous TRPM3 in DRG neurons from mice and isolated β-cells. By drinking grapefruit juice naringenin could be consumed in 412 concentrations that are sufficiently high enough to block TRPM3 activity in vivo. In sensory neurons, TRPM3 may exert similar functions as TRPV1. Thus, TRPM3 blocker could bear a therapeutic potential for analgesic treatment. Induction of Oxidative Stress and Genomic Damage by Insulin Othman E. M., Stopper H. Universität Würzburg Toxikologie, Versbacher Str. 9, 97078 Würzburg, Germany 415 Type 2 diabetes mellitus (DM2) is a growing health problem affecting more than 150 million people worldwide. It is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. Cardiac cysteine-rich LIM-only protein 4 (CRP4) exerts anti-fibrotic effects of Epidemiological studies clearly indicate that the risk of several types of cancer (including cGMP/cGMP kinase I signaling pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is Straubinger J., Majer M., Ruth P., Lukowski R. increased in diabetic patients. Uni Tübingen Pharmakologie, Toxikologie und Klinische Pharmazie, Auf der Diabetic patients are exposed to oxidative stress which plays a pivotal role in the Morgenstelle 8, 72076 Tübingen, Germany pathogenesis of both micro- and macro-vascular complications. This is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive Background: Myocardial hypertrophy, morphologically characterized by enlarged oxygen species (ROS). Since the insulin resistance in DM2 leads to hyperinsulinemia cardiomyocytes and fibrosis, is a fatal outcome of many cardiovascular disorders. we studied the cellular consequences of the elevated insulin level and showed that it Clinical studies and functional analysis of genetically modified mice have established generates superoxide anions (O2-) and DNA damage by a NADPH oxidase dependent that natriuretic peptides (NP) and their cardiac receptors diminish development of mechanism in cultured cells. In addition, we found elevated genomic damage in the pressure and volume induced heart hypertrophy and fibrosis via cGMP. However, lymphocytes of diabetic patients as well as oxidative stress and genomic damage in signaling downstream of cGMP is not well understood. Aiming towards the identification kidneys of diabetic rats. of anti-hypertrophic and anti-fibrotic pathways, we studied the role of CRP4, a novel This effect of insulin may contribute to the pathogenesis and progression of DM2 cardiovascular target of cGMP kinase I which phosphorylates CRP4 at Ser104. complications including the elevated cancer risk. CRP3/MLP, a homologue of CRP4, causes hypertrophic cardiomyopathies in mice and men when mutated, but is not phosphorylated by cGMP kinase I. Methods: Gene-targeted CRP4 knockout (KO) mice and their heterozygous (HET) and wild type (WT) littermates were subjected to a chronic pressure-dose of angiotensin II (AngII). Cardiac hypertrophy, as defined by the heart-to-body weight ratios (HW/BW), was determined in AngII treated mice and compared to littermate animals that received S94
saline. Changes in hypertrophy marker genes and putative effects of AngII on CRP analysis foci in cell nucleus were counted by eye down using a fluorescence levels and components of the NP/cGMP pathway were analyzed in total protein and microscope. Each experiment was performed at least four times. mRNA isolated from the ventricles. These experiments were corroborated by the localization of CRP4 in the myocardium and Sirius red staining as a quantitative In the XTT test following EC50 values of substances were found (mmol/L;mean +/-sem): a, b b, c a, c measure of fibrosis. TMP(EO)9TA 0.087 ± 0.011; 1,6-HDDMA 4.500 ± 0.700; ETMA ; 12.000 ± Results: Under basal conditions and upon AngII infusion CRP4 mRNA and protein were 1.100; a significantly (p < 0.05) differently to 1,6-HDDMA, b significantly (p < 0.05) c detectable in the myocardium from WT mice, whereas CRP4 was absent from KO differently to ETMA, significantly (p < 0.05) differently to TMP(EO)9TA. hearts and significantly reduced in HET mice. HW/BW ratios of all three genotypes were not different at baseline, but increased in HET and KO in response to infusion of AngII. After six hours of exposure with TMP(EO)9TA at 0.00348 mM there were induced 0.55 γ- Sirius red staining and quantitative RT-PCR experiments revealed an increase in H2AX foci-formations in HGFs, at 0.00870 mM 0.67 foci, at 0.02900 mM 0.86 foci and at interstitial fibrosis and a diminished production of anti-fibrotic factors such as BNP in 0.08700 mM 0.97 foci. After exposure with 1,6-HDDMA at 0.180 mM there were induced HET and KO hearts. 0.45 γ-H2AX foci, at 0.450 mM 0.64 foci, at 1.500 mM 0.94 foci and at 4.500 mM 1.32 Conclusion: The susceptibility of KO hearts to AngII at a pressure dose revealed that foci. After exposure with ETMA at 0.48 mM there were induced 0.43 γ-H2AX foci, at 1.2 beneficial effects of cGMP/cGMP kinase I signaling to oppose heart growth and fibrosis mM 0.50 foci, at 4.0 mM 0.61 foci and at 12 mM 0.71 foci. The negative controls DMSO induced by Gαq may be mediated by CRP4. and medium cultures displayed 0.31 – 0.34 γ-H2AX foci/cell. It was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-HDDMA > TMP(EO)9TA > ETMA.
416 These results show that dental composite components can induce DSBs in primary oral cells and therefore these substances demonstrate a genotoxic potential.
A novel mouse model of cardiomyocyte-specific thrombospondin 1 overexpression Stümpel F. T., Schulte J. S., Schmitz W., Müller F. U. 418 Universitätsklinikum Münster - Institut für Pharmakologie und Toxikologie, Domagkstr. 12, 48149 Münster, Germany Effects of antioxidants on the DNA-toxicity of dental (co)monomers in human Thrombospondin 1 (TSP-1) is an extracellular glycoprotein involved in the inhibition of gingival fibroblasts 1 2 1 1 3 3 angiogenesis, activation of TGFβ1, and de-adhesion. Its expression in the adult healthy Styllou P. , Scherthan H. , Styllou M. , Urcan E. , Durner J. , Heinrich R. , Reichl F. - 1,3 heart is low, but increases in response to injury or stress. We now created a mouse X. model of TSP-1 overexpression under control of the cardiomyocyte-specific αMHC 1Walther-Straub-Institut für Pharmakologie und Toxikologie der Ludwig-Maximilians- promoter to further analyse its role in the heart. TSP-1 transgenic (TG) mice are viable Universität München Toxikologie, Nußbaumstraße 26, 80336 München, Germany and born in an equal percentage as their wild-type (WT) littermates, and their survival is 2Institut für Radiobiologie der Bundeswehr Radiobiologie, Neuherbergstr. 11, 80937 unaltered. TSP-1 protein was abundant in TG cardiomyocytes, whereas it was not München, Germany detectable in WT cardiomyocytes, in line with a successful overexpression of TSP-1 in 3Department of Operative/Restorative Dentistry Ludwig-Maximilians-University of TG hearts. In-vivo left-ventricular catheterisation basally (bas) and under acute Munich Periodontology and Pedodontics, Goethestr. 70, 80336 Munich, Germany stimulation with the β-adrenoceptor agonist isoprenaline (iso) showed a reduced heart rate (HR) and a reduced left-ventricular pressure (LVP) as well as a diminished effect of Unreacted (co)monomers can be released from restorative dental materials and may iso stimulation on contraction velocity (dP/dtmax), relaxation velocity (dP/dtmin), stroke show biologic activity after ingestion in the human organism. In previous studies the volume (SV), cardiac output (CO), stroke work (SW), and ejection fraction (EF) in TG mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) animals. (All data are mean±SEM; n=10 per group; 2-way-ANOVA on genotype [+: on the human DNA was demonstrated. In this study the effects of the antioxidants p<0.05 vs. WT], iso dose [*: p<0.05 vs. bas], and interactions [‡: p<0.05]) vitamin C and N-acetylcysteine on the DNA toxicity of the (co)monomers triethylen- glycol-dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) was WT bas WT iso TG bas TG iso investigated. The induction of DNA double-strand breaks with (co)monomers alone and HR (min-1) 416±12 587±6 * 393±15 + 569±6 +* in combination with antioxidants was investigated in human gingival fibroblasts (HGF). HGF were incubated with substances without or with antioxidants for a period of 6 hours. LVP (mmHg) 76.6±2.0 78.2±1.3 69.5±2.6 + 67.5±1.5 + Induced DNA double-strand breaks (DSBs) were tested by the γH2AX focus assay, dP/dt 6038±409 11055±290 * 6045±408 8519±318 +*‡ which is a direct marker for DSBs using anti γH2AX antibodies. For quantitative analysis max of the γ-H2AX test, foci were counted by the same investigator by eye down the dP/dtmin -5466±301 -6971±196 * -5181±223 -5811±319 +*‡ fluorescence microscope. Each experiment was performed at least four times. The half- maximum effect concentration EC50 (mmol/l) of triethylenglykol dimethacrylat (TEGDMA) SV (RVU) 3.94±0.41 6.96±0.66 * 4.67±0.42 6.26±0.65 *‡ and 2-hydroxyethyl methacrylat (HEMA) was taken from of a previous study after using CO (RVU/min) 1620±158 4108±420 * 1855±187 3567±376 *‡ XTT-based cell viability assay.
SW (RVU·mmHg) 261±26 486±49 * 295±32 384±47 *‡ TEGDMA induced significantly (p < 0.05) higher DSBs compared to HEMA (1.91 ± 0.04 vs 1.66 ± 0.02). The mean number of cells scored and the standard deviation (SD) were EF (%) 56.9±3.7 83.6±1.9 * 64.1±4.2 73.7±4.6 *‡ calculated. Body weight (g, WT 28.2±1.8, TG 27.3±1.1), heart weight (mg, WT 121.4±6.0, TG 118.5±4.6), and heart-to-bodyweight-ratio (mg/g, WT 4.34±0.09, TG 4.37±0.14) were When cells were exposed to TEGDMA in combination with the antioxidant vitamin C an unaltered. We conclude that TSP-1 attenuates cardiac response to β-adrenergic increase of DSBs was observed (2.02 ± 0.06), compared to TEGDMA alone. When cells stimulation and hypothesise that this might exert a protective effect in situations of were exposed to HEMA in combination with vitamin C an increase of DSBs was cardiac injury or overload. Our animal model will provide further valuable insights into observed (1.89 ± 0.07), compared to HEMA alone. the function of thrombospondin 1 in heart physiology and pathophysiology. (Supported by the DFG.) When cells were exposed to TEGDMA in combination with the antioxidant N- acetylcysteine a decrease of DSBs was observed (1.64 ± 0.04), compared to TEGDMA alone. When cells were exposed to HEMA in combination with N-acetylcysteine a decrease of DSBs was observed (0.76 ± 0.02), compared to HEMA alone. 417 These results show that dental (co)monomers can induce DSBs in primary oral cells. It also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant N-acetylcysteine. Induction of DNA double-strand breaks of dental composite components in human gingival fibroblasts Styllou M.1, Scherthan H.2, Styllou P.1, Urcan E.1, Durner J.3, Heinrich R.3, Reichl F. - 1,3 X. 419 1Walther-Straub-Institut für Pharmakologie und Toxikologie der Ludwig-Maximilians- Universität München Toxikologie, Nußbaumstraße 26, 80336 München, Germany 2Institut für Radiobiologie der Bundeswehr Radiobiologie, Neuherbergstr. 11, 80937 Negligible effects of eNOS-dependent vascular oxidative stress on neointima München, Germany 3 formation Department of Operative/Restorative Dentistry Ludwig-Maximilians-University of Munich Periodontology and Pedodontics, Goethestr. 70, 80336 Munich, Germany Suvorava T., Nagy N., Dao V. T. - V., Fischer J. W., Kojda G. Heinrich-Heine-University Institute of Pharmacology and Clinical Pharmacology, Co-monomers released from dental composites have been shown to elicit cytotoxic Moorenstr. 5, 40225 Düsseldorf, Germany responses in human cells. From following composite components the cytotoxic effects were tested in human gingival fibroblasts (HGF): ethoxylated trimethylolpropane (9) Purpose: We aimed to investigate the role of superoxide and peroxynitrite generated by triacrylate [TMP(EO)9TA], 1,6-hexadiol dimethacrylate [1,6-HDDMA], and ethyltriglycol genetically destabilized eNOS for the development of endothelial dysfunction and methacrylate [ETMA]. vascular remodelling. Methods: A mutant of bovine eNOS in which Cys 101 was replaced by Ala (C101A) XTT-based cell viability assay was used to determine the half-maximum effect resulting in destabilization of eNOS has been generated (eNOS-C101A). Transgenic concentration (EC50) for the investigated composite components in HGF. Following mice carrying C101A were generated on a C57Bl/6 background using the endothelium- concentrations of substances were used to determine the induced double strand DNA specific tie-2 promoter. By breeding these mice with eNOS knockouts (eNOS-KO), mice 1 1 1 breaks (DSBs): /25× EC50, /10× EC50 , /3× EC50, and 1× EC50. Each experiment was that express eNOS-C101A (eNOS-KO/eNOS-C101A-Tg) exclusively in the endothelium performed at least four times. were obtained. Unilateral common carotid artery ligation experiments were performed in HGF were incubated with various concentrations of substances for a period of 6 hours. C57Bl/6, eNOS-KO, and eNOS-KO/ eNOS-C101A-Tg to study a role of destabilized Induced DNA double-strand breaks (DSBs) were tested by the γH2AX focus assay, eNOS for vascular lesion formation. which is a direct marker for DSBs using anti γH2AX antibodies. For quantitative γH2AX S95
Results: Western blot analysis confirmed the expression of eNOS in eNOS-KO/eNOS- inflammation, as indicated by a significant increase in sputum neutrophils, we did not C101A-Tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and detect a significant estimated treatment effect adjusted for period on cardiovascular myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of eNOS on measurements. Resting heart rate (clean air: 59±2, ozone 60±2 bpm), blood pressure Ser1176/79 (470±47%) as compared to C57Bl/6 (p<0.05, n=8). Endothelium-specific (clean air: 121±3/71±2 mmHg; ozone: 121±2/71±2 mmHg), cardiac output (clean air: overexpression of destabilized eNOS induced a large increase in superoxide and 7.42±0.29 mmHg; ozone: 7.98±0.60 l/min), and plasma norepinephrine levels (clean air: peroxynitrite formation in the aorta and the heart of eNOS-KO/eNOS-C101A-Tg (P<0.05, 213±21 pg/ml; ozone: 202±16 pg/ml), were similar on both study days. No difference of n=5-8), which was abolished by NOS-inhibitor L-nitroarginine (L-NA) suggesting eNOS- resting MSNA was observed between ozone and air exposure (air: 23±2, ozone: 23±2 C101A as a source of elevated radical generation. Endothelium-specific introduction of bursts/min). Maximum MSNA obtained at the end of apnea (air: 44±4, ozone: 48±4 eNOS-C101A at ~ 35% of C57Bl/6 level almost completely restored aortic endothelium- bursts/min) and during the phase II of the Valsalva maneuver (air: 64±5, ozone: 57±6 dependent relaxation. Experiments with L-NA, soluble guanylyl cyclase inhibitor ODQ, bursts/min) was similar. Our study suggests that acute ozone-induced airway PEG-catalase and NO-scavenger Fe(DETC)2 indicated that endothelium-dependent inflammation does not increase resting sympathetic nerve traffic in healthy subjects, an relaxation in eNOS-KO/eNOS-C101A-Tg is NOS- and cGMP-dependent and NO- observation that is relevant for environmental health. However, we can not exclude that mediated. chronic airway inflammation may contribute to sympathetic activation. Four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (P<0.05, n=4-7). Consistent with vasoprotective roles of eNOS, neointima formation was accelerated in eNOS-KO (n=4-7, P<0.05). Despite significantly higher vascular levels of 422 nitrotyrosine and peroxynitrite, neointima formation in eNOS-KO/eNOS-C101A-Tg was substantially lower then in eNOS-KO and tended to be similar to C57Bl/6. Conclusions: Increased vascular superoxide and peroxynitrite formation caused by Gαq/11 signaling tonically modulates nociceptor function and contributes to destabilization of eNOS does not induce endothelial dysfunction in healthy mice and has activity-dependent sensitization negligible effect on neointima formation. 1 2,3 4 5 2 Tappe-Theodor A. , Constantin C. , Tegeder I. , Lechner S. , Langeslag M. , Lepcynzsky P.1, Wirotanseng R.1, Kurejova M.1, Agarwal N.1, Nagy G.6, Todd A.6, Wettschureck N.1,7, Offermanns S.1,7, Kress M.2, Lewin G.5, Kuner R.1 1Universität Heidelberg Pharmakologisches Institut, Im Neuenheimer Feld 366, 69120 420 Heidelberg, Germany 2Innsbruck Medical University Department of Physiology and Medical Physics, Division of Physiology,, Innsbruck, Austria Fenton reactivity as a determining parameter for the interaction of manganese 3Universität Freiburg Department of Physiology, Freiburg, Germany oxide nanoparticles with lung epithelial cells 4Pharmazentrum Frankfurt, Klinikum der Goethe-Universität, Frankfurt, Germany 1 1 1 2 2 1 Sydlik U. , Bieschke C. , Stöckmann D. , Gotic M. , Music S. , Unfried K. 5Max-Delbrueck-Center for Molecular Medicine Department of Neurosciences, Berlin, 1Leibniz-Institut für umweltmedizinische Forschung Partikel-Zell-Interaktion, Auf´m Germany Hennekamp 50, 40225 Düsseldorf, Germany 6University of Glasgow Spinal Cord Group, Glasgow, Great Britain 2Ruder Boskovic Institut Materialchemie, Bijenička cesta 54, 10000 Zagreb, Croatia 7Max-Planck-Institut for Heart and Lung Research Department of Pharmacology, Bad Nauheim, Germany Nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and Numerous mediators released in inflammatory and neuropathic pain states activate G- diagnostics. Therefore, the interaction of such nanoparticles with human target cells is of protein-coupled receptors (GPCRs) and modulate nociception via activation of Gs, Gi/o, particular interest for the success of nanomedical approaches but also with regard to G12/13, or Gq/11 G proteins. Each of the G protein-coupled receptor pathways is involved unintended side effects. To address this problem, we tested different kinds of in nociceptive modulation and pain processing, but the relative contribution of the manganese nanoparticles (MnNP) in an in vitro system which we earlier evaluated for individual signaling proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles pathways in vivo has not yet been worked out. (CNP). The Gq/11 signaling branch is of particular interest in pain research because it leads to MnNP were synthesized by hydrothermal treatment of manganese salt solutions. The the activation of phospholipase C, protein kinase C, and the release of calcium from particles were subsequently characterized by scanning electron microscopy and intracellular stores. dynamic light scattering. Biological and toxic effects of the generated particles were Using a conditional gene-targeting approach we generated double-deficient mice lacking studied in comparison to carbon nanoparticles (CNP) in experiments with rat and human Gaq and Ga11 selectively in nociceptors to investigate the contribution of the entire Gq/11- lung epithelial cells (RLE-6TN and 16HBE14o-). Cytotoxicity was determined as signaling pathway in nociceptors towards the regulation of pain. measures of membrane damage (lactate dehydrogenase release) and metabolic activity We observed that mice lacking Gq/11 in nociceptive neurons show normal development of (water soluble tetrazolium conversion). The oxidative capacity of the particles as well as the nociceptive circuitry. The nociceptor-specific loss of Gq/11 results in reduced pain the generation of intracellular oxidative stress was monitored using dichlorofluorescein hypersensitivity following paw inflammation or spared nerve injury. Surprisingly, our diacetate in cell free experiments and flow cytometry assays (FACS), respectively. The behavioral and electrophysiological experiments also indicated defects in basal particle-specific phosphorylation of src family kinases (SFK) and mitogen activated mechanical sensitivity in Gq/11 deficient mice, suggesting a novel function for Gq/11 in tonic protein kinases Erk1/2 were investigated using Western Blot techniques. modulation of acute nociception. Patch-clamp recordings revealed changes in voltage- After physico-chemical characterization, a set of three MnNP consisting of Mn3O4 or dependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in MnO2 with significant differences in size and shape were selected. According to the nociceptors upon a loss of Gq/11, whereas potassium currents remained unchanged. different oxidation stages of manganese, the particles showed significant differences in Our results indicate that the functional role of the Gq/11 branch of G-protein signaling in Fenton reactivity in the cell free system. These data did not reflect the capacity of the nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, particles to induce intracellular oxidative stress. The characteristic to trigger membrane- but is also operational in tonic modulation of basal nociception and acute pain. dependent signaling processes, however, was correlated to the intrinsic oxidative capacity of MnNP than to the ability to induce intracellular ROS. Furthermore, the metabolic activity (WST) was negatively correlated with intracellular ROS, indicating a link between mitochondrial activity and ROS generation. None of the particles had 423 effects on the membrane integrity of the cells. The data demonstrate that MnNP, unlike other poorly soluble nanoparticles (e.g. CNP), mainly trigger adverse health effects through ROS production via the Fenton reaction. Provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes Tekook M., Fehrmann E., Schulte J. S., Schmitz W., Müller F. U. Westfälische Wilhelms-Universität Institut für Pharmakologie und Toxikologie, 421 Domagkstraße 12, 48149 Münster, Germany
Rationale: Acute ozone induced airway inflammation does not effect resting human AP duration and Ca2+ cycling are altered in cardiomyocytes of different genetic mouse sympathetic nerve traffic models. Here, we systematically tested various protocols to study the inducibility of 1 2 1 2 3 4 4 Tank J. , Biller H. , Heusser K. , Holz O. , Diedrich A. , Framke T. , Koch A. , arrhythmic events in mouse cardiomyocytes. 4 2 2 1 2 Grosshennig A. , Koch W. , Krug N. , Jordan J. , Hohlfeld J. 1Medizinische Hochschule Hannover Institut für Klinische Pharmakologie, Carl Neuberg Methods and Results: Str. 1, 30625 Hannover, Germany Adult ventricular cardiomyocytes were isolated from wildtype (WT) mice by enzymatic 2Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM Klinische digestion and subsequently tested to trigger arrhythmic events within 6 hours after Atemwegsforschung, Nikolai Fuchs Str. 1a, 30625 Hannover, Germany isolation. 3Vanderbilt University Division of Clinical Pharmacology, 1161 21st Avenue South, In patch clamp experiments (perforated patch, whole cell current clamp) APs were Nashville, TN 37232-2195, United States stimulated for 1 second (5Hz; 10Hz; 5Hz + S2-stimulus after 50-80ms) followed by a 4s 4Medizinische Hochschule Hannover Institut für Biometrie, Carl Neuberg Str. 1, 30625 rest period. The resting-membrane-potential (RMP) was observed over 30 cycles. We Hannover, Germany observed RMP-fluctuations of different length (amplitude <5 mV; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), Ozone concentrations in ambient air are related to cardiopulmonary perturbations in the spontaneous depolarizations (>5 mV; 92%, 5.1) and spontaneous APs (62%, 1.9). aging population. Increased central sympathetic nerve activity induced by local airway Intracellular Ca2+ transients and sarcomere shortening were measured after loading inflammation may be one possible mechanism. cardiomyocytes with indo-1/AM. After preconditioning (10 min/1 Hz) cells were We performed a randomized, double-blind, cross-over study, including 14 healthy measured under basal and continuous isoprenaline (10-6M) stimulation (ISO). A 4 min subjects (3 females, age 22-47 years), who underwent a 3 h exposure with intermittent pacing period was followed by a 1 min interval of no pacing. Pacing frequency was exercise to either ozone (250 ppb) or clean air. Induced sputum was collected 3 h after reduced after each cycle (1 Hz, 0.5 Hz, 0.25 Hz and 0.12 Hz). Arrhythmic events exposure. Nineteen to 22 hours after exposure, we recorded ECG, finger blood occurred in all observed WT cardiomyocytes under both conditions (% of 43 events pressure, brachial blood pressure, respiration, cardiac output, and muscle sympathetic basal/ 63 events ISO, Ca2+ oscillations 14%/63%, spontaneous Ca2+ releases 77%/24%, nerve activity (MSNA) at rest, during deep breathing, maximum-inspiratory breath hold, untriggered Ca2+ releases 9%/13%, observed events in 4 cardiomyocytes). and a Valsalva maneuver. While the ozone exposure induced the expected airway S96
Conclusion: viabilitiy was observed. This protective effect was particularly revealed at high α-AMA Arrhythmic events were provocable with stimulation/rest protocols both by field concentrations (0,5 µM and 1 µM). stimulation and direct stimulation via patch pipette. However, low stimulation frequencies In conclusion, our data suggest that benzylpenicillin in monotherapy is more effective seem to lead to distinct destabilization of cardiomyocytes probably due to Ca2+ overload. than in combination with silibinin or silibinin alone. We conclude that the tested stimulation protocols are able to provoke arrhythmic events even in WT single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. (supported by the IZKF Münster) 426
Derived values and databases for non-cancer endpoints- considerations and 424 concerns Tluczkiewicz I., Batke M., Mangelsdorf I., Escher S. Fraunhofer Institut für Toxikologie und Experimentelle Medizin Chemikalienbewertung, Visualization of cGMP in living cells and tissues of transgenic mice. Nikolai-Fuchs-Straße 1, 30625 Hannover, Germany 1 1,2 1 3 4 1 Thunemann M. , Fomin N. , Hillenbrand M. , Ott T. , Russwurm M. , Feil R. 1Universität Tübingen Interfakultäres Institut für Biochemie, Tübingen, Germany In 1996 Munro et al. derived TTC values for three toxicological classes that discriminate 2Universität Tübingen Graduate School of Cellular & Molecular Neuroscience, Tübingen, compounds of low, moderate and high toxicity according to the Cramer decision tree Germany (Cramer et al. 1978) using study results of oral repeated-dose toxicity tests with 611 3Universität Tübingen IZKF - Transgene Tiere, Tübingen, Germany chemicals. The thresholds for oral, chronic exposure are 1800 µg/person/d for Cramer 4Ruhr-Universität Bochum Pharmakologie MA Nord1, Bochum, Germany class 1 (low toxicity); 540 µg/person/d for class 2 (moderate toxicity), and 90 µg/person/d for class 3 (high toxicity). cGMP is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the Since 1996 the TTC concept was evaluated, further developed and TTCs for other nervous system. However, it is not fully understood how cGMP regulates these and endpoints than oral, chronic exposure were derived. other processes on a mechanistic level. In particular, the existence and functional relevance of global and local cGMP signaling domains is not clear. Recently, highly Escher et al. (2009) showed that TTC values based on the database RepDose are specific genetically-encoded optical biosensors for cGMP have been developed. These similar to the values derived by Munro, thus confirming the general approach with a cGMP indicators are either based on fluorescence resonance energy transfer (FRET), broader database. This analysis further revealed an overlap of the NOEL distribution in with cGMP-binding domains sandwiched between fluorescent proteins with overlapping the three Cramer classes. A combined DB TTC RepDose including Munro, ToxRef, spectra, or they consist of a single fluorescent protein fused to cGMP-binding domains. Toxbase and RepDose DB was used to improve TTC values and evaluate the selectivity With these cGMP indicators, the spatiotemporal dynamics of cGMP signals, which result of the Cramer decision tree (Tluczkiewicz et al. 2011). from the interplay between cGMP-producing guanylyl cyclases, cGMP-binding effectors, and cGMP-degrading phosphodiesterases (PDEs), can be monitored in living cells. For the inhalation route Carthew et al. 2009 and Escher et al. 2010 derived TTC values Here, we report the generation of transgenic mice expressing the FRET-based cGMP by using databases of 92 and 203 chemicals respectively. indicators cGi500 and cGi6000 with apparent cGMP affinities of 500 nM and 6000 nM, respectively. One mouse line expresses cGi6000 driven by a CMV promoter in neural In 2008 Safford, RJ derived Dermal Sensitization Thresholds DSTs for rinse-off products cells. FRET experiments were performed with isolated cerebellar granule neurons, and for leave-on products by using a database of 167 sensitizing chemicals with results hippocampal neurons, and astrocytes. We observed nitric oxide (NO)-induced cGMP of local lymph node assays. transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to Several working groups analysed TTC values for reproductive toxicants. Bernauer et al. induce cGMP responses. In another mouse line, the SM22alpha promoter directs (2008) used 91 compounds to derive a fertility/developmental TTC. Ravenzwaay et al. cGi500 expression specifically to smooth muscle cells (SMCs). FRET experiments have 2011 derived a TTC value for combined maternal and developmental toxicity based on been performed with SMCs isolated from aorta, bladder and colon, as well as with intact 93 studies. In 2011 Laufersweiler et al. also derived TTC values for the reproductive or vessels in the retina and cremaster muscle of transgenic animals. In primary SMCs we developmental endpoint using a database of 283 chemicals. studied responses to NO, atrial and C-type natriuretic peptide (ANP,CNP). In different All TTC values for reproductive toxicants are in the range of Cramer class 3 derived by SMC types we observed differences in the overall ability to react to these stimuli and in Munro. the kinetics of the induced cGMP transients. We also studied the effects of PDE Usually the structure of a compound has to be clearly identified when using the TTC inhibitors on the NO-, ANP-, and CNP-induced cGMP signals. Importantly, we were able concept. However, Rennen et al. 2011 and Koster et al. 2011 propose to use a stepwise to detect cGMP transients upon NO stimulation in intact vessels of the retina and approach to derive TTC values for substances with an unknown chemical structure cremaster. We conclude that the cGi transgenic mouse lines are valuable tools to found in food. visualize cGMP signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. For future work it should be continued to derive and apply endpoint specific TTC values but broader databases are necessary to derive robust thresholds and to broaden the chemical domain. Further there is a need to improve the classification of chemicals into appropriate toxicity classes. 425 References Munro, I.C., Ford, R.A., Kennepohl, E. and Sprenger, J.G. 1996. Correlation of a Intoxication with alpha-amanitin: Comparison of commonly used clinical antidotes structural class with noobserved-effect levels: a proposal for establishing a threshold of benzylpenicillin and silibinin in human hepatocyte culture concern. Food and Chemical Toxicology, 34,829-867 1 1 2 2 1 1 Cramer, G.M., Ford, R.A. and Hall, R.L. 1978. Estimation of toxic hazard - a decision Tischler A. , Steinritz D. , Eyer F. , Zilker T. , Thiermann H. , Schmidt A. tree approach. Food and Cosmetic Toxicology, 16, 255-276 1 Institut für Pharmakologie und Toxikologie der Bundeswehr, Neuhergerstraße 11, Escher, S., Mangelsdorf, I. 2009. Evaluation of the TTC concept with the database 80937 München, Germany RepDose Toxicology Letters 189: S10 2 Klinikum rechts der Isar Toxikologische Abteilung der II. Medizinischen Klinik, Tluczkiewicz, I., Buist, H.E., Martin, M.T., Mangelsdorf, I., Escher, S.E. 2011. Ismaninger Str. 22, 81675 München, Germany Improvement of the Cramer classification for oral exposure using the database TTC RepDose - a strategy description, Regul Toxicol Pharmacol 61; 340-350 Alpha-amanitin (α-AMA) is a polycyclic oligopeptide and the main toxin of Amanita Carthew, P., Clapp, C., Gutsell, S. 2009. Exposure-based waiving: The application of the phalloides (death cap) and its subspecies (A. virosa and A. verna). toxicological threshold of concern (TTC) to inhalation exposure for aerosol ingredients in Hepatocellular uptake of α-AMA leads to inhibition of RNA polymerase II, causing consumer products. Food Chem Toxicol 47:1287–1295 decrease in DNA transcription and protein synthesis in hepatocytes. Thus, it tends to Escher, S.E., Tluczkiewicz, I., Batke, M., Bitsch, A., Melber, C., Kroese, E.D., Buist, result in disordered cell metabolism, inducing apoptosis and necrosis. If liver H.E., Mangelsdorf, I. 2010. Evaluation of inhalation TTC values with the database regeneration during symptomatic treatment does not occur, organ transplantation RepDose. Reg Toxicol Pharmacol 58:259–274 remains the only alternative. Safford, R.J. 2008. The dermal sensitisation threshold—a TTC approach for allergic Current research data dealing with pharmacotherapy of α-AMA intoxication shows a contact dermatitis. Regul Toxicol Pharmacol 51:195–200 particularly high variability regarding the protective effect of silibinin. The aim of this Bernauer, U., Heinemeyer, G., Heinrich-Hirsch, B., Ulbrich, B., Gundert-Remy, U., 2008. study was therefore to evaluate the influence of the frequently used clinical antidotes Exposure-triggered reproductive toxicity testing under the REACH legislation: a proposal benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated to define significant/relevant exposure. Toxicol. Lett. 176, 68–76. with α-AMA. van Ravenzwaay, B., Dammann, M., Buesen, R., Schneider, S., 2011. The threshold of Cytotoxicity and apoptosis testing were performed after two and five days of toxicological concern for prenatal developmental toxicity. Regul. Toxicol. Pharmacol. 59, simultaneously exposure to α-AMA and/ or tested antidotes. To quantify apoptosis, 81–90. necrosis and cell viability, we used Cell Death Detection Elisa plus®, ToxiLight® Laufersweiler , M.C., Gadagbui, B., Baskerville-Abraham, I.M., Maier, A., Willis, A., Bioluminescence Assay and Cell Proliferation Kit II (XTT). Furthermore, we analysed the Scialli, A.R., Carr, G.J., Felter, S.P., Blackburn, K., Daston, G. 2011. Correlation of ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 chemical structure with reproductive and developmental toxicity as it relates to the use of and 9, activated caspase 3, and AIF). the threshold of toxicological concern Reg Toxicol and Pharmacol, available online Exposure of hepatocytes to α-AMA at concentrations of 0,2 µM, 0,5 µM and 1 µM Rennen, M.A.J., Koster, S., Krul, C.A.M, Houben, G.F. 2011. Application of the threshold resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared of toxicological concern (TTC) concept to the safety assessment of chemically complex with unexposed hepatocytes. food matrices. Food and Chemical Toxicology 49 (2011) 933–940 In hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µM and 1mM, Koster, S., Boobis, A.R., Cubberley, R., Hollnagel, H.M., Richling, E., Wildemann, T., silibinin at 50 µM and 100 µM and a combination of both (30 µM benzylpenicillin and 50 Würtzen, G., Galli, C.L. 2011. Application of the TTC concept to unknown substances µM silibinin, 1mM benzylpenicillin and 100 µM silibinin), ToxiLight® values in the found in analysis of foods. Food and Chemical Toxicology 49, 1643–1660 supernatant and XTT values were not significantly different from untreated cultures. Simultaneous exposure to α-AMA (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-AMA alone (exept 50 µM silibinin at 0,2 µM α- AMA); however, in both groups dosed with benzylpenicillin the highest hepatocyte S97
427 These findings gave rise to the concept of personalized antiplatelet therapy – i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (HCPR). Contribution of uncoupled endothelial nitric oxide synthase to dexamethasone- GRAVITAS was the first study to test this concept by comparing double-dose clopidogrel induced oxidative stress? to standard-dose clopidogrel in patients with HCPR. GRAVITAS failed to correct HCPR 1 1 2 1 1 1 1 consistently in the study arm, which coupled with a low overall event rate precluded Tobias S. , Habermeier A. , Daiber A. , Xia N. , Closs E. I. , Förstermann U. , Li H. demonstrating a substantial benefit from improved platelet inhibition. The TRIGGER-PCI 1Institut für Pharmakologie, Obere Zahlbacher Straße 67, 55131 Mainz, Germany 2 trial tested the effectiveness of the more potent thienopyridine prasugrel versus II. Medizinische Klinik und Poliklinik Labor für Molekulare Kardiologie, Obere clopidogrel in patients with HCPR after elective PCI with implantation of drug-eluting Zahlbacher Straße 63, 55101 Mainz, Germany stents (DES). Switching from clopidogrel to prasugrel in patients with HCPR afforded effective platelet inhibition. However, given the low rate of adverse ischemic effects Glucocorticoids (GCs) are important hormones in the regulation of metabolic using contemporary DES after PCI in stable ischemic heart disease, the clinical utility of homeostasis. Synthetic GCs, such as dexamethasone (Dex), play a fundamental role in this strategy could not be demonstrated and the study was terminated prematurely for the treatment of inflammatory diseases. There are numerous side effects of a Dex futility. therapy, e.g. the development of hypertension. In the pathogenesis of hypertension Multiple studies have shown that both heterozygotes and homozygotes for loss-of- oxidative stress is a crucial factor. Glucocorticoid-induced hypertension has been shown function CYP2C19 alleles have higher rates of adverse cardiovascular events as to be associated with an imbalance between nitric oxide (NO) and superoxide. However, compared with noncarriers on approved maintenance dosing of clopidogrel (75mg QD), the source of this elevated superoxide production is unknown. We hypothesize that an albeit carriage of CYP2C19 loss-of-function alleles accounted for only a minor proportion uncoupling of the NO synthase (eNOS), a key mediator of vascular homeostasis, may of the variability in on-clopidogrel platelet reactivity. Results of ongoing studies with contribute to Dex-induced oxidative stress. antiplatelet treatment stratified by CYP2C19 genotyping are awaited to assess the Incubation of human endothelial cells (EA.hy 926) with dexamethasone led to a clinical benefit of this approach. decrease in eNOS expression at mRNA and protein levels. This effect of Dex was time- and concentration-dependent. Since the major cause of eNOS uncoupling is a deficiency of its co-factor tetrahydrobiopterin (BH4), we analyzed the amount of BH4 in EA.hy 926 by HPLC. A concentration-dependent reduction of BH4 and also BH2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. 430 BH4 can be synthesized endogenously by two different pathways – the de novo pathway (from GTP with GTP cyclohydrolase I, GCH1, acting as the rate-limiting enzyme) and THP-1 represent a useful model to study OCTN2 expression and function in the salvage pathway (conversion of sepiapterin to BH4 involving dihydrofolate reductase, DHFR). Treatment of EA.hy 926 cells with Dex decreased mRNA and protein expression immune cells 1 2 1 2 1 1 of both GCH1 and DHFR. Because BH4 is the major “coupling switch”, an eNOS Triebel I. , Penski J. , Jedlitschky G. , Siegmund W. , Kroemer H. K. , Grube M. uncoupling is likely to occur in Dex-treated cells. 1Institut für Pharmakologie Allgemeine Pharmakologie, Felix-Hausdorff-Straße 3, 17487 In summary, we showed that Dex treatment led to a reduced availability of the important Greifswald, Germany 2 co-factor BH4 which could lead to eNOS uncoupling. The uncoupled eNOS may possibly Institut für Pharmakologie Klinische Pharmakologie, Felix-Hausdorff-Straße 3, 17487 contribute to glucocorticoid-induced vascular oxidative stress. Greifswald, Germany
The organic cation transporter novel type 2 (OCTN2/SLC22A5) represents a high affinity uptake system for carnitine. Besides metabolic disease like severe system carnitine 428 deficiency, genetic variants within the SLC22A5 gene have been associated with inflammatory diseases like colitis ulcerosa. Against this background, we characterized OCTN2 expression in peripheral blood cells thereby identifying its expression in all cell Involvement of c-Fos/AP-1 in the regulation of nucleotide excision repair upon types. In the present work we studied OCTN2 expression in monocytes and THP-1 cells UVC-induced DNA damage as an in vitro model for this cell type. In addition we examined transcriptional regulation of the carnitine transporter in LPS activated THP-1 and investigated the effect of Tomicic M., Reischmann P., Rasenberger B., Kaina B., Christmann M. carnitine and its analog mildronate on the respective cytokine response. Universitätsmedizin Mainz Institut für Toxikologie, Obere Zahlbacher Str. 67, 55131 OCTN2 expression was characterized on monocytes and THP-1 cells on mRNA and Mainz, Germany protein level. Transporter mRNA expression could be shown in both cell types by real- time PCR. However, the protein expression was analyzed by western blot, flow The cellular oncoprotein c-Fos is a major component of the heterodimeric transcription cytometry and immunofluorescence microscopy demonstrating OCTN2 specific signals factor AP-1 and has been commonly found over-expressed in tumors and cancer cells of as well as a localization in the plasma membrane. Following THP-1 cells were activated different origin. Previous work showed that mouse cells lacking the immediate-early using LPS (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as gene c-fos are hypersensitive to ultraviolet (UVC) light. Here we demonstrate that in demonstrated by increased TNFα (24fold induction) and IL-1β (37fold induction) mRNA human telomerase-immortalized VH10tert foreskin fibroblasts (behaving like primary levels. In addition, OCTN2 expression was analyzed identifying an initial reduction of cells) and SV40-immortalized GM637 fibroblasts, UVC-triggered induction of c-Fos around 60% compared to untreated cells. In parallel activated THP-1 cells were protein is a delayed and long-lasting event. Sustained up-regulation of c-Fos went along coincubated with increasing concentrations of the OCTN2 substrate carnitine or its with transcriptional stimulation of the nucleotide excision repair (NER) gene xpf, carrying analog resulting in reduced cytokine release as shown by ELISA for TNFα. Here, the an AP-1 binding site in the promoter. c-fos mRNA was induced in a biphasic manner. An TNFα effect was diminished by 64% in the presence of 50mM carnitine. This effect does immediate c-fos mRNA expression (30-90 min after exposure) was not translated into not rely on a direct neutralization of LPS by carnitine since it was also present in cells the protein, the second wave of transcription (4-24h after UVC exposure) resulted in c- only preincubated with carnitine. Fos protein expression, 18-48h post-UV. The stress-activated/mitogen-activated protein In the present work we could show that THP-1 cells represent a useful model to study kinases (JNK, p38K and ERKs) were immediately induced upon UVC exposure and OCTN2 expression and function. In addition, we demonstrate immunosuppressive stayed active for at least 24h. Inhibitor experiments revealed that c-Fos was effects of OCTN2 substrates like carnitine. Further experiments will be necessary to phosphorylated by ERKs and JNK. The activation of c-Fos preceded re-synthesis and identify the underlying mechanism of this observation. the induction of xpf mRNA, which was observed 24-40h post-UVC, resulting in the increased expression of the XPF protein. Cells over-expressing c-Fos showed an accelerated induction of xpf mRNA, and consequently a faster repair of cyclobutane pyrimidine dimers (CPDs). siRNA-mediated silencing of c-Fos (transient c-Fos knock- down) resulted in abrogated UVC-triggered induction of XPF, attenuated repair of CPDs 431 and increased apoptosis. Finally, we observed that the removal of CPDs but not of (6-4) photoproducts was significantly faster when cells were pre-exposed to a low UVC dose, indicative of an adaptive response to DNA damage. Castor oil induces laxation and uterus contraction through ricinoleic acid The work was financed by Deutsche Forschungsgemeinschaft (DFG CH 665/2-1). activating EP3 receptors 1 1 2 3,4 1,4 Tunaru S. , Althoff T. , Nüsing R. M. , Diener M. , Offermanns S. References 1Max-Planck Institut für Herz- und Lungenforschung, Ludwigstrasse 43, 61231 Bad Tomicic et al., Cell. Mol. Life Sci. (2011) 68 :1785-1798 Nauheim, Germany 2Institute for Clinical Pharmacology, J.W. Goethe University Frankfurt, Theodor-Stern- Kai 7, 60590 Frankfurt, Germany 3Institute for Veterinary Physiology, Justus Liebig University, Frankfurter-Str. 100, 35392 429 Giessen, Germany 4Medical Faculty, J.W. Goethe University Frankfurt, Theoder-Stern-Kai 7, 60590 Frankfurt, Germany Personalizing antiplatelet treatment: Phenotyping versus genotyping Castor oil has been used for more than 3000 years for its laxative effects and also to Trenk D. induce labor in pregnant women. Despite its wide-spread use, the mechanism of action Herz-Zentrum Bad Krozingen Klinische Pharmakologie, Südring 15, 79189 Bad remained unknown. The active metabolite of castor oil is ricinoleic acid which is released Krozingen, Germany from castor oil by intestinal lipases. We have found that exposure of MEG-01 cells to 2+ ricinoleic acid caused an increase in [Ca ]i, an effect which was dose-dependent and The addition of clopidogrel to aspirin reduces ischemic events in patients with acute abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a coronary syndrome and in those undergoing percutaneous coronary intervention (PCI). G-protein coupled receptor. To search for a putative receptor, we determined ricinoleic However, recurrent ischemic event occurrence during dual antiplatelet therapy remains 2+ acid-induced [Ca ]i increases in cells transfected with a siRNA library directed against a major concern. Variability in the pharmacodynamic response to clopidogrel is well human GPCRs. In this way, we identified prostaglandin E2 receptors EP3 and EP4 as recognized, and patients with higher platelet reactivity while receiving clopidogrel are at mediators of ricinoleic acid-induced effects. To test if EP3 and EP4 receptors mediate increased risk of ischemic cardiovascular events. Clopidogrel is an inactive prodrug pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by requiring biotransformation to form the platelet inhibiting metabolite. Interindividual -/- -/- castor oil in wild-type (WT) mice, EP3-deficient (EP3 ) or EP4- deficient mice (EP4 ). differences in clopidogrel metabolism are the major source of variability in antiplatelet -/- -/- While EP4 mice responded similarly to the WT mice, EP3 animals were totally response. Polymorphically expressed cytochrome P450 (CYP) enzymes play a critical insensitive to castor oil-induced laxation. Moreover, mice lacking the EP3 receptor only role in the metabolism of clopidogrel. in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack S98
EP3 receptor only in epithelial cells of the intestinal mucosa. Similarly, ricinoleic acid- 434 induced contractions of isolated ileal segments were absent in segments lacking EP3, consistent with a preferential expression of the EP3 receptor in the longitudinal muscle layer of the intestine. Also, ricinoleic acid-induced contractions of isolated uteri were Insulin effects on hyaluronan production - a possible link between diabetes and dependent on the expression of EP3 receptor in the myometrium. These findings identify cancer? the cellular and molecular mechanism underlying the effects of castor oil and indicate a Twarock S., Fischer J. W. role of the EP3 receptor as a pharmacological target to induce laxative effects. Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany
Background: Epidemiological studies have shown an elevated incidence of certain 432 tumor entities in diabetes type 1 and type 2 patients. To reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. Abundant production of hyaluronic acid (HA) in the vicinity The antidiabetic drug sitagliptin inhibits a late component of Ito induced by DPP10 of gastrointestinal cancer cells is a hallmark in tumor development. Esophageal cancer 1 2 1 1 1 Turnow K. , Radicke S. , Christ T. , Ravens U. , Wettwer E. is a rare but severe kind of gastrointestinal cancer which is differentiated in 1Institut für Pharmakologie und Toxikologie, Fiedlerstraße 42, 01307 Dresden, Germany adenocarcinoma and squamous cell carcinoma (SCC) of the esophagus. 2Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16-18, 04107 Methods & Results: We studied the effects of increased glucose levels on HA Leipzig, Germany production in an SCC cell line (OSC1). In starving and full media, elevated glucose concentrations increased the production of HA secreted to the medium in 24h as Dipeptidyl-peptidase like protein DPP10 is an accessory β-subunit of the Kv4.3-encoded measured by an HA-binding protein linked assay (starved, 0g/L glucose: 100±4.7%; transient outward current (Ito) channel complex in the human heart. DPP10 increases Ito 1g/L: 185.7±44.8%; 4g/L: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; current density and accelerates time course of inactivation and recovery from 422.7±32.0%). Surprisingly, total HA concentrations were about 2.2-2.8 fold higher inactivation. DPP10 is a type II transmembrane protein homologue to the serine under starved conditions. To investigate whether this effect might be due to insulin protease DPPIV which is reversibly inhibited by binding of the antidiabetic drug actions, starved cells were treated with 10 mg/L insulin for 24h. We observed a dose- sitagliptin to the active site of the large extracellular domain. We hypothesize that the dependent decrease in HA production following insulin treatment (control vs insulin, 1g/L extracellular domain of DPP10 though lacking enzymatic activity may serve as ligand glucose: 100±6.5% vs 67.83±4.4%; 4g/L: 100±2.2% vs 71.8±6.6%). This finding might binding site and regulate Ito. suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing Here, the influence of sitagliptin was studied on human right atrial action potentials and HA synthesis. A premise to this assumption is the ability of OSC1 cells for insulin on ionic currents, in particular on Ito in isolated cardiomyocytes and heterologous independent glucose uptake. To verify this thesis, mRNA expression levels of insulin- expression systems (CHO-cells) using standard microelectrode and voltage clamp independent and insulin-dependent glucose transporters (GLUT1, GLUT4) were techniques. analyzed by qRT-PCR. The relative abundance was 24.32±3.49 in favor of GLUT1 Sitagliptin concentration-dependently prolonged APD20 in human atrial trabeculae indicating the presence of insulin-independent glucose transport. indicating an effect on Ito. In addition, sitagliptin prolonged the APD90, depolarized the Conclusion: In OSC1 the absence of insulin actions caused increased HA production resting membrane potential and decreased dV/dtmax as well as action potential which might be due to diminished insulin driven glycolysis, thus leading to the use of amplitude. Patch clamp experiments in human atrial cardiomyocytes documented early glucose metabolites for HA production instead of energy gain. This finding could be blocking effects on IK1, INa and Ito. In CHO cells expressing the Ito channel complex important in the context of diabetes type 2, where insulin actions are also diminished KV4.3/KChIP2 plus DPP10, we identified a slowly inactivating component (“Ito-late”) four because of insulin resistance. Since increased HA production is of critical importance for fold larger than in control cells which did not express DPP10. Sitagliptin (1mM) cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to decreased Ito-peak current in CHO cells independent of DPP10 expression, but HA anabolism could therefore indicate a possible link between diabetes type 2 and significantly reduced the Ito-late component by 30% only in cells expressing DPP10. cancer progression. We provide evidence that sitagliptin reduced a DPP10 specific late current component of Ito suggesting that the agent interacts specifically with the extracellular domain of DPP10. Future studies will have to address whether the extracellular domain of DPP10 possesses affinity for physiological ligands modulating Ito characteristics. The binding 435 site may serve as a potential drug target for modulation of cardiac Ito.
Biomarkers and Molecular Tumour Classification for Non-genotoxic Carcinogenesis 433 Schwarz M., Unterberger E. Universtität Tübingen, Institut für klinische und experimentelle Pharmakologie und Toxikologie Abteilung Toxikologie, Wilhelmstraße 56, 72074 Tübingen, Germany Uncertainty Regarding Health Information – Current Challenges For The Drug Information Service Chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating 1 1 1 2 Tuttas K. , Goltz L. , Kahnt M. , Kirch W. mutation in a gene encoding an important cell-regulatory protein. Tumour initiation may 1Institut für Klinische Pharmakologie, TU Dresden Arzneimittelberatungsdienst, be caused by genotoxic substances which directly interact with the DNA, causing Fiedlerstr. 27, 01307 Dresden, Germany mutations. Cells carrying permanent mutations experience clonal expansion which may 2Institut für Klinische Pharmakologie, TU Dresden, Fiedlerstr. 27, 01307 Dresden, be accelerated by exposure of the experimental animals to tumour promoters during the Germany following step of tumour promotion. It has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the Introduction: Patients seek health information from various sources. They are facing model tumour promoter phenobarbital which, amongst others, activates constitutive the challenge to differentiate between reliable and untrustworthy sources and at the androstane receptor (CAR). Since these tumour promoters do not seem to directly same time identify the best drug therapy for them. Furthermore generalised health interact with DNA causing mutations they can be regarded as non-genotoxic information confuses more than they benefit or rather unsettle. Patients are not carcinogens. However, the molecular mechanisms of non-genotoxic carcinogenesis are necessarily qualified to assess the evidence of statements properly. There is thus a still widely unknown which also poses a major problem in preclinical drug-development. need for providing competent drug information, which is offered by the independent drug The aim of the MARCAR (BioMARkers and Molecular Tumour Classification for Non- information service at the Institute of Clinical Pharmacology in Dresden, Germany. genotoxic CARcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by Methods: For the present descriptive evaluation we selected 5 drugs (Arimidex®, a regimen including model tumour promoters such as phenobarbital. The ultimate aim is Cipralex® Pentalong®, Onbrez® and Pradaxa®), that were affected by new reference- to differentiate spontaneous liver tumours from tumours generated by non-genotoxic price formation, generic registration, warnings or directions in 2011. In specified time carcinogens. This molecular profile includes mutational analyses, immunostaining for frames we assessed the increase in and the cause of enquiries. Deductively we draw known tumour-specific markers, phospho-proteome analyses, genome wide and conclusions for a perspicuous presentation of patient information. promoter-specific DNA methylation analyses, as well as miRNA analyses. Mutation analyses were carried out with mouse and rat tissue from phenobarbital Key findings: Since generic registrations of the aromatase inhibitor Arimidex® enquiries promoted liver tumours to identify mutations which phenobarbital provides a growth on side effects of this drug were stable, but 17 additional consultations were held on advantage for. Furthermore, real time PCR measurements show that the expression of a generic changeovers. The antidepressant Cipralex® as well as the long-acting β-agonist particular non-coding RNA and miRNA precursor is up-regulated in tissue isolated from Onbrez® were assigned to reference-price groups, which resulted in an 8-times phenobarbital promoted mouse liver tumours. Additional in-situ-hybridisation (Cipralex®: 5 → 40) and 5-times (Onbrez®: 2 → 10), respectively, increase in enquiries. experiments demonstrated the localisation of this transcript in Ctnnb1-mutated tumours. Main aspect was to give background information on reference prices and point out therapeutic alternatives (Cipralex®: 34 of 40; Onbrez®: 10 of 10). An additional amount of 22 conversations were carried on the fictive registered drug Pentalong® after health insurance companies advised practitioners to avoid recourse by not prescribing this 436 organic nitrate. Notable insecurity was aroused by media reporting on lethal bleeding after taking Pradaxa® for anticoagulation. Every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). Larch-derived diterpenes are potent and selective TRPC6 blockers
Urban N.1, Kübler W.2, Schaefer M.1 Conclusion: Patients are confronted by current changes, but often do not get enough 1 background information from their health care providers to become acquainted with the Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr. 16-18, 04107 Leipzig, Germany tidings. Health seekers may find eligible data from media coverage. However the 2 individual assessment as well as the risk-benefit-relation may not be feasible for them. Charité - Universitätsmedizin Berlin Physiologisches Institut, Thielallee 71, 14195 The drug information service for patients is a convenient helpline to reduce lack of Berlin, Germany knowledge and uncertainties and therefore support shared decision making. 2+ The transient receptor potential channel TRPC6 is a poorly Ca -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (DAG). Consistent with the major sites of TRPC6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung S99
edema, chronic obstructive lung disease, allergic airway disease, and focal segmental these tissues in mice developing neuropathic pain. Importantly, we observed that spinal glomerulosclerosis. Amongst various plant extracts, conifer oils and resins are delivery of recombinant Serpina3n inhibits mechanical allodynia in a mouse model of traditionally used to treat pulmonary ailments. Therefore, we reasoned that they may neuropathic pain. We identified a novel serine protease substrate for Serpina3n, which is contain constituents with a biological activity to modulate TRPC6 activity. The true upregulated in the spinal cord in mice undergoing neuropathic pain (‘Enzyme E’). turpentines, oils and resins of various coniferous genera were tested with respect to a Recombinant Enzyme E delivered intrathecally to the spinal cord of mice elicited rapid possible inhibition of DAG- or receptor-induced activation of TRPC6 and TRPC3. and long-lasting allodynia, which was fully blocked by concomitant administration of Indeed, turpentines and resins, but not coniphere oils blocked TRPC6 and TRPC3 in a Serpina3n. Our results suggest that serine protease-serpin signaling modulates spinal concentration-dependent manner. Interestingly, the larch-derived turpentine exerted a neuronal and glial cell networks involved in processing pain and that activity-induced TRPC6-prevalent inhibition. We identified larixol and its mono- and diacetates as the spinal release of Serpina3n constitutes an endogenous defence mechanism against specific compounds that are contained in larch resin and give rise to a TRPC6-selective establishing chronic pain hypersensitivity. These data have important implications for the block. Larixol acetates displayed an IC50 towards the DAG- or receptor-stimulated pathophysiology of pathological pain and potentially hold therapeutic relevance. TRPC6 activity of about 0.3-1 µM, but did not strongly inhibit a number of other TRP channels, including TRPV1, TRPM2, TRPM3, TRPM8, or TRPA1. Selectivity for TRPC6 compared to its closest relative, TRPC3, was about 30-fold. Unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no 439 significant cellular toxicity at concentrations that are required to block TRPC6. Electrophysiological analysis confirmed the highly potent block, which was voltage- independent and reversible. In a murine hypoxia-induced pulmonary vasoconstriction Gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy (HPV) model, larixol acetate abrogated the Euler-Liljestrand mechanism and, thus, mimicked the phenotype of TRPC6-/- mice. We conclude that TRPC6 blockers and, more Vidal M., Lohse M. J., Lorenz K. specifically, larixol-related derivatives may provide novel therapeutic strategies to treat Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 or prevent pulmonary diseases. Würzburg, Germany
Introduction. Activated β1-adrenergic receptors and their G protein Gαs induce the development of cardiac hypertrophy. However, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, cAMP or PKA, are controversely 437 discussed. Recently, a hypertrophic pathway involving a G protein βγ subunit induced phosphorylation of the mitogenic kinases Erk1/2 at threonine 188 (ErkThr188- phosphorylation) has been described to mediate Erk-induced hypertrophy. This study Identification of protein complexes involved in calcium and iron homeostasis after aims to investigate whether ErkThr188-phosphorylation is involved in cardiac hypertrophy TCDD exposure triggered by β-adrenergic receptors. Verma N., Pink M., Rettenmeier A. W., Schmitz-Spanke S. Methods and Results. ErkThr188-phosphorylation was detected in HEK cells Universitätsklinikum Essen Institut für Hygiene und Arbeitsmedizin, Hufelandstr. 55, overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after 45122 Essen, Germany isoprenaline treatment. We performed [3H]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. Neonatal rat cardiomyocytes (NRCMs) Introduction overexpressing wild-type Erk2 showed a significant increase in [3H]-isoleucine Dioxin is an environmental contaminant, believed to affect basic biological equilibria incorporation after isoprenaline treatment. In contrast, NRCMs transfected with such as calcium and iron homeostasis. However, the molecular mechanisms underlying ErkThr188-phosphorylation deficient mutants (Erk2T188A and Erk2T188S) or pretreatment these effects are still largely unknown. This strongly hampers the estimation of the with the Erk inhibitor, PD98059, significantly attenuated cardiomyocyte hypertrophy. For hazard to humans associated with dioxin exposure and necessitates further studies in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and aimed at the clarification of these mechanisms. It has been suggested that nearly all transgenic mice overexpressing either wild-type Erk2T188T or Erk2T188S. biological and biochemical processes are mediated by protein complexes. The most Echocardiography and histological analyses revealed that ErkT188S mice developed less commonly used technology for monitoring changes in the expression of complex protein left ventricular hypertrophy than control mice. Hypertrophic target proteins of Erk (e.g. mixtures is still 2D gel electrophoresis, but this method suffers from poor expression of Elk1) are located in the nucleus. Western blot and confocal microscopy analyses low or moderately abundant proteins. Blue native PAGE and subcellular fractionation showed that overexpressed Erk2T188A or Erk2T188S are retained in the cytosol and form an ideal partnership when it comes to enrichment and analysis of intracellular prevented Elk1- phosphorylation after isoprenaline stimulation. Co-immunopreciptation organelles and low abundant multiprotein complexes. The aim of the study is to identify assays in HEK cells and NRCMs underlined the direct involvement of G protein βγ/Erk and characterize multiprotein complexes by Blue native PAGE to elucidate the network interaction upon isoprenaline stimulation. In line with this finding, direct activation of of protein-protein interactions that regulate protein function after dioxin exposure. adenylyl cyclase by forskolin did not lead to Gβγ induced ErkThr188-phosphorylation. Conclusion. Taken together, Gβγ-subunits participate in β1-adrenergic receptor Methodology mediated hypertrophy by enhancing ErkThr188-phosphorylation. These findings add Sample preparation and subcellular fractionation important insight to the molecular signaling of G proteins in cardiac hypertrophy. RT4 cells were cultured in McCoy’s 5A medium. Cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. First dimension (BN-PAGE) 440 50 mg of protein sample was mixed with 5% of Coomassie blue G-250 (CBB G-250) and loaded in each lane of 4–15% polyacrylamide native gradient gels. Second-dimension (SDS-PAGE) and mass spectrometry The protein tyrosine kinase Src and its role upon alpha-toxin stimulation of The lanes from the first dimension were cut into individual strips and were placed into a human platelets 12% SDS gel. The gels were stained with Coomassie and the spots were picked up for 1 2 1 mass spectrometry. Vogel K. , Burke M. , Presek P. 1 Martin-Luther University Halle-Wittenberg Department of Pharmacology and Results and Conclusion Toxicology, Division of Clinical Pharmacology, Magdeburger Str. 4, 06097 Halle (Saale), Germany 2 BN/SDS-PAGE combined with MS led to the identification of proteins involved in the Martin-Luther University Halle-Wittenberg Department of Internal Medicine III, Medical regulation of both calcium and iron homeostasis in dioxin-exposed cells. These results Faculty and University Hospital Halle, Ernst-Grube-Str. 40, 06120 Halle (Saale), demonstrate for the first time that dioxin exposure simultaneously affects calcium and Germany iron metabolism. Since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may Introduction: be associated with dioxin-dependent cell-fate decisions. Alpha-toxin, a 34 kDa calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of Staphylococcus aureus infections. Alpha–toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). In the present paper, we focused our interest on the protein tyrosine kinase Src, the 438 most abundant member of the Src-family kinases present in platelets (2). By the use of various inhibitors, we studied Src and its role in α–toxin-induced platelet aggregation.
The Murine Protease Inhibitor Serpina3n Inhibits Mechanical Allodynia in a Model Methods: of Neuropathic Pain Isolated human platelets from healthy volunteers were stimulated with α-toxin in the 1,2 1,2 1 1 1 3 Vicuna L. , Simonetti M. , Bali K. K. , Husainie D. , Kurejova M. , Costigan M. , presence or absence of the Src-family member inhibitors PP1, PP2 or SU6654 (referred 4 3 1,2 Devor M. , Woolf C. , Kuner R. as Src inhibitors). Src and autophosphorylation of Src were analyzed by SDS-PAGE and 1Universität Heidelberg Pharmakologisches Institut, INF 584, 69120 Heidelberg, Western blotting using specific antibodies against Src and Tyr-416-phospho-Src from Germany Calbiochem and Cell Signaling, respectively (3). Furthermore, calpeptin, an inhibitor of 2Cluster of Excellence CellNetworks, Heidelberg, Germany the calcium-dependent protease calpain, was used. Platelet aggregation was measured 3Children's Hospital Boston Kirby Neurobiology Center, 300 Longwood Avenue, Boston, by the method of Born. MA 02115, United States 4Institute of Life Sciences, Hebrew University of Jerusalem Department for Cell and Results: Animal Biology, Givat Ram, 91904 Jerusalem, Israel Staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 - 3.0 µg/ml of toxin). Pre-incubation with 3 Src inhibitors (PP1, PP2 or Several chronic diseases are accompanied by strong, long-lasting pain. A majority of SU6654) reduced α-toxin-induced platelet aggregation by about 50%. Similar inhibitory chronic pain diseases are not well understood yet and cannot be controlled by effects have been observed by the use of calpeptin that acts as an inhibitor of the Src conventional analgesics or non-pharmacological approaches. Therefore, there is a major degrading protease calpain. need to develop novel therapeutic principles. Using a genetic screen, we identified With respect to Src itself, a-toxin induced autophosphorylation at Tyr-416 followed by a Serpina3n, a serine protease inhibitor, which is homologous to human a1-anti- fast and complete dephosphorylation within 10 min. While calpeptin modified the time chymotrypsin, to be a determinant of low neuropathic pain. We found that Serpina3n is course of dephosphorylation, only little effect of the Src inhibitors has been seen on Tyr- expressed in the dorsal root ganglia (DRG) and spinal cord and that it is upregulated in 416 phosphorylation/dephosphorylation. S100
The typical calpain-dependent degradation of Src can be blocked by calpeptin (1µM), specific modes of action. The EpiAirwayTM model has proven to be robust, showing high but also by depletion of extracellular calcium indicating that it is a calcium-dependent reproducibility between pre- and main-tests as well as in the concurrent controls but it process. will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. Conclusion: Taken together, our data demonstrate that α-toxin of Staphylococcus aureus induces platelet aggregation accompanied by Src degradation and autophosphorylation at Tyr- 416 typically observed in activated platelets. Inhibition of the cellular tyrosine kinase Src 443 as well as the protease calpeptin reduces aggregation indicating an important role of Src and/or other Src-family members in α-toxin-induced platelet stimulation. Remodeling of intracellular Ca2+ handling and cyclic AMP-dependent signaling in References atrial myocytes from patients with chronic atrial fibrillation. (1) Bhakdi S, Walev I, Husmann M and Valeva A: Staphylococcal alpha-toxin. In: 1 1 2 1 Microbial Protein Toxins (eds. Schmitt MJ, Schaffrath R), Topics in Current Genetics, Voigt N. , Abu-Taha I. , Wieland T. , Dobrev D. 1 Vol. 11, pp. 91-110, Spinger, Berlin - Heidelberg, 2005 Medizinische Fakultät Mannheim, Universität Heidelberg Sektion Experimentelle (2) Presek P, Martinson EA: Platelet Protein Tyrosine Kinases. In: Platelets and Their Kardiologie, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany 2 Factors. Handbook of Experimental Pharmacology (eds. Bruchhausen F, Walter U), Vol. Medizinische Fakultät Mannheim, Universität Heidelberg Institut für Experimentelle 126, pp. 263-296, Springer, Berlin, 1997 Pharmakologie und Toxikologie, Maybachstr. 14, 68169 Mannheim, Germany (3) Nass N, Vogel K, Hofmann B, Presek P, Silber RE, Simm A (2010) Glycation of 2+ 2+ PDGF results in decreased biological activity. Int J Biochem Cell Biol. 42: 749-54 Background: In atrial myocytes Ca entry through L-Type Ca channels (ICa,L) triggers 2+ 2+ a larger Ca release (Ca transient,CaT) from the sarcoplasmic reticulum activating contractile myofilaments. Reduced ICa,L is a hallmark of atrial remodeling in chronic atrial fibrillation (cAF) and is supposed to contribute to action potential shortening and contractile dysfunction. However, the coupling efficiency between ICa,L and CaT and its 441 regulation by cAMP-dependent signaling in cAF patients are unexplored. Methods: ICa,L (voltage-clamp) and CaT (Fluo-3) were measured simultaneously in right- atrial myocytes from sinus-rhythm (Ctl) or cAF patients. A saturating concentration of the Evidence for a differential role in muscarinic receptor activation of the conserved non-selective β-adrenoceptor (AR) agonist isoprenaline (ISO, 1µM) and the non- epitope tryptophan 7.35 selective phosphodiasterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX, 10µM) Vogel L., Kebig A., Mohr K., Janßen N. were used to increase cellular cAMP content. cAMP content was assessed with Institute of Pharmacy Pharmacology and Toxicology, Gerhard-Domagk-Str. 3, 53121 immunoassay. Bonn, Germany Results: In cAF amplitudes of ICa,L (3.3±0.3pA/pF, n=12/6 [myocytes/patients] vs 6.2±0.8 pA/pF, n=15/10, p<0.01) and CaT (182.2±26.8nM vs 307.5±30.9nM, p<0.01) were lower 2+ The five subtypes of muscarinic acetylcholine receptors belong to the superfamily of G- than in Ctl myocytes, whereas diastolic [Ca ]i levels were unchanged (cAF, protein coupled receptors. The even-numbered subtypes M2 and M4 prefer coupling to 312.3±56.7nM; Ctl, 305.3±43.6nM). The coupling efficiency between ICa,L and CaT was Gi proteins, whereas the odd-numbered receptors M1, M3 and M5 prefer coupling to Gq similar in Ctl and cAF. Application of ISO increased ICa,L amplitude to 10.4±2.0pA/pF proteins. (n=7/4) in cAF and to 14.3±1.7pA/pF (n=9/7) in Ctl. The corresponding CaTs increased With respect to ligand binding and M2 receptor activation, the conserved epitope Trp to 493.0±132.3nM in cAF and to 545.0±79.0nM in Ctl. Although the amplitudes of ICa,L 7.35 at the beginning of TM7 displays remarkable functional features. It is located at the and CaT also increased after PDE inhibition with IBMX, the magnitude of these junction between the orthosteric and the allosteric binding site of the M2 receptor [1]. In increases was smaller than the ISO-induced enhancements. Both ISO and IBMX had no 2+ the inactive M2 receptor, it provides subtype-independent baseline affinity for allosteric effect on diastolic [Ca ]I and coupling efficiency. However, the relative ISO-induced antagonists [1]. In the active receptor, M2 Trp 7.35 affords binding affinity for the full increases in ICa,L (cAF, +202.3±47.3% vs Ctl, +98.4±16.1%, p<0.05) and CaT (cAF, agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2]. +215.4±57.8% vs Ctl, +101.9±20.3%, p=0.06) were significantly higher in cAF compared To study the role of Trp 7.35 for M3 receptor activation, agonist-induced formation of D- to Ctl myocytes and a similar tendency was found for IBMX. Basal cAMP levels were myo-inositol-monophosphate was measured in CHO-cells transfected with the higher in cAF compared to Ctl (cAF, 9.9±1.5pmol/mg, n=6 vs Ctl, 5.0±0.6pmol/mg, n=7, respective human receptor-cDNA. Surface receptor expression measured by radioligand p<0.05), pointing to an increased cAMP-dependent signaling in cAF patients. binding was similar in hM3 wild-type-cells and hM3 Trp 7.35→Ala-cells, amounting to Conclusions: These data point to remodeling of cAMP-dependent signaling in cAF 6 0.07 and 0.11x10 receptors per cell, respectively. patients which likely contributes to the stronger relative increases of ICa,L and CaT The intrinsic efficacy of acetylcholine was not influenced at M3 Trp 7.35→Ala relative to amplitudes after β-AR stimulation and PDE inhibition. Remodeling of cAMP-dependent M3 wild-type, whereas potency was reduced about tenfold. These findings resemble signaling might be a novel contributor to AF pathophysiology. those made previously in M2 and the corresponding mutant. In the case of pilocarpine, replacement of Trp 7.35 by alanine in M3 did not reduce intrinsic efficacy. This finding is in contrast to M2, where the corresponding mutation induced a loss of pilocarpine’s intrinsic efficacy. The potency of pilocarpine was 444 diminished about tenfold at the M3 Trp 7.35→Ala mutant relative to M3 wild-type. This finding is also in contrast to M2, at which pilocarpine’s potency was not sensitive to the Trp 7.35→Ala mutation. Direct visualisation of G-protein-coupled receptors and heterotrimeric G-proteins Taken together, the diverging sensitivity of pilocarpine to the Trp 7.35→Ala mutation using single-molecule microscopy between the M3 and the M2 receptor suggests that the role of this epitope for receptor 1,2 2 1,2 1,2 function may differ between even- and odd-numbered muscarinic acetylcholine Wagner J. , Zabel U. , Lohse M. J. , Calebiro D. 1 receptors. Universität Würzburg Rudolf-Virchow-Zentrum, Josef-Schneider-Str. 2, 97078 Würzburg, Germany 2 References Universität Würzburg Institut für Pharmakologie und Toxikologie, Versbacher Str. 9, [1] Prilla, S. et al. (2006) Mol. Pharmacol. 70: 181-196 97078 Würzburg, Germany [2] Jäger, D. et al. (2007) J. Biol. Chem. 282: 34968-34976 G-protein-coupled receptors (GPCRs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. Although the basic mechanisms of GPCR signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. 442 In this study, we developed new methods for labelling GPCRs and G-protein subunits based on SNAP- and CLIP-tags and visualise them with single-molecule sensitivity. The SNAP-tag is a mutant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase Respiratory toxicity in vitro: Comparison of lung 3D-model and monolayer cell that reacts with fluorescent benzylguanine derivatives, whereas the CLIP-tag is reacting lines specifically with O2-benzylcytosine derivatives. These tags allow labelling proteins Vogel S., Hess A., Kolle S., van Ravenzwaay B., Landsiedel R. directly in living cells with very high specificity and low background. SNAP/CLIP-tagged BASF SE Experimental Toxicology and Ecology, Carl-Bosch-Str. 38, 67056 receptors and G-proteins were covalently labelled with small organic fluorophores and Ludwigshafen, Germany visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath In vivo experiments for inhalation toxicity are time and animal consuming. Thus several the cell surface. Particles were automatically analysed with previously published as well in vitro methods aim to replace or reduce and refine the in vivo experiments. Human 3D- as newly developed algorithms. The results indicated that both receptors and G- tissue models are commercially available reconstructed from different donors (normal, proteins, although diffusing with high speed on the cell surface (diffusion coefficients: smokers, chronic obstructive pulmonary diseases), which show a normal human receptors ~ 0.05 mm2/s, G-proteins ~ 0.1mm2/s), can be visualised and correctly bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli tracked. A variable fraction of receptors and G-proteins are immobile or show hop and cilia on the apical surface of the cultures. The presence of tight junctions and mucus movements, possibly suggesting their interaction with cytoskeletal or other membrane- secretion has also been confirmed comparable to the in vivo situation. These 3D-models bound proteins. Our data also suggest the feasibility of performing two-colour analyses are cultured on a porous membrane as air-liquid interface. Test substances can be with SNAP- and CLIP-tagged proteins aimed at directly visualizing transient interactions applied apically, either as solution or with an aerosol-inducer. between receptors and G-proteins or among G-protein subunits.
In our in house validation to test the strengths, handling and reproducibility of such 3D- References model systems as well as determining the correlation between in vivo inhalation data, Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K. (2003) A general we have assessed the EpiAirwayTM model from MatTek, USA. A set of 20 substances method for the covalent labeling of fusion proteins with small molecules in vivo. Nat were selected with known in vivo toxicity data and mode of action. The substances were Biotechnol. 21:86-9. tested in the EpiAirway model an in parallel, in 3T3 and A549 cell lines to assess Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, Danuser G. putative unspecific cytotoxic effects of the test substances. (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods. 5:695-702. A comparison of toxicity data from the 3D-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with S101
pleckstrin homology domain of PLCγ2, which is essential for its interaction with activated Rac2, is dispensable for the inhibitory effect of RhoH. In summary, our results indicate, that RhoH acts as a PLC-isozyme-specific negative regulator of the activity of PLCβ2 and PLCγ2, both of which are specifically expressed in hematopoietic cells. These findings suggest a novel mechanism of PLC isozyme regulation by RhoH. The results also suggest that RhoH plays an important role in B cell maturation, function, and leukemogenesis by modulating B-cell-receptor-mediated PLCγ2 activation.
447
Effect of Rac1 inhibition on doxorubicin mediated cell response Wartlick F., Fritz G. Heinrich-Heine-Universität Düsseldorf Institut für toxikologie, Universitätsstrasse 1, Figure 1.: 40225 Düsseldorf, Germany Single G-Proteins on cell surface (a) CHO cell expressing SNAP-tagged Gs-proteins, labelled with SNAP-TMRStar, visualised by TIRF microscopy. (b) Tracking of a single Background: The small GTPase Rac1 is a well characterized member of the Ras- G-protein moving on cell surface, stopping for several frames before resuming homologous (Rho) family. Rac1 is not only a key regulator of the actin cytoskeleton but movement. also regulates the activity of NADPH oxidase, stress kinases and transcription factors (e.g. NF-κB, AP1). Furthermore, Rac1 can translocate into the nucleus and interacts with topoisomerase type II (Topo II). Yet the general nuclear function of Rac1 is still unclear. Here, we address the question how Rac1 influences the genotoxicity of the Topo II poisons doxorubicin and etoposide. 445 Methods: To study the function of Rac1, human hepatoma cells were pretreated with the Rac1-inhibitor EHT 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (IR). Identification of a cell amount indicator to normalize in vitro metabolomics data To check the influence of Rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the WST-assay, Western blot (WB), Walk T.1, Ramirez T.2, Ahlbory-Dieke D.1, Fux E.1, Looser R.1, Kamph H.2, van 2 co-immunoprecipitation experiments, FACS-analysis and the alkaline comet-assay. Ravenzwaay B. Results: As compared to the control, cells that have been pretreated with the Rac1 1 Metanomics GmbH, 10589 Berlin, Germany inhibitor showed a higher viability, less phosphorylation of H2AX (S139) and a reduced 2 BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany DNA damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with IR. Furthermore inhibition of Rac1 In-vitro screening systems are particularly well suited to preclinical toxicology testing at resulted in a reduced phosphorylation of Topo IIα (S1106) and an increased interaction an early stage of drug development as they have the advantage of being fast and of Topo IIα with Hsp90 in doxorubicin treated cells. requiring only a small amount of test substance. The demands for in-vitro screening Conclusion: The data indicate that inhibition of Rac1 protects human hepatoma cells assays for systemic toxicity are multiple and include the need of organ specific cell against Topo II poisons due to interference with Topo IIα function. systems, the use of optimal cell numbers, cell passages and incubation times. Even minimal changes in the conditions of the test system may lead to significant changes of the biological system. Therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological 448 system. BASF has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. The in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. Herein, we Development of a MDCK cell in vitro model for the assessment of BCRP mediated compare five different normalization strategies referring to their suitability in the drug transport in the lactating mammary gland of dairy animals 1 1 1 2 1 application to in-vitro metabolite profiling data. The strategies comprise statistical Waßermann L. , Lindner S. , Halwachs S. , Honscha K. U. , Honscha W. approaches, approaches referring to reference values from each individual sample or 1Universität Leipzig Institute of Pharmacology, Pharmacy and Toxicology, An den samples generated in dependent batches. Best results were achieved by an individual Tierkliniken 15, 04103 Leipzig, Germany strategy using a new reference value correlating well over a large range of cell counts 2Universität Leipzig Institute of Veterinary Physiology, An den Tierkliniken 7, 04103 previously used for generating corresponding cell extracts. Statistical analysis revealed Leipzig, Germany the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. The presence of drugs or other potential toxic substances in milk has enormous Generation and application of this new reference value and the corresponding toxicological and nutritional consequences for consumers of dairy products. The ATP- normalization strategy will be presented the first time. Validation will be featured on the binding cassette (ABC) transport protein Breast Cancer Resistance Protein (BCRP; basis of extracts of the human hepatocellular carcinoma cell line Hep G2. ABCG2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. BCRP is known to play a major role in the active secretion of a variety of xenobiotics into human milk. So far there is little information about the transport activity and substrate specificity of dairy BCRP. Therefore we aimed to establish a MDCK cell in vitro model 446 expressing BCRP of dairy animals. BCRP mRNA was isolated from bovine, caprine and ovine mammary gland. Full-length clones were generated using RACE (rapid amplification of cDNA ends) PCR. The final full-length bovine, ovine and caprine ABCG2 cDNA-clone Molecular mechanisms of the inhibitory function of RhoH in phospholipase C- sequences were submitted to the NCBI genebank (EU570105, GQ141082 and GQ241418). mediated signalling Stable transfection of BCRP in MDCK cells was performed and the subcellular localization of BCRP at the apical plasma membrane was identified by confocal laser scanning Walliser C., Löschmann Y., Ziegler V., Kühne E., Schilling P., Rasonabe Z., Bühler A., microscopy. BCRP-mediated transport of the substrate Hoechst 33342 was measured and Vatter P., Gierschik P. the selectivity was determined by the BCRP inhibitor Ko143. Inhibition studies using Universitätsklinikum Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein- Hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic Allee 11, 89081 Ulm, Germany agents like oxfendazole as substrates of bovine, caprine and ovine BCRP. To further characterize BCRP carrier activity, bidirectional transport studies were performed with Rho GTPases are a subfamily of Ras GTPases regulating diverse signalling pathways, Transwell® filter inserts that allow studying drug transport between an apical and for example those regulating the reorganisation of the actin cytoskeleton. Among them, basolateral compartment. Cell monolayer integrity was checked by measuring TEER values Rac2 and RhoH show an expression restricted to the hematopoietic lineage. RhoH is as well as by measuring the paracellular flux marker atenolol by LC/MS. Bidirectional constitutively active, because it carries mutations in two positions (S13 and N62) known transport studies with Enrofloxacin were performed to characterize the BCRP transporter to be important for GTP hydrolysis. Hence, RhoH is controlled on the level of protein activity. Our results may contribute to increase the understanding of carrier associated drug expression and, possibly, by tyrosine phosphorylation. RhoH has been implicated in transport into the milk of dairy cattle and therefore enlarge consumer protection. human malignancies, since the gene is subject to somatic hypermutation in its non- coding regions and to translocation to the gene encoding LAZ3/BCL6 or to other genes in human B-cell lymphomas. Furthermore, RhoH is overexpressed in primary human chronic lymphocytic leukemia (CLL) cells. These findings suggested that RhoH is 449 involved in the initiation and/or progression of CLL. We previously showed that RhoH acts as a potent inhibitor of both Rac2-mediated phospholipase C-β2 (PLCβ2) and PLCγ2 activation in intact cells. The aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of RhoH on PLC Acrolein and acrylamide: excretion of mercapturic acids after consumption of activity. The results showed that RhoH directly inhibited the activity of constitutively potato chips Watzek N., Scherbl D., Berger F., Feld J., Eisenbrand G., Richling E. active variants of PLCγ2, PLCβ2, and PLCδ1, but that it had little or no effect on the activity of PLCγ1 and PLCε. The amino acid residues S13 and N62, likely to be the Technische Universität Kaiserslautern Fachbereich Chemie; Fachrichtung cause for the GTPase-deficiency of RhoH, are not required for the inhibitory function of Lebensmittelchemie & Toxikologie, Erwin-Schrödinger-Str. 52, 67663 Kaiserslautern, RhoH. Furthermore, the switch-I and switch-II regions of RhoH are not necessary for the Germany inhibitory effect of RhoH, since RhoH mutants carrying switch-I or switch-II regions of Rac2 caused inhibitory effects on Rac2-mediated PLCβ2 and PLCγ2 stimulation Acrolein (AC) and acrylamide (AA) may be formed from food constituents during heating indistinguishable from wild-type. Interestingly, RhoH seems to interact with regions of of food. AC is supposed to be generated via heat induced formation from PLCγ2 distinct from those which are necessary for Rac2 interaction, as the split glycerides/glycerol, AA is known to arise during the Maillard reaction from asparagine S102
and reducing carbohydrates. AC also has also been suggested to be formed by h after seeding. Analysis of real time PCR (TaqMan®) experiments, using β-actin as a endogenous metabolism as a side product of carbohydrate and/or amino acid turnover housekeeping gene, showed a significant increase of EGR1 expression in a time and or by oxidative desamination of polyamines [1]. As an α,b-unsaturated aldehyde, AC concentration dependent manner. These effects were specific for stimulation with cCMP- forms 1,4-Michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino AM but not the control phosphate trisacetoxymethyl ester. HeLa cells were also cultured groups. In the organism, AC and AA are preferentially conjugated to glutathione and are in serum free resting medium (MCDB 153, Sigma) that induces growth arrest, one to excreted as mercapturic acids (MA), N-acetyl-S-(3-hydroxypropyl)-cysteine (3-HPMA), eight hours prior to stimulation. Here, even higher EGR1 expression levels through N-acetyl-S-(carboxyethyl)-cysteine (CEMA), (N-acetyl-S-(2-carbamoylethyl)-cysteine cCMP-AM stimulation could be seen. These results suggest that cCMP could function as (AAMA), and (N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA). a second messenger just as cAMP and cGMP do. Studies are in progress to further Data on human exposure to AC and its occurrence in the diet are scarce. In general, examine the mechanisms of the cCMP-AM effects on EGR1 expression in HeLa cells. contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2]. Nevertheless, in a pilot study in humans urinary 3- HPMA excretion of 14 non-smokers References was reported to be about three fold higher, as compared to AAMA [3]. [1] Burhenne H et al., Naunyn-Schmiedeberg’s Arch Pharmacol 383, Suppl 1: P096 In the present human intervention study we monitored the excretion of MAs in five (2011) healthy volunteers (male) after ingestion of commercially available potato chips (175 g), [2] Wolter S et al., Biochem Biophys Res Commun (2011) in press equivalent to an uptake of 44 µg AA (absolute amount), together with an as yet unknown [3] Ravni A et al., Mol Pharmacol 73, 1688 (2008) amount of acrolein [4]. Urinary MA contents were monitored by HPLC-MS/MS following solid phase clean-up of urine for up to 24 h after test meal uptake. The results demonstrated kinetics of 3-HPMA and CEMA excretion in human urine to be clearly related to ingestion of the potato chip meal. On the basis of AUC values, total excretion 452 of 3-HPMA plus CEMA exceeded that of AAMA plus GAMA by a factor of about four. The results confirm earlier findings on urinary MAs, suggesting markedly higher human exposure to dietary AC / potential AC precursors than to AA. It is an as yet unresolved Mitochondrial structure and function is affected by the epigenetic regulator question, whether and to what extent concomitant substantial AC exposure may methyl-CpG-binding protein 2 in the heart influence toxicology of such dietary heat- induced toxicants. Weiss S.1, Gilsbach R.1, Preißl S.1, Schwarzer M.2, Schrepper A.2, Kretz O.3, Schwan 1 2 1 References C. , Doenst T. , Hein L. 1 [1] Stevens, J.F. and Maier, C.S. (2008) Molecular Nutrition & Food Research 52; 7 Universitätsklinikum Freiburg Institut für Experimentelle und Klinische Pharmakologie [2] Osorio, V. M. and de Lourdes Cardeal, Z., (2011) Journal of Chromatography A 1218; und Toxikologie, Albertstr. 25, 79104 Freiburg, Germany 2 3332 Universitätsklinikum Jena Klinik für Herz und Thoraxchirurgie, Erlanger Allee 101, [3] Schettgen, T., Musiol, A., and Kraus, T., (2008) Rapid Communications in Mass 07747 Jena, Germany 3 Spectrometry 22; 2629 Universitätsklinikum Freiburg Institut für Anatomie und Zellbiologie, Albertstraße 17, [4] Ewert, A., Granvogl, M., and Schieberle, P., (2011) Lebensmittelchemikertag 2011 79104 Freiburg, Germany
Introduction Methyl-CpG-binding protein 2 (MeCP2) recognizes methylated DNA, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. We have 450 previously shown that expression of MeCP2 is diminished in murine and human heart failure. Prevention of MeCP2 downregulation in transgenic mouse models aggravated + cardiac hallmarks of heart failure. In patients with Rett syndrome, which is caused by Protective effects of increased NAD levels in human peripheral blood mutations in the MeCP2 gene, mitochondrial function was found to be altered in the mononuclear cells exposed to DNA damaging agents central nervous system. As the impact of MeCP2 on mitochondrial function in the heart Weidele K., Beneke S., Bürkle A. is unknown, the aim of the present study was to characterize the significance of MeCP2 University of Konstanz Molecular Toxicology Group, Department of Biology, Jacob- of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or Burckhardt-Str.31, 78457 Konstanz, Germany ablation of MeCP2. Methods and results The DNA damage-activated enzyme poly(ADP-ribose) polymerase 1 (PARP-1) acts as a In order to investigate the cardiac function of MeCP2, two genetically modified mouse nick sensor and modifies target proteins by covalent attachment of poly(ADP-ribose) models were previously generated, including mice with inducible transgenic expression [PAR] using NAD+ as substrate. The intracellular levels of PAR and NAD+ are important of MeCP2 in cardiac myocytes under control of the tetracycline-system (MeCP2-Tg) and parameters for biological responses to genotoxic stress and influence diverse cellular mice with targeted ablation of MeCP2 in myocytes (MeCP2MLCCre). These mice were functions including DNA repair or maintenance of genomic stability. Notably, loss of analyzed under basal conditions and after chronic transverse aortic constriction (TAC). genomic stability is a hallmark of both carcinogenesis and the ageing process. Here we At baseline, cardiac-specific overexpression of MeCP2 did not cause any difference in analysed the impact of elevated NAD+ levels in human blood peripheral mononuclear cardiac function as compared to control mice using Millar catheterization. Isolated cells (PBMC) with regard to (i) poly(ADP-ribose) formation, (ii) cell death, (iii) initial DNA interfibrillar mitochondria showed a decrease in citrate synthase activity. After chronic damage and subsequent repair, as well as the influence on (iv) genomic stability under pressure overload, the decrease in cardiac MeCP2 expression could be completely genotoxic stress. After ex vivo supplementation of PBMC with low concentration of NAD+ prevented by the MeCP2 transgene. Cardiac contractility and relaxation were precursor nicotinic acid (NA) intracellular NAD+ level significantly increased up to 2 fold significantly decreased in MeCP2-Tg animals. Upon electron microscopical in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. After DNA damage infliction, investigation, transgenic MeCP2 expression was associated with a significant reduction PARP activity was dramatically increased in supplemented cells, necrotic cell death was of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and reduced and DNA strand break repair was significantly affected. Furthermore the smaller mitochondrial cross sections as compared with control specimens. In contrast, frequency of micronuclei decreased significantly after irradiation damage, emphasizing cardiac myocyte-specific ablation of MeCP2 caused a rightward shift in the size the fundamental role of adequate NAD+ levels in maintaining genomic integrity. distribution of mitochondria as compared with MeCP2-Tg hearts. Conclusion References Epigenetic processes, including the recognition of DNA methylation by MeCP2, may 1. Weidele, K., et al., Ex vivo supplementation with nicotinic acid enhances cellular play an important role in the control of mitochondrial gene expression, structure, poly(ADP-ribosyl)ation and improves cell viability in human peripheral blood subcellular localization and function in the heart. Thus, precise control of MeCP2 mononuclear cells. Biochem Pharmacol, 2010. 80(7): p. 1103-12. expression and function is essential to prevent deterioration of metabolic function during chronic heart failure.
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Stimulation of HeLa cells with cCMP acetoxymethyl ester and its impact on EGR1 gene expression levels Normalisation of blood pressure does not prevent angiotensin II-induced DNA Weiler F.1, Wolter S.1, von der Ohe J.1, Schwede F.2, Seifert R.1 damage in kidney and heart of ren2 rats 1Medizinische Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Straße 1, Weissenberger S., Hey V., Lau D., Schupp N. 30625 Hannover, Germany Universität Würzburg Institut für Pharmakologie und Toxikologie, Versbacher Strass 9, 2Biolog, Flughafendamm 9a, 28071 Bremen, Germany 97078 Würzburg, Germany
The cyclic purine nucleotides adenosine 3’:5’ monophosphate (cAMP) and guanosine Increased activity of the renin angiotensin system (RAS) with enhanced levels of 3’:5’ monophosphate (cGMP) are well-examined second messengers with many proven angiotensin II (AngII) leads to oxidative stress with endothelial dysfunction, hypertension biological functions. In a recent study, using a highly sensitive and specific mass and atherosclerosis. Epidemiological studies revealed a higher cancer mortality and an spectrometry method, we have shown that cyclic 3’:5’ cytidine monophosphate (cCMP), increased kidney cancer incidence in hypertensive patients. We could show in vitro and in a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1]. cCMP vivo that AngII causes structural DNA damage dose-dependently in kidney cells and in the activates both cAMP- and cGMP-dependent protein kinases with low potency [2] but the kidney. Elevated AngII levels therefore might contribute to carcinogenesis of the kidney. physiological function of cCMP is still very poorly understood. In an effort to delineate In a model of high AngII organ levels, the transgenic ren2 rat, carrying an additional the function of cCMP, we analyzed expression of the early response gene EGR1. We renin gene, DNA damage in the kidney was analysed in animals of 13 and 32 weeks. chose this gene because it is regulated by numerous stimuli including cAMP [3]. In our Untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. first study, we showed that dibutyryl-cCMP and cCMP failed to increase EGR1 gene Therefore, the line is kept on angiotensin I converting enzyme inhibitor therapy, which expression levels after stimulation of KB cells under various experimental conditions normalizes blood pressure and kidney function to values of control Sprague Dawley rats. using real-time PCR (TaqMan®). We have now changed the experimental set-up using Despite this normalized blood pressure of the ren2 animals, a significant higher HeLa cells and the new cCMP analogue, cCMP-acetoxymethyl ester (cCMP-AM), still superoxide production could be observed in kidneys already in 13 week old animals. focusing on gene expression of EGR1. Esterification of the negatively charged cyclic Also a higher frequency of structural DNA damage and double strand breaks could be phosphate of cCMP allows better transport of the nucleotide across the cell membrane, detected in the comet assay and with an antibody against the double strand break thus augmenting possible intracellular effects. HeLa cells were stimulated in cell culture marker γ-H2AX in kidneys. Further, fittingly, an increased DNA repair activity exists in medium with extracellularly applied cCMP-AM (3, 10, 33, 100 µM) over 15 to 240 min 24 kidneys of ren2 rats compared to control rats. As another organ affected by hypertension S103
the heart of the ren2 animals was analysed for oxidative DNA damage. Although only a 456 marginal increase of superoxide production could be found, also in the heart a significant higher frequency of DNA double strand breaks and cells positive for DNA repair activity could be observed. Epigenetic regulation of ABCC2 drug transporter: Differences of miRNA Our data let us conclude that normalization of blood pressure in a state of activated RAS interactions with distinct ABCC2 haplotypes is not sufficient to prevent AngII-induced genotoxicity. This further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of Werk A., Halekotte J., Haenisch S., Bruckmüller H., Cascorbi I. elevated AngII levels. Institut für klinische und experimentelle Pharmakologie, Arnold-Heller-Str. 3 Haus 30, 24105 Kiel, Germany
Background: ABCC2 is adjacent to P-Glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several 454 compartment barriers. By increasing evidence it is shown that the ABCC2 polymorphisms are of clinical significance. The aim of our study is to analyze the epigenetic regulation of distinct ABCC2 haplotypes by the influence of microRNAs. The D541A pore mutation leads to complete inactivation of TRPV6 channels in Methods: ABCC2 cDNA clones containing five distinct haplotypes were generated by epididymis site-directed mutagenesis, cloned into pIRES-ZsGreen expression vectors and 1 1 1 1 2 1 Weißgerber P. , Kriebs U. , Tsvilovskyy V. , Olausson ,. J. , Kretz O. , Störger C. , transfected into different cell lines for further functional analysis. One modified vector set 1 1 1 3 1 Mannebach S. , Wissenbach U. , Vennekens R. , Middendorff R. , Flockerzi V. , contained a short 3’UTR sequence of ABCC2 whereas the other contained the full 4 Freichel M. length (fl) sequence. miRNAs potentially interacting with ABCC2 were identified form an 1Universität des Saarlandes Experimentelle und Klinische Pharmakologie und miRNA array after rifampicin stimulation of HepG2 cells. Toxikologie, 66424 Homburg, Germany Results: We could demonstrate that there is no difference in the basal protein 2Universität Freiburg Zentrum für Neurowissenschaften, Albertstrasse 23, 79104 expression level comparing the two vector types (fl 3’UTR vs. mod. 3’UTR) concerning Freiburg, Germany the -24C/1249G/3972C (CGC) ABCC2 WT in HepG2 cells. Using the fl vector construct, 3Justus Liebig Universität Gießen Institut für Anatomie und Zellbiologie, Aulweg 123, the expression level of CAC haplotype protein was increased (136,15% ± 20%). 35385 Gießen, Germany Transfection assays with the miR-397, which was identified as a candidate miRNA 4Universität Heidelberg Pharmakologisches Institut, Im Neuenheimer Feld 366, 69120 targeting ABCC2 mRNA, and the two different vector constructs harboring the CAC or Heidelberg, Germany the CGC (WT) haplotype, confirmed that miR-379 was able to down regulate the ABCC2 protein expression. There was no significance in downregulation for one ABCC2 Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 haplotype, respectively (modified 3’UTR: CGC 22-34%; CAC 20-40%, fl 3’UTR: CGC channels in mice disrupts Ca2+ absorption by the epididymal epithelium resulting in 20-25%; CAC 17-44% down regulation compared to miR-negative control). abnormally high Ca2+ concentrations in epididymal luminal fluid and in a dramatic but Discussion: Differences of ABCC2 protein expression level are due to the epigenetic incomplete loss of sperm motility and fertilisation capacity raising the possibility of regulation of ABCC2 haplotypes. To further characterize the effect of miR-379 on TCG, residual activity of channels formed by TRPV6D541A proteins (Sci Signal 4, ra27, 2011). It TGT and CGT ABCC2 haplotypes, transfection assays are currently performed using is known from other cation channels that introducing pore mutations even if they largely cell cultures as well human primary leukocyte cultures. affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. To gain insights whether the TRPV6D541A pore mutant still contributes to residual channel activity and/or channel- independent functions in vivo, we compared important fertility-parameters between 457 Trpv6-/- and Trpv6D541A/D541A mice: The fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either Trpv6 mutation as well as the Sex differences affect the pathophysiology and pharmacology of leukotriene motility, in vitro fertilization capacity and viability of sperm were reduced to the same biosynthesis extent in both genotypes. Also, no differences were observed in copulatory behavior -/- D541A/D541A 2+ Werz O. between Trpv6 and Trpv6 males. The profound reduction in Ca uptake by the epididymal epithelium was identical in Trpv6-/- and Trpv6D541A/D541A males. This direct Friedrich-Schiller-University Jena, Institute of Pharmacy, Philosophenweg 14, 07743 comparison of these parameters indicate that deleting TRPV6 does not further Jena, Germany aggravate the phenotype observed in Trpv6D541A/D541A mice, and - in our opinion - allows the conclusion that the D541A pore mutant of the TRPV6 protein leads to complete Inflammatory diseases affect more females than males. Thus, women suffer more often inactivation of the TRPV6 channel activity or channel-independent scaffolding functions from asthma, rheumatoide arthritis, Alzheimer´s Disease, and many autoimmune in epididymal epithelium. diseases than men. Of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. We have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (LTs) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. Interestingly, testosterone downregulates LT biosynthesis and 455 also causes a sex bias in the efficiency of LT synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-LTs. We found that certain inhibitors of LT biosynthesis were more efficiently in females than in males, and that Characterization of a naturally occurring C-terminal mutation (N996I) on hERG androgens are responsible for these gender differences. In fact, the FLAP inhibitor channel function in HEK293 cells MK886 effectively reduced LTB pleural levels in female but not in male rats treated with 1 2 1 1 4 Sellmaier V. , Moretti A. , Engelhardt S. , Welling A. carrageenan, and MK886 increased survival only of female mice in the LT-related 1Technische Universität München Pharmakologie und Toxikologie, Biedersteinerstr. 29, disease model of PAF-induced lethal shock. Administration of testosterone to female 80802 München, Germany mice abolished the protective effects of MK886. In view of the current active 2Technische Universität München I. Medical Department – Molecular Cardiology, development of LT synthesis inhibitors as therapeutics in respiratory and cardiovascular Ismaninger Strasse 22, 81675 München, Germany diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. Long-QT-syndromes (LQTS) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. In affected individuals, the electrocardiogram shows a prolongation in the QT interval, due to an unstable repolarization of the action potential. Acquired forms of LQTS are often the result of 458 treatment with medications that block cardiac potassium channels, such as class III antiarrhythmic drugs or antihistamines. Inherited LQTS are caused by mutations in cardiac ion channels. Congenital long QT syndrome 2 (LQT2) is caused by loss-of- Inhalation and Instillation Cause Different Pulmonary Toxicity: A Case Study with function mutations in the human ether-á-go-go-related gene HERG (also known as Amorphous Silica Nanoparticles KCNH2 or Kv11.1). HERG encodes the pore-forming α-subunit of the rapid delayed Lan M. - H.1, Wiench K.2, Wohlleben W.3,1, Strauss V.1, Treumann S.1, van Ravenzwaay rectifier potassium current IKr, whose physiological role is to repolarize the late phase of 2 4 5 1 cardiac action potentials. HERG channel α-subunits exist as 2 isoforms (1a and 1b) that B. , Brill S. , Wiemann M. , Landsiedel R. 1BASF SE Experimental Toxicology and Ecology, 67063 Ludwigshafen am Rhein, are identical except for structurally divergent N termini. Native cardiac IKr channels are tetraheteromers containing 2 of each α-subunit types. A loss-of-function can be due to Germany 2BASF SE Product Safety, GV/TB, 67063 Ludwigshafen, Germany either defects in a) channel opening (gating), b) ion permeation or c) protein maturation 3 and trafficking. BASF SE Polymer Physics, 67063 Ludwigshafen am Rhein, Germany 4BASF SE Occupational Medicine, 67063 Ludwigshafen am Rhein, Germany We have identified a so far uncharacterized dominant missense mutation in the HERG1 5 gene (N996I) in a patient with LQT. Both hERG1a and hERG1b subunits were cloned ibe R&D gGmbH, Münster, Germany from a human heart cDNA library and the specific N996I mutation introduced by site directed mutagenesis. Considering the complexity of deposition and kinetics of air-borne nanomaterials in the HEK 293 cells were transiently transfected with equal amounts of mutated hERG1a and lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by wild type (WT) hERG1b cDNAs and the resulting potassium current compared to hERG1a/b inhalation studies. Those studies demand special equipment and large quantities of test WT. Whole-cell patch-clamp analysis showed similar current densities for WT versus material. Intratracheal instillation appears as a simple and low substance-consuming mutated channels. Also the voltage-dependence of activation was unchanged with a half- alternative, although bolus dosing and the more central distribution of the particles in the maximal activation at –27 mV for WT and –29 mV for the mutated channel assembly. lung are a well known trade-off. Differences were found for the deactivation and inactivation kinetics. The deactivation was We compared the inflammatory response of the lung to amorphous silica (AS) after instillation and inhalation. For inhalation the established short-term protocol for faster in the mutated channels with tfast = 62 ms and tslow = 480 ms versus tfast = 90 ms and nanomaterials was employed (Ma-Hock et al. Inhalation Toxicology, 21:102, 2009): Male tfast = 620 ms in WT channels, determined from the tail current at –40 mV after a 5-second pulse to + 50 mV. The half-maximal steady-state inactivation of the tail current was shifted Wistar rats were exposed to the test items for 6 h/day on five consecutive days. The by 20 mV to the depolarizing direction in the mutated channel compared to the WT. A defect lungs were evaluated by analysis of bronchoalveolar lavage fluid (BALF) and by in channel gating appears to be the most likely explanation. histopathology three days and three weeks after the end of the exposure. In a parallel study, rats were intratracheally instilled and equally evaluated three days after S104
instillation. Assuming a deposition rate of 10%, the instilled dose corresponded to the database. It also suggests that acute oral toxicity categories can be used as a predictor aerosol concentration of 10 mg/m3 used for inhalation. for the overall hazard potential of a substance. Results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. This References difference may be due to the bolus effect inevitable linked to instillation. Instillation Munro, I.C. et al. (1996) Correlation of Structural Class with No-Observed-Effect Levels: stuldies with amorphous silica may, therefore, be of limited value with respect to dose- A Proposal for Establishing a Threshold of Concern. Food Chem. Toxicol. 34: 829-867 response assessment.
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Comparison of different in-vitro models for inhalation toxicology with respect to Zinc oxide – nanosize does not change the toxicological profile the effects of cigarette smoke total particulate matter Wiench K.1, Schulte S.1, Schneider S.2, Lan M. - H.2, van Ravenzwaay B.1, Monteiro- Wiese J.1, Schumann B.1, Glahn F.1, Thomas S.1, Schön I.2, Sandner A.2, Foth H.1 3 4 2 Riviere N. , Creutzenberg O. , Landsiedel R. 1Martin-Luther-University Institute of Environmental Toxicology, Franzosenweg 1a, 1BASF SE Product Safety, GV/TB, 67063 Ludwigshafen, Germany 06097 Halle / Saale, Germany 2BASF SE Experimental Toxicology and Ecology, 67063 Ludwigshafen am Rhein, 2Martin-Luther-University Department of Otorhinolaryngology, Head and Neck Surgery, Germany Magdeburgerstr. 12, 06097 Halle / Saale, Germany 3North Carolina State University, Raleigh, 27601, United States 4Fraunhofer ITEM, Hannover, Germany Cell cultures of various origins and complexity have become commonly used models in inhalation toxicology. We compared two human tumor cell lines (H322, A549), primary Sunscreen products containing UV filters protect consumers from the harmful effects of cultures of normal human bronchial epithelial cells (NHBEC), peripheral lung cells (PLC) UV exposure. Pigmentary grades of metal oxides like ZnO result in an opaque and so called mini organ cultures derived from nasal turbinates (N-MOC), bronchus (B- whiteness as a result of scattering visible light, whereasnanoparticles result in MOC) or peripheral lung tissue (L-MOC) with respect to the effects of total particulate transparent products for better consumer acceptance and thus improved protection of matter (TPM) of cigarette smoke (2R4F). human skin against UV-induced damage. In addition scatter UV light is most efficiently B- and L-MOC can be cultivated up to 70 days without loss of viability, as determined by reflected at a nanosize of 60–120 nm. resazurin-assay. Viability of cell cultures was determined by MTT-assay after incubation In the last 2 years the toxicological properties of nanoZnO in comparison with with increasing doses of TPM. Exposure of H322 to TPM (30 mg/l) reduced viability to pigmentary ZnO were examined, the results of these comprehensive studies are 95% or 83% after 24 or 72h, respectively. In A549 viability was 67% after 72h with TPM presented. All tests were performed according to OECD guidelines, which were (30 mg/l). The same dose of TPM lead to a decrease in viability to 82% (24h) or 25% modified, especially in regard of substance preparation where appropriate. (72h) in NHBEC and to 74% (24h) or 28% (72h) in PLC. Nanosized ZnO showed no acute toxicity after dermal application, in the BCOP assay as As a marker of oxidative stress the level of intra-cellular glutathione (GSH) was well as in the Epiderm assay it showed no corrosion / irritation potential. determined by HPLC. In both the tumor cell lines analysed GSH-level was increased by Nanosized ZnO does not penetrate the intact as well as the sunburned skin. A dermal TPM (5 mg/l). In H322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. absorption test in rats (OECD 427) with 65Zn-labelled test item as well as penetration While in A549 it was 1.3 (24h) and 1.4 fold (72h). In NHEC and PLC TPM (5 mg/l) did tests in weanling pigs after UV radiation did not show a penetration of the ZnO not have a significant effect on GSH-levels after 72h. In N-MOC TPM (5 mg/l) also did nanoparticles through the skin. not modulate GSH after 24h, but it diminished GSH-level by 0.5 fold upon prolonged Genotoxicity was tested in vitro in the Ames Test, in the Chromosomal Aberration Test contact (72h). In B-MOC GSH concentrations also decreased to 0.6 or 0.8 fold the level in V79 cells, both showing negative results whereas the Mouse lymphoma mutation test of controls after 24 or 72h incubation. / L5178Y/TK+/- cells was positive. In vivo no mutagenic effect was detected in two The results presented show that in-vitro models of varying complexity and origin within Mouse Micronucleus tests, on with intraperitoneally application and another after the respiratory tract clearly differ in their response to TPM, which was used a model repeated inhalation. inhalative toxicant. The tumor cell lines used seem to be better adapted to chemical Nanosized ZnO was tested in 5-days, 14-days and 90-days inhalation studies, in all stress, while the models closer to the in-vivo situation are more vulnerable. studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a NOAEC of 1.5 mg/m3 in the 90-day study. In a Prenatal Developmental Toxicity Study according to OECD TG 414, with repeated inhalation exposure to female Wistar rats from gestation day 6 through 19, maternal 462 toxicity was observed by increase lung weights and inflammations in the lungs. But no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) The nongenomic effects of the mineralocorticoid receptor in transgenic mouse and no increase in external and soft tissue malformations and variations could be heart detected. 1 2 1 2 2 1 The overall result of all the toxicological studies with nanosized and pigmentary ZnO is Winter S. , Schreier B. , Gergs U. , Grossmann C. , Gekle M. , Neumann J. 1 that the toxicological profile of both is very similar. Institute for Pharmacology and Toxicology Medical Faculty, Martin-Luther-University Halle-Wittenberg, Magdeburgerstr. 4, 06112 Halle (Saale), Germany 2 References Julius-Bernstein-Institute of Physiology Medical Faculty, Martin-Luther-University Halle- Studies were sponsored partly by CEFCI LRI and partly by BASF SE. Wittenberg, Magdeburgerstr. 6, 06112 Halle (Saale), Germany
Within the renin-angiotensin-aldosterone system (RAAS), the mineralocorticoid receptor (MR) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. However, survival of patients with severe heart failure is 460 increased when MR blockage is combined with standard therapy suggesting aldosterone, the MR ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. In recent years, evidence Use of REACh Registration Data for Improving Thresholds of Toxicological accumulated that besides its function as a hormone-activated transcription factor the MR Concern (TTC) also functions via nongenomic pathways. Wieneke N., Dorn S., Jakupoglu C., Schäfer C., Sica M., Wiegand C., Mostert V. To investigate the function of the nongenomic effects of the MR in cardiovascular DR. KNOELL CONSULT GmbH, Marie-Curie-Str. 8, 51377 Leverkusen, Germany dysfunction, we generated a transgenic (TG) mouse model expressing a truncated human MR lacking the DNA-binding site (hMRDEF) under control of the cardiac specific α The Threshold of Toxicological Concern (TTC) concept is utilised to identify human myosin heavy chain promoter (αMHC), a model for nongenomic effects of the MR in the exposures that are so low that in-depth toxicological investigations are expendable. This heart. In this mouse model no enhanced mortality could be observed. Body weight (BW), is called "exposure-based waiving". heart weight and relative heart weight were not different compared to wild type (WT) Exposure-based waiving serves to focus available resources on substances with while left atrial weight/BW was increased by 20 % (WT 0,25 ± 0,01 mg/g vs. TG 0,30 ± relevant human exposure potential. 0,02 mg/g, p<0.05, n=12). Important work into establishing TTC values has been published by Munro et al. (1996). Compared to WT mice neither surface electrocardiographic experiments nor The initial report used a database of 613 organic substances compiled from publicly echocardiographic experiments revealed modified parameters for TG mice under basal available sources. In total, 2941 NOELs were collected in this fashion. The Munro (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol concept used the Cramer classification to categorise substances according to their (ISO, 100 µl 1 mM ISO intraperitoneally applied). hazard potential. To uncover the role of aldosterone in the development of cardiovascular diseases We broadened the TTC database by including NOAELs published on the EChA website treatment with aldosterone and high-salt diet (1%) was performed. After 28 days cardiac as per 3 November 2011, containing data for more than 3900 registrations. Only non- function and heart dimensions were analyzed, surface electrocardiographie uncovered gaseous mono-constituent substances with oral NOAELs were included in the TTC increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and QTc interval (61 ± 2 ms database. Organophosphates and genotoxic substances were excluded from the vs. 50 ± 3 ms, p<0.05) in TG (n=7) compared to WT (n=5) animals. These findings database as well as NOAELs obtained for surrogate substances. NOAELs for all probably indicate more sensitive conduction pathways to aldosterone in TG mice. systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. Where appropriate, default assessment factors of up to 6 were used to establish chronic NOAELs for each substance. For every eligible substance, we collected the published CLP category for acute oral toxicity as a potential predictor of overall hazard potential. This gives rise to five categories of acute oral toxicity. A TTC is calculated from the 5th percentile of NOAELs in each of these categories using the REACh rules for establishing DNELs for workers and the general population. This poster presents the preliminary results for more than 1000 substances. The results indicate that the TTC concept becomes more robust when using the very broad EChA
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463 in the control of a variety of cellular processes. PKA exists as an inactive tetramer of a dimeric regulatory (R2) and two catalytic (C) subunits that releases the active C-subunits upon binding of cAMP. Oligomerization is important for regulation of phospholamban activity Stimulation of the mouse T-lymphoma cell line S49 wild-type (wt) with dibutyryl (DB)- cAMP induces apoptosis by an intrinsic, mitochondria-dependent mechanism. Apoptosis Wittmann T., Lohse M. J., Schmitt J. P. - induced by DB-cAMP occurs via a PKA-dependent mechanism, since S49 kin cells Institut für Pharmakologie und Toxikologie, Versbacher Straße 9, 97078 Würzburg, lacking the catalytic subunit of PKA are resistant to DB-cAMP-mediated cell death. DB- Germany cAMP is cleaved by esterases into the biologically active compound N6-MB-cAMP and into 2'-O-MB-cAMP. Phospholamban (PLN) is a heart specific protein located in the membrane of the 2+ sarcoplasmic reticulum. It inhibits the Ca -ATPase SERCA2a, thereby decelerating Other cyclic nucleotides (cNMPs) in addition to cAMP, like cCMP and cUMP can also cytosolic Ca2+ clearance during diastole of the cardiac cycle. Upon phosphorylation the 2+ function as second messengers and activate PKA and cGMP-dependent protein kinase inhibitory activity of PLN on myocyte Ca transport is attenuated. Further, it is believed (PKG)1. Therefore, we investigated the effects of a series of membrane-permeable that phosphorylation favors the formation of (rather inactive) pentamers and that PLN analogues of cAMP, cGMP, cCMP and cUMP in S49 wt und S49 kin- cells on apoptosis. pentamers itselves were an inferior substrate for phosphorylation compared to monomers. This would suggest an important role of PLN oligomerization in the Stimulation with DB-cCMP or DB-cGMP induced neither apoptosis in S49 wt nor in S49 regulation of PLN activity. To prove this hypothesis, we are investigating the patterns kin- cells. Interestingly, we observed apoptosis in S49 wt and S49 kin- cells after and kinetics of PLN phosphorylation in the context of alterations in PLN structure. incubation with membrane-permeable nucleotide acetoxymethyl ester (AM)-analogues The introduction of specific point mutations into the transmembrane region of PLN of cGMP, cCMP, cUMP and also cAMP. Induction of apoptosis occurs via PKA- yielded mutants that are purely monomeric (L37A, I40A and C41F) or favor pentamer dependent and also PKA-independent pathways. A potential role of PKG and of the formation (I45A, V49A). Transfected HEK293 cells expressing these mutants or wild- Exchange protein activated by cAMP (Epac) in the induction of apoptosis is unsolved type PLN were stimulated with forskolin to induce PLN phosphorylation before lysis of and will be explored by specific PKG- and Epac-activators in this system. Investigations cells and Western blot analysis using antibodies directed against phosphorylated PLN. are on the way to identify the targets, the involved signal transduction pathways and the Surprisingly, phosphorylation was increased for both monomeric and pentameric PLN mechanisms of pro-apoptotic actions mediated by cNMP-AMs. after stimulation with 0.25µM forskolin for less than one minute. At increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and References pentamers. For measurement of phosphorylation kinetics stimulation of cells with 2.5µM (1) Wolter S, Golombek M, Seifert R (2011) Differential activation of cAMP- and cGMP- forskolin was stopped at different time points. We found phosphorylation of both PLN dependent protein kinases by cyclic purine and pyrimidine nucleotides. Biochem monomers and pentamers within seconds of stimulation. Differences in phosphorylation Biophys Res Commun. In press patterns became more pronounced when assays were performed at low temperature (14°C). Intriguingly, preliminary analyses suggest that PKA dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. Our data suggest that both PLN pentamers and monomers are suitable substrates for PKA dependent PLN phosphorylation. Unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of PLN monomers in transfected HEK293 cells suggesting an important role of PLN pentamers in the regulation of PLN activity.
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Regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish Wolf N. M.1,2, Abu-Taha I.2, Spiger K.2, Lutz S.3, Meder B.1, Katus H. A.1, Rottbauer W.1, Hippe H. - J.1, Wieland T.2 Apoptosis in S49 cells : 1University Hospital Heidelberg Department of Internal Medicine III, Im Neuenheimer Induction of apoptosis in S49 wt and in S49 cells lacking the catalytic subunit of PKA Feld 410, 69120 Heidelberg, Germany (S49 kin-) with A) DB-cNMPs and B) cNMP-AMs for 72h. 2Institute of Experimental and Clinical Pharmacology and Toxicology University of Heidelberg, Maybachstraße 14, 68169 Mannheim, Germany 3Department of Pharmacology University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany 466 In the heart, nucleoside diphosphate kinases (NDPKs) can interact with heterotrimeric G proteins, thus regulating cAMP synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. We further investigated the interaction of Nebivolol reduces vascular inflammation in spontaneously hypertensive rats NDPK isoforms with heterotrimeric G proteins in the heart in vivo and in vitro using Wu Z., Xia N., Förstermann U., Li H. zebrafish embryos and embryonic fibroblast from NDPK A/B double knockout mice (NDPK A/B KO MEFs). In zebrafish the morpholino-mediated knockdown of NDPK A did Institut für Pharmakologie, Obere Zahlbacher Str. 67, 55131 Mainz, Germany not lead to an obvious phenotype, although the total NDPK activity was reduced. Depletion of NDPK B caused a cardiac phenotype characterized by severely impaired Nebivolol is a third generation β1 receptor blocker with additional effects on endothelial atrial and ventricular contractility and insufficient blood flow. The depletion of NDPK B nitric oxide production. The aim of the present study is to investigate the anti- was associated with a significant decrease of protein levels of the heterotrimeric G inflammatory effects of Nebivolol in vivo. 48 spontaneously hypertensive rats (SHR) were divided into two groups: control or nebivolol treatment group. Nebivolol treatment protein subunits Gβγ, Gαs and Gαi. The knockdown of NDPK C led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in atrium was unaffected. In accordance to the reduced cardiac pumping function, cAMP SHR. The drug had no effect on coagulation. Aorta from nebivolol-treated rats showed levels were significantly diminished in the NDPK B and NDPK C morphants. Similar significantly improved endothelial function. Nebivolol did not change the expression findings were obtained in NDPK A/B KO MEFs. The absence of NDPK A and B resulted levels of aortic NF-kB, but significantly reduced its DNA binding activity. Furthermore, nebivolol decreased the expression of adhesion molecules (e.g. ICAM-1, VCAM-1) and in a decrease of the plasma membrane content of Gβ and Gαs and a significant reduction in cAMP synthesis. The protein expression of the isoform NDPK C was also pro-inflammatory cytokines (e.g. IL-6). In conclusion, nebivolol reduces vascular significantly reduced in the NDPK A/B KO MEFs. The re-expression of NDPK B but not inflammation in experimental hypertension. It inhibits NF-kB activity, decreases the NDPK A rescued the basal cAMP production and the membrane content of G proteins. expression of adhesion molecules and pro-inflammatory cytokine, and improves Interestingly, the overexpression of NDPK C led to a 5-fold enhancement of the cAMP endothelial function. level and a significant increase of the membrane content of Gβ and Gα, and thus rescued the knockout phenotype. Our data indicate, that the NDPK isoforms B and C are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including NDPK B, NDPK C and heterotrimeric G proteins. The 467 isoform NDPK C, with its N-terminal hydrophobic region, might serve as a membrane anchor for the NDPK/G protein complex. Characterization of the cellular activity of PDE4 inhibitors using two novel PDE4 reporter cell lines Wunder F., Quednau R., Barg M., Tersteegen A. 465 Bayer Pharma AG Lead Discovery Wuppertal, Aprather Weg 18a, 42096 Wuppertal, Germany
Induction of apoptosis via PKA-dependent and PKA-independent pathways by Cyclic nucleotide-specific phosphodiesterases (PDEs) play an essential role in cellular cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines signal transduction by regulating the intracellular levels of cAMP and cGMP and, Wolter S.1, Golombek M.1, Schwede F.2, Seifert R.1 therefore, are important pharmacological targets. 1Medizinische Hochschule Hannover Institut für Pharmakologie, Carl-Neuberg-Str. 1, We report here the generation and pharmacological characterization of two novel PDE4 30625 Hannover, Germany reporter cell lines. Plasmid constructs encoding human PDE4B1 or PDE4D3 were stably 2BIOLOG Life Science Institute, Flughafendamm 9a, 28199 Bremen, Germany co-transfected with the beta1-adrenoceptor in a parental cAMP reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for cAMP is a second messenger that plays an important role in intracellular signal intracellular cAMP. In this reporter cell line, cAMP levels can be monitored in real-time transduction of various hormones and neurotransmitters. A major function of cAMP in via aequorin luminescence stimulated by calcium influx through the CNG channel. eukaryotes is the activation of cAMP-dependent protein kinase A (PKA). PKA is involved S106
By using different PDE4 and non-PDE4 inhibitors, we could show that our novel Dexrazoxane (DRZ, ICRF-187) is the only approved drug shown to protect against PDE4B1 and PDE4D3 reporter cell lines specifically monitor PDE4 inhibition with high anthracycline-induced heart failure. The protection is usually ascribed to the iron- sensitivity. PDE4-selective inhibitors alone did not increase basal luminescence levels in chelation by DRZ, which is thought to reduce anthracycline-induced oxidative stress. this experimental setting. However, these inhibitors induced concentration-dependent However, similarly to anthracyclines, DRZ is also an inhibitor of the anthracycline target luminescence signals in combination with the adrenoceptor agonist isoproterenol. In Topoisomerase II (TOP2). We hypothesized that the cardioprotective effects of DRZ are contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant PDE4 mediated by the prevention of the anthracycline-induced DNA damage mediated by expression, PDE4 inhibitors had no effect on isoproterenol-stimulated luminescence TOP2B, the dominant cardiac TOP2 isoform. This was investigated using TOP2B signals. mutants resistant to DRZ, which were expressed in cells depleted of wild-type TOP2 We compared the cellular activity of different PDE4 inhibitors with the in vitro inhibition of isoforms. TOP2B-mediated double-strand DNA breaks were assessed as γ-H2AX. The full-length and truncated (catalytic domain) PDE4D3 from cell lysates. Two different levesl of DSB generated by the TOP2B mutants in response to the anthracycline groups of PDE4 inhibitors could be identified. The first group, including the allosteric doxorubicine (DOX) was indistinguishable from that mediated by a wild-type TOP2B. inhibitors PMNPQ and D159153, showed high cellular activity and much better inhibition Preincubation with DRZ depleted wild-type TOP2B and this was accompanied by a of full-length versus truncated PDE4D3. The second inhibitor group, including classical decrease in the DNA damage following a subsequent exposure to DOX. In contrast, competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably neither TOP2B depletion nor the reduction of DSB by DRZ was seen in DRZ-resistant lower cellular activity and similar inhibitory activity on full-length and truncated PDE4D3. TOP2B mutants. Furthermore, the cardially ineffective DRZ analog ICRF-161, capable The results imply that these novel PDE4 reporter cell lines are well-suited for the of iron chelation but not of TOP2 binding, affected neither the stability of TOP2B, nor the characterization of the cellular activity of PDE4 inhibitors and may also support a better DOX-induced DNA damage mediated by this enzyme. These results indicate that DRZ understanding of the complex PDE4 pharmacology. may exert its cardioprotective effects by reducing the DNA damage mediated by DOX- poisoned TOP2B rather than by iron chelation. They also suggest a cardioprotective function of TOP2B, which is currently under investigation using cardiomyocyte-specific TOP2B mouse knockouts. 468
Plexin-B2 is required for kidney regeneration after acute renal failure 471 Xia J.1, Gröne H. - J.2, Offermanns S.1, Worzfeld T.1 1Max-Planck-Institut für Herz- und Lungenforschung Pharmakologie, Ludwigstr. 43, 61231 Bad Nauheim, Germany Effects of Aminoglycoside Antibiotics on Protein Expression in SerW3-Cells, a Rat 2Deutsches Krebsforschungszentrum Heidelberg Zelluläre und Molekulare Pathologie, Sertoli Cell Line INF 280, 69120 Heidelberg, Germany Zabel R., Horvath A., Stahlmann R. Charité Universitätsmedizin Berlin Institut für Klinische Pharmakologie und Toxikologie, Acute renal failure is a common clinical problem with unsatisfactory therapeutic options Luisenstr. 7, 10117 Berlin, Germany and high mortality in humans. Therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal Aminoglycosides are important antibiotics in the treatment of life-threatening infections, injury. Plexin-B2 belongs to a family of transmembrane receptors which mediate the especially those caused by Gram-negative bacteria. Their nephrotoxic and ototoxic cellular effects of semaphorins. While plexins have first been described in the context of potential is well-known, but little is known about the effects of aminoglycosides on the axon guidance, several recent studies have established them as key regulators of male reproductive system. We studied the effects of four aminoglycosides on Sertoli- organogenesis, the immune system and cancer. We have recently found that Plexin-B2 cells in vitro. Rat Sertoli-cells from the cell line SerW3 were cultivated for 3, 6, and 9 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are days in DMEM supplemented with three different concentrations of amikacin, most sensitive to acute ischemic injury. To study the role of Plexin-B2 during kidney streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, regeneration we generated mice lacking Plexin-B2 specifically in tubular epithelial cells. 100 mg/l). We determined the expression of two junctional proteins (connexin 43, N- Under physiological conditions, these mice displayed normal kidney morphology and cadherin) and one protein of the cytoskeleton (vimentin) by Western blot. Cells were function. In contrast, following ischemia/reperfusion injury, Plexin-B2 conditional solubilized in lysis buffer. Lysates were separated by SDS-PAGE and electroblotted on a knockout mice exhibited severely impaired kidney regeneration. While the renal function PVDF-membrane. After incubation with primary antibodies overnight and horseradish of control mice fully recovered within 3 weeks after injury, Plexin-B2 knockout mice had peroxidase-conjugated secondary antibody the visualization was achieved by a strongly elevated serum creatinine and urea levels associated with increased morbidity chemiluminescence-detection system. In addition, proteins were detected by and mortality. This was accompanied by hyperproliferation of tubular epithelial cells and immunohistochemistry. obstruction of tubular lumina. We conclude that Plexin-B2 is required for regeneration After three days in culture amikacin caused the most pronounced effect. At the lowest after acute ischemic renal injury and that pharmacological interventions activating concentration tested (30 mg/l) connexin 43 and N-cadherin were reduced to 55±19% Plexin-B2 might represent a new therapeutic strategy in acute renal failure. and 92±10% of the controls (n=6). No change was recognized for vimentin (102±16%). Effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). Similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 469 43: 88±15% and 81±15%; N-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; N-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). After incubation for 6 and 9 days the SIRT1 inhibits Rac1-mediated activation of NADPH oxidase effects occurred in the same range. The substances showed no influence on the viability 1 2 3 1 1 1 1 of SerW3 Sertoli-cells up to 300 mg/l in the MTT assay. By immunohistochemistry we Xia N. , Tenzer S. , Daiber A. , Reifenberg G. , Kleinert H. , Förstermann U. , Li H. showed that the localisation of the proteins - connexin 43 and N-cadherin at the cell 1 Institut für Pharmakologie, Obere Zahlbacher Str. 67, 55131 Mainz, Germany membrane and vimentin in the cytoplasm – was not influenced by the aminoglycosides. 2 Institut für Immunologie, Langenbeckstr.1, 55131 Mainz, Germany 3II. Medizinische Klinik, Langenbeckstr.1, 55131 Mainz, Germany
The NADPH oxidase enzyme complex consists of two membrane-bound catalytic subunits (a Nox protein and p22phox) and several cytosolic regulatory components 472 including p47phox, p67phox, p40phox and the small GTPase Rac1. We have previously shown that treatment of apolipoprotein E knockout mice with resveratrol led to a down- regulation of Nox2 and Nox4 in the heart. Our recent data demonstrated that resveratrol Palmitoylation controls BK channel regulation by protein kinase C 1 2 3 4 5 6 1 also reduced the enzymatic activity of cardiac NADPH oxidase. Because activation of Zhou X. - B. , Wulfsen I. , Shipston M. J. , Ruth P. , Wieland T. , Korth M. , Dobrev D. NADPH oxidase enzyme complex is induced by translocation of the regulatory subunits, 1Division of Experimental Cardiology Medical Faculty Mannheim, University of we studied whether the reduced enzymatic activity is due to an inhibition of such a Heidelberg, Mannheim, Germany translocation. Indeed, resveratrol treatment prevented Rac1 membrane translocation 2School of Life Science Hamburg gGmbH University Medical Center Hamburg- from cytosol. Resveratrol is known as an activator and expression enhancer of the Eppendorf, Hamburg, Germany longevity gene sirtuin 1 (SIRT1). We then wanted to find out whether the effect of 3Centre for Integrative Physiology College of Medicine and Veterinary Medicine, resveratrol on Rac1 was mediated by SIRT1. SIRT1 is a histone/protein deacetylase. In University of Edinburgh, Edingburgh, Great Britain vitro incubation of Rac1 with SIRT1 led to a reduction of lysine acetylation. Deacetylation 4Department of Pharmacology and Toxicology Institute of Pharmacy, University of of Rac1 on lysine 166 could be identified by Mass spectrometry analyses. The lysine Tübingen, Tübingen, Germany 166 lies within the p67phox-binding region of Rac1. Consistently, in vitro incubation of 5Institute of Experimental and Clinical Pharmacology and Toxicology Medical Faculty Rac1 with SIRT1 markedly reduced its binding activity to p67phox. In conclusion, we Mannheim, University of Heidelberg, Mannheim, Germany provide evidence that Rac1 is a direct target molecule of SIRT1. SIRT1 deacetylates 6Department of Experimental Pharmacology and Toxicology University Medical Center Rac1 on lysine 166 and thereby inhibits its interaction with p67phox. This is a novel Hamburg-Eppendorf, Hamburg, Germany mechanism of NADPH oxidase inhibition by SIRT1/resveratrol. Large conductance calcium- and voltage-gated potassium (BK) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. The stress-regulated exon (STREX) adds to the BK 470 channel C terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase A (PKA) from excitatory to inhibitory. Here we investigated the mechanisms by which the STREX insert influences BK channel regulation by protein kinase Mutational analysis of the effects of the cardioprotective drug dexrazoxane on C (PKC). Topoisomerase II beta in vitro Activity of BK channels without STREX insert (BK-ZERO), transiently expressed in HEK cells, was inhibited by PKC in inside-out membrane patches (~ 50% inhibition). BK channels Yan T., Deng S., Frensch I., Gödtel-Armbrust U., Wojnowski L. with STREX insert (BK-STREX), however, were insensitive to PKC. Phosphomimetic UNIVERSITÄTSMEDIZIN der Johannes Gutenberg-Universität Mainz Institut für mutation of a PKC phosphorylation site (S700E) in BK-STREX, resulted in a ~50% Pharmakologie, Obere Zahlbacher Straße 67, 55101 Mainz, Germany reduction of basal channel activity, whereas the S700A mutant retained normal activity. To examine whether palmitoylation, and thus association of the STREX domain with the plasma membrane, prevents PKC inhibition of BK channel gating, palmitoylation was S107
abolished by either site-directed mutagenesis (C12:13A) or by pharmacological inhibition of 475 palmitoyl transferases with 2-bromopalmitate (2-BP). Both, mutation and pretreatment with 2-BP resulted in the expression of BK-STREX channels which were sensitive to PKC (~ 50% inhibition of channel activity). No inhibitory PKC effect was observed in patches of the Release of metals from different sections of domestic drinking water installations BK-STREX S700A channel mutant pretreated with 2-BP. In a clonal rat somatomammotroph pituitary cell line (GH3B6), in which PCR products without (ZERO) and Zietz B. P., Richter K., Laß J., Suchenwirth R., Huppmann R. with the 174 bp STREX exon could be identified, the PKC activator PMA blocked channel Governmental Institute of Public Health of Lower Saxony Division of Environmental activity by ~25 %. This inhibition was increased to over 50% when GH3B6 cells were Medicine and Environmental Epidemiology, Roesebeckstraße 4-6, 30449 Hanover, pretreated with 2-BP, indicating that both channel isoforms were functionally active. Germany In summary, the present study demonstrates that palmitoylation of STREX prevents BK channel regulation by PKC, which is mediated by phosphorylation of Ser700, probably Different metals were used as important piping materials in the drinking water supply for by steric hindrance. Our results provide further evidence for a cross-talk between a long time. Due to corrosion metals can leach into the tap water. Of special importance palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic is the toxic element lead. However other heavy metals in drinking water such as copper, regulation of ion channels. nickel and cadmium can also give reason for health concerns. In this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. For the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. After stagnation ten sample 473 volumes were collected in series. Existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. The elements Al, As, Cd, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Sb, Se, U and Zn were Human pleural mesothelial MeT-5A cells are a limited in vitro model system in detected by means of ICP-MS. In total 16 water pipe strands of 11 domestic installation determining potential asbestos-like genotoxic effects of multiwall carbon systems were examined. They comprised 379 single water samples and 5306 single nanotubes parameters. Depending upon the type of plumbing different courses and concentration 1 1 2 1 1 Ziemann C. , Reamon-Büttner S. , Schlichting A. , Brockmeyer H. , Rahmer H. , ranges of the elements could be measured in the tap water samples. Terminal taps or 2 Bellmann B. installation parts were frequently responsible for a release of nickel and in several cases 1Fraunhofer ITEM Genetic Toxicology, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany of cadmium. The concentration courses of the element zinc proved as a good indicator 2Fraunhofer ITEM Inhalation Toxicology, Nikolai-Fuchs-Str. 1, 30625 Hannover, for the allocation of the emission source to a brass containing section of the installation Germany (zinc as an alloy component of brass). One can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable Multiwall carbon nanotubes (MWCNT) are nanomaterials with important technological non-destructive method for drinking water-hygienic investigations of domestic impact. Depending on their diameter, length, and biopersistence, however, some installations. MWCNT seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. Thus, a project funded by the German Federal Ministry of Education and Research (BMBF contract No. 03X0109A) focuses on potential adverse biological effects of different types of MWCNT to enlarge the knowledge base about toxicity 476 determining parameters. This project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. As mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human SV40- Novel interaction partners of the murine TRPC4 protein transformed, non-malignant pleural mesothelial cell line MeT-5A was chosen as the main in vitro model in this project. In the present study part, MeT-5A cells were Zimmermann J., Beck A., Flockerzi V. characterized concerning their usefulness as an in vitro model to study potential Universität des Saarlandes Experimentelle und Klinische Pharmakologie und asbestos-like cytotoxic and genotoxic effects of different MWCNT varieties. Using an Toxikologie, Kirrberger Str. 1, 66421 Homburg, Germany MWCNT-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos In this work novel interaction partners of the murine protein Transient Receptor Potential (2, 10, and 20 µg/cm2) was demonstrated in MeT-5A cells. Cells also showed asbestos- Canonical 4 (mTRPC4) were identified. The TRPC4 protein is the major subunit of a induced increase in DNA-strand breaks and oxidative DNA-damage in the hOGG1- cation channel, residing in the plasma membrane. It comprises six trans-membrane modified comet assay. Thus, MeT-5A cells were responsive to asbestos treatment. domains and cytosolic amino and carboxyl termini. Two major splice variants of the Owing to asbestos potential to induce aneugenic effects and spindle fiber damage, TRPC4 gene exist, TRPC4a (974 aa) and TRPC4b (890 aa), TRPC4b lacks aa 781 to micronucleus induction, determination of numerical chromosome aberration, and altered 864 of the TRPC4a variant. Both TRPC4 variants are co-expressed in endothelial cells, meta-, ana-, and telophase morphology were planned as in vitro endpoints. MeT-5A intestinal smooth muscle and brain. cells were thus initially characterized in this regard and were found to exhibit highly To identify TRPC4-interacting proteins a yeast two-hybrid system, CytoTrap®, which variable chromosome numbers with lower than 10% cells exhibiting a normal diploid allows identification of protein-protein interactions within the cytosol was used. A chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as premade mouse brain cDNA library was screened by the cytosolic amino and carboxyl compared to polychromatic bone marrow erythrocytes in rodents, and a profound terminal parts of mouse TRPC4a (aa 1 to 324; aa 622 to 974). For the carboxyl terminal number of aberrant meta-, ana- and telophases with bridges, lagging chromosomes and part fourteen proteins were identified. To independently prove the interaction, the full- multipolar divisions. In conclusion, MeT-5A cells are of only limited value as an in vitro length cDNAs of all fourteen proteins were cloned, fused to a FLAG-Tag and co- model system to study potential asbestos-like effects of MWCNT and also biopersistent expressed with TRPC4 in HEK 293 cells. Co-immunoprecipitations were performed for fibers. The cells are indeed responsive to asbestos, but unfortunately demonstrate all candidates using both the anti-FLAG-antibody and the antibody for TRPC4. In marked genomic instability and thus limited significance concerning genotoxic effects. addition, changes of cytosolic calcium were monitored and TRPC4 currents were recorded in HEK 293 cells expressing the candidate cDNAs and stably expressing the TRPC4a or TRPC4b and the muscarinic receptor type 2 cDNAs. The Tarbp2 protein, one of the candidates shown to interact with TRPC4, changed calcium influx when co- expressed with TRPC4. In order to identify the domains of TRPC4 responsible for its 474 interaction with the Tarbp2 protein, six TRPC4-GST-fusion proteins covering the carboxyl terminal 353 aa of TRPC4a were expressed in E. coli and used for pull-down experiments. By this approach two domains of TRPC4 could be identified to interact with Waixenicin A inhibits cell proliferation through magnesium-dependent block of Tarbp2. One of these domains is well conserved within the TRPC5 protein, TRPM7 channels corresponding to the result, that TRPC5 and Tarbp2 effectively co-immunoprecipitate, 1,2 3 1 3 3 1 3 Zierler S. , Yao G. , Zhang Z. , Kuo W. C. , Pörzgen P. , Penner R. , Horgen F. D. , too. The Tarbp2 protein has been shown to be a component of the RISC loading 1 1 Fleig A. , Fleig A. complex, also known as the micro-RNA loading complex which is composed of DICER1, 1The Queen’s Medical Center and John A. Burns School of Medicine University of EIF2C2/AGO2 and Tarbp2 (Chendrimada et al. 2005). By its interaction it may link Hawaii, Center for Biomedical Research, Honolulu, Hawaii 96813, United States TRPC4 to pre-microRNA processing. 2Ludwig-Maximilians-University Munich Walther-Straub-Institute for Pharmacology and Toxicology, Goethestrasse 33, 80633 Munich, Germany References 3Hawaii Pacific University Department of Natural Sciences, Laboratory of Marine Chendrimada, T. P., R. I. Gregory, E. Kumaraswamy, J. Norman, N. Cooch, K. Nishikura Biological Chemistry, Kaneohe, Hawaii 96744, United States und R. Shiekhattar (2005). TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436(7051): 740-744. Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. These characteristics are the basis for recent 477 interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native Increased levels of Angiotensin II provoke DNA damage and have influence on TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free DNA repair in mouse kidneys magnesium (Mg2+) concentration. Mutating a Mg2+-sensitive site on the TRPM7 kinase Zimnol A., Brand S., Schupp N. domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg2+. Waixenicin A failed to inhibit the closely homologous Universität Würzburg Institut für Toxikologie, Versbacherstr. 9, 97078 Würzburg, TRPM6 channel and did not significantly affect TRPM2, TRPM4, and CRAC (calcium Germany release activated calcium) channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 The renin–angiotensin system (RAS) plays a crucial role concerning the blood pressure, inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat electrolyte balance and cardiovascular homeostasis. Angiotensin II (Ang II), the active basophilic leukemia cells. Based on the compound’s ability to inhibit cell proliferation hormone of the RAS, in higher concentrations leads to vasoconstriction, oxidative stress through Mg2+-dependent block of TRPM7, waixenicin A or structural analogs may have and hypertension. Hypertensive patients have an increased risk to develop cancer, cancer-specific therapeutic potential, particularly since certain cancers accumulate especially kidney cancer. We have shown in vitro and in vivo, that Ang II is capable to cytosolic Mg2+. cause an elevation of blood pressure as well as DNA damage dose-dependently. 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To investigate whether the high blood pressure or the enhanced levels of Ang II are product into the culture medium following drug exposure for up to 7 days. Efficacy of a responsible for DNA damage, male C57BL/6-mice were equipped with osmotic 0.05% OxBu solution proved close to or even better when compared to both a 0.1% 5- minipumps, delivering Ang II in a concentration of 600ng/kg · min during 28 days. fluorouracil and 0.025% aphidicolin solution. The former being the gold standard of Additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, current AK therapy, the latter is a frequently used inhibitor of human polymerase alpha with the Ang II receptor antagonist candesartan, the vasodilator hydralazine, and the and delta, however, failed to be introduced into clinical use. - In fact, in monolayer antioxidant tempol. DNA damage was analysed with the comet assay. We measured the cultures aphidicolin proved most toxic for normal human keratinocytes which was not base excision repair (BER)-related DNA repair in the kidney with a comet-based in vitro true with OxBu[4]. - repair assay. Furthermore, the distribution and expression of the Ang II-type 1 (AT1) Therefore, these 3D tumour constructs offer a new approach to pre-clinical drug receptor in the kidney was analyzed by immunohistochemistry. assessment and may be added to other 3D models of skin diseases currently gaining Treatment with Ang II led to a significant increase of blood pressure, whereas the increased interest as test platforms. medication with candesartan decreased the systolic blood pressure. The intervention with hydralazine lowered the blood pressure only for a short time. The other substances References had no effect at all on the blood pressure. Genomic damage, quantified with the comet [1] STOCKFLETH, E. & KERL, H. 2006. Eur J Dermatol, 16, 599-606. assay, was augmented by Ang II and improved by all interventions, particularly by [2] RICHARTZ, A., J Enzyme Inhib Med Chem, 23, 94-100. candesartan and tempol. Beyond that, Ang II showed a tendency to reduce DNA repair. [3] ZDRAZIL, B., J Enzyme Inhib Med Chem, 26, 270-9. Treatment with candesartan, hydralazine and tempol increased the repair capacity. [4] SCHWANKE, A., Int J Pharm, 397, 9-18. Furthermore, Ang II tended to result in a downregulation of the AT1 receptor in kidney [5] HOELLER OBRIGKEIT,. Photochem Photobiol, 85, 272-8. tubule cells. Candesartan and ramipril, especially were able to augment the expression of the AT1 receptor, whereas hydralazine achieved the opposite. These results demonstrate that Ang II leads to DNA damage in the kidney independent of blood pressure. Apparently elevated levels of Ang II affect DNA repair and expression 480 of AT1 receptor. To confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in DNA repair. IMA910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple CD8+ and CD4+ T-cell responses associated with improved survival Walter S.1, Kuttruff S.1, Mayer A.1, Schoor O.1, Ludwig J.1, Mayer F.2, Hitre E.3, Nowara 478 E.4, Torday L.5, Rössler B.1, Maurer D.1, Vass V.1; Lindner J.1, Bronte V.6, Pawlowski N.1, Trautwein C.1, Dengjel J.1, Hilf N.1, Weinschenk T.1, Frisch J.1, Reinhardt C.1, Singh H.1 Regulation of HCN channel activity by Cyclic Cytidine 3´, 5´-Monophosphate 1immatics biotechnologies, Tuebingen, Germany Zong X., Krause S., Chen C. - C., Gruner C., Cao-Ehlker X., Fenske S., Wahl-Schott C., 2University of Tuebingen, Tuebingen, Germany Biel M. 3National Institute of Oncology, Budapest, Hungary LMU München, Department Pharmazie, Pharmakologie für Naturwissenschaften Center 4Centrum Onkologii Instytut im. Marii Skłodowskiej-Curie, Gliwice, Poland for Integrated Protein Science Munich (CIPSM), Butenandtstr. 5-13, 81377 München, 5University of Szeged, Szeged, Hungary Germany 6University of Verona, Verona, Italy
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a key role in IMA910 is a novel peptide-based vaccine consisting of 10 HLA-A*02 and 3 HLA-DR controlling cardiac pacemaker activity and are essential for normal function of neuronal binding synthetic peptides that were identified based on natural presentation on human circuits. HCN channels are principally gated by voltage but are coactivated by the cyclic colorectal cancer (CRC) samples. nucleotides cAMP and cGMP which directly bind to a C-terminal binding domain. IMA910 was characterized in a phase I/II trial in advanced/metastatic CRC patients Recently, cyclic CMP (cCMP) was shown to be present in various cell lines and tissues being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. All at concentrations that are comparable to cellular cGMP levels. Moreover, there is recent patients received a single application of low-dose cyclophosphamide for evidence that cCMP can activate cAMP- and cGMP-dependent protein kinases in vivo. immunomodulation. This was followed by repeated IMA910 vaccinations in combination Here, we examined whether cCMP exerts effects on HCN channels. To this end, we with low-dose GM-CSF (cohort 1; n=66) or IMA910 / GM-CSF plus topically applied recorded HCN channel-mediated currents (Ih) in HEK 293 cells that stably express imiquimod (cohort 2; n=26). HCN1, HCN2, HCN3 or HCN4, respectively. Currents were measured either with a Before and post vaccination, patients were analyzed by HLA-multimer assay and intra- standard patch-clamp setup or by employing the planar patch-clamp technology. In cellular cytokine (ICS) assay for CD8+ T-cell responses and by ICS assay for CD4+ T- HCN2 and HCN4 channels, cCMP shifted the membrane potential for half maximal cell responses. As immune status biomarkers, 6 phenotypically defined myeloid derived activation (V0.5) to more positive values. In addition, cCMP accelerated activation while it suppressor cell populations (MDSC1-6) were analyzed prior to immunotherapy. Tumor slowed down deactivation kinetics. The EC50 for cCMP was 30 µM which is about 5 status of patients was monitored repeatedly by CT/MRI according to RECIST and times higher than the EC50 of cGMP. Cyclic CMP is a partial agonist of HCN channels corresponding tumor scans were reviewed centrally. Clinical assessment included since it activates only 65 % of the maximal current obtained with cAMP or cGMP. To disease control rate (DCR), time to progression (TTP), progression-free survival (PFS) identify in vivo effects of cCMP we recorded Ih of murine sinoatrial node (SAN) cells in and overall survival (OS). the presence and absence of 1 mM cCMP. Like for heterologously expressed HCN IMA910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% + + channels, cCMP shifted V0.5 of Ih by about +5 mV. Importantly, the steepness of the of patients mounting multiple CD8 and CD4 T-cell responses, respectively. Patients diastolic depolarization of SAN pacemaker potentials which is mainly determined by the that received the immunomodulator imiquimod presented with significantly more multiple + amplitude of Ih was profoundly increased by cCMP, compared to control conditions. As a CD8 cell responses as detected by ICS (p=0.016). + + consequence of the upregulation of Ih the frequency of SAN pacemaker potentials was Multiple CD8 and multiple CD4 responses were individually associated with increased by about 20 % in the presence of cCMP. Our results suggest that cCMP is a significantly better clinical outcome. The association was most pronounced for patients physiological regulator of HCN channel activity. with both multiple CD8+ and multiple CD4+ responses. These patients had significantly higher DCR at 6 months (p=0.002), improved TTP (p=0.006) and improved PFS (p=0.009) than other patients. Most importantly, a trend for prolonged OS was also observed in these patients (p=0.088, hazard ratio 0.53). 479 In the study population, levels of 5 different MDSC phenotypes were significantly increased as compared to age/gender matched controls. High MDSC levels were associated with fewer immune responses and for MDSC4 and MDSC5 high frequencies A 3D actinic keratosis like construct for assessment of innovative tumour were associated with shorter OS (p=0.007 and p=0.019, respectively). therapeutics To summarize, both HLA-A*02 and HLA-DR restricted peptides in IMA910 were immunogenic. A significantly better clinical outcome of multi-TUMAP responders in Zoschke C.1, Mohamed Ali von Laue C. M. O.1, Höller Obrigkeit D.2, Merk H. F.2, 3 1 comparison to patients with one/no TUMAP response strongly indicates clinical activity Korting H. C. , Schäfer-Korting M. of IMA910. 1 Freie Universität Berlin, Institut für Pharmazie Pharmakologie und Toxikologie, Königin- Luise-Str. 2+4, 14195 Berlin, Germany 2RWTH Aachen, Universitätsklinikum Hautklinik, Pauwelsstr. 30, 52074 Aachen, Germany 3LMU München Klinik und Poliklinik für Dermatologie und Allergologie, Frauenlobstr. 9– 481 11, 80337 München, Germany
The incidence of Actinic Keratosis (AK) has increased dramatically in the last decades Toxicology of the thermally generated dietary genotoxic carcinogen acrylamide 1 1 1 1 1 1 1 and it is considered the most frequent carcinoma in situ today. Especially Eisenbrand G. , Watzek N. , Böhm N. , Feld J. , Scherbl D. , Berger F. , Merz K. H. , 2 2 3 3 1 1 immunosuppressed patients are at high risk to develop invasive squamous cell Lampen A. , Reemtsma T. , Tannenbaum S. R. , Skipper P. L. , Baum M. , Richling E. carcinoma (SCC)[1] which asks for most efficient and well-tolerated AK therapy. Yet, 1Department of Chemistry, Division of Food Chemistry and Toxicology, University of current measures do not fit with these demands. Kaiserslautern Nucleotide analogues, recently identified by molecular modelling[2,3], outperformed the 2Federal Institute for Risk Assessment, Berlin, Germany current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the 3Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, tumour cell line SCC25, in normal human keratinocytes and fibroblasts[4]. As next step in Massachusetts, United States of America pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue OxBu in reconstructed human tumour skin. Based on the 3D Acrylamide (AA), a genotoxic carcinogen (IARC class 2A) is formed in food by thermal construct of SCC developed by Höller and co-workers[5] for start we introduced several treatment from different precursors. After oral ingestion, AA is metabolically epoxidized adaptations with respect to keratinocyte, fibroblast and SCC12 seeding to grow an AK- in the liver by CYP450 2E1 into glycidamide (GA). GA binds to DNA, forming covalent like construct with SCC12 cells forming nests in particular in the epidermis. In addition, adducts, primarily at N7 of guanine (N7-GA-Gua). Both, AA and GA undergo conjugation first experiments with the OxBu on the AK like constructs showed promising results for to glutathione (GSH) to be excreted via urine as mercapturic acids (MA), namely N- an efficient treatment of actinic keratosis. Efficacy was derived from immunohistology acetyl-S-(2-carbamoylethyl)-cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2- (marker for proliferation: Ki-67, marker for SCC: cytokeratin-10, AxL, marker for carbamoylethyl)-cysteine (GAMA). invasion: MMP2, marker for apoptosis: caspase-7, nuclei were stained with DAPI) as In a dose response study, encompassing the dosage range from human dietary well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage exposure levels up to 10 mg/kg bw, female Sprague Dawley (SD) rats on a diet devoid S109
of detectable AA content were gavaged with single doses of AA. Formation of urinary MAs and of N7-GA-Gua DNA adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of N7-GA-Gua was reached. The untreated control group was found to excrete about 0.8 nmol (AAMA plus GAMA) in the urine (16 h), indicating a background of endogenous AA formation. Compared to untreated control, the lowest dosage of 0.1 µg AA/kg bw neither resulted in significantly enhanced MA excretion, nor in a detectable N7-GA-Gua adduct levels in any organ tested (limit of detection, LOD, 0.2 adducts/108 nucleotides). At the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/108 nucleotides) and lung (< 1 adduct/108 nucleotides), but not in liver. At 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg AA/kg bw (about 1- 2 adducts/108 nucleotides). The results of this in vivo study and of further recent research on AA toxicology will be discussed with respect to risk assessment. Exposure of rats to single doses of AA in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to N7-GA-Gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background DNA lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. Thus, the question of significant impact on human background DNA damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment.
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Pharmaco-economic impact of price, volume and demographic development Böcking W., Kirch W. Institut für Klinische Pharmakologie, Medizinische Fakultät der TU Dresden, Fiedlerstr. 27, 01307 Dresden
Health Insurance costs in Germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn EUR in 2009. While costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the Statutory Health Insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. Therefore, the question was asked how pharmaceutical expenses in a large German private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. The data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. Additionally, the two largest indication groups are analyzed in detail. As a result, both price and volume effects drive an overall cost increase. These effects are even stronger in older age groups. This cost increase is not sustainable for the German health insurance system over a longer period of time and will even further increase due to the ageing of the German population.
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A novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development Scheider J.1,2, Winter P.3, Oehlmann J.1 1Dept. Aquatic Ecotoxicology, University of Frankfurt/Main, Germany 2ECT Oekotoxikologie GmbH, Flörsheim/Main, Germany 3GenXPro GmbH, Frankfurt/Main, Germany, E-mail: [email protected]
Alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. However, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. Here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. Chicken embryos were inoculated with different amounts of tributyltin (TBT) and bisphenol-A (BPA). Embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. From identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using SuperTag Digital Gene Expression (ST-DGE, SuperSAGE) profiling. Male and female gonadal tissues both revealed histological aberrations in response to TBT and BPA. Female gonads became masculinized in response to TBT and, vice- versa, BPA-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. In both chemicals mortality increased especially in the most affected sex (TBT: females, BPA: males). The expression profiles of more than 60 million mRNAs revealed massive effects of both chemicals, TBT and BPA, on important cellular signaling pathways. Gene expression differences were most pronounced in the phenotypically most affected sex. Our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. Together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sex- specific gene expression of embryonic development. The project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations.
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Index Behnke F...... 017 Brüning T...... 303 Belkacemi A...... 035 Brunner T...... 168 A Bellmann B...... 473 Brunskole I...... 064, 319 Belz G. T...... 139 Büch T. R. H...... 348 Aasland D...... 001 Bender-Sigel J...... 036 Buchborn T...... 065 Abd Alla J...... 310 Benedikt S...... 126 Büchele B...... 399 Abdel-Aziz H...... 002, 167, 187, 189 Beneke S...... 037, 243, 450 Buchholzer M. - L...... 066 Abel J...... 115 Benkler T...... 037 Büchter C...... 004, 067 Abstiens K...... 078 Berg C...... 346 Buechse A...... 202 Abu-Taha I...... 003, 443, 464 Berger F...... 449, 481 Buesen R...... 200 Ackermann D...... 004, 067, 271 Bergmann M...... 021 Bühler A...... 068, 446 Adam D...... 325 Bernau M...... 362 Buhrke T...... 069 Adler M...... 187 Bert B...... 061 Bumke Scheer M...... 070, 245 Agarwal N...... 278, 422 Besche V...... 017 Bumnaran B...... 042 Agouri S...... 005 Beste K...... 032, 038 Bünemann M...... 006, 044, 255, 288, 306, 330 Ahlbory-Dieke D...... 445 Beuerlein K...... 141 Burhenne H...... 032, 033, 064, 071, 219, 319 Ahles A...... 006 Beyer S...... 213 Burk O...... 384 Ahmadian M. R...... 390 Bhatti A...... 277 Burkart V...... 291 Ahmedat A...... 313 Biel M...... 073, 199, 253, 292, 478 Burke M...... 440 Ahnert-Hilger G...... 334 Bien S...... 174, 121 Burkhalter M...... 072 Aigner A...... 007, 348 Bieschke C...... 420 Burkhardt B...... 376 Akdeli N...... 020 Biller A...... 187 Bürkle A...... 037, 081, 243, 450 Aktories K...... 027, 179, 184, 293, 324, 385, 393 Biller H...... 421 Buschauer A...... 064 Albarrán-Juárez J...... 011 Birk A...... 330 Büscher A...... 142 Alber J...... 133 Birnbaumer L...... 009, 085, 246 Butenschön V. M...... 108 Albrecht A. E...... 008 Birod K...... 355 Albrecht C...... 171, 204, 291, 357 Bisha M...... 005 Allgayer J...... 190, 191 Bittner H...... 193 C Almering J...... 009 Blaich A...... 304 Alpdogan S...... 087 Blanke K...... 039 Calebiro D...... 444 Al-Rawi M...... 010 Blaszkewicz M...... 268, 269, 289 Calzada-Wack J...... 180 Althaus F...... 037, 041 Blatz V...... 040 Camacho Londono J. E...... 246 Althoff T...... 011, 431 Blenn C...... 041 Cambien F...... 342 Alvers M. R...... 218, 378 Blodow S...... 248 Campbell K. P...... 087 Amann K...... 056 Blömeke B...... 080, 089, 353 Campos V...... 268 Ambrosio M...... 012 Bloßfeld M...... 042 Cao-Ehlker X...... 073, 253, 478 Ammer H...... 013 Blume K...... 043 Caron M...... 072 Andersson P. L...... 233, 272 Bock H...... 221 Carrier L...... 309 Andresen K...... 233, 272 Böcking W...... 482 Cascorbi I...... 062, 063, 138, 151, 456 Angela W...... 014 Bodmann E. - L...... 044 Cassee F...... 171 Anger L. T...... 015 Bodor C...... 218 Castonguay J...... 074, 240 Angioni C...... 402 Boeck M...... 213 Celner J...... 247 Antkowiak B...... 388 Boehmert L...... 045 Chai X...... 221 Appel K. E...... 153 Boere A...... 171 Chang C. C...... 070 Arlt O...... 016 Böhm A...... 046, 113 Chen C...... 129 Art J...... 017 Böhm N...... 481 Chen C. - C...... 087, 478 Asbe-Vollkopf A...... 384 Böhme A...... 047, 048, 392 Chen X...... 229 Aumann A...... 018 Böhmer G...... 270 Chhibber A...... 138 Autschbach R...... 362 Böker I...... 049 Chiamvimonvat N...... 225 Bollmann F...... 050 Chovolou Y...... 004, 067, 075, 076, 077 Christ T...... 305, 432 B Bollmann P...... 051 Bolz S. - S...... 014 Christmann M...... 001, 428 Chubanov V...... 078 Baarlink C...... 132 Bonaterra G. A...... 110 Bonifacio E...... 052 Chung B...... 079, 177 Bach A...... 019 Clemens J...... 080 Bacher M...... 225 Bonifas J...... 089 Boosen M...... 053, 097 Closs E. I...... 036, 126, 427 Bachmann H. S...... 020 Collins M...... 176 Bader J...... 041 Bopp A...... 054 Borchert P...... 316 Conrad A...... 146, 147 Bakhaus K...... 021 Constantin C...... 422 Bali K. K...... 022, 438 Bordet T...... 096 Boris B...... 093 Costigan M...... 438 Balszuweit F...... 023 Cramer B...... 269 Banerji S...... 206 Börner C...... 207 Bornscheuer U...... 213 Creed M...... 214 Bantz C...... 186 Creutzenberg O...... 459 Baranowska-Kuczko M...... 239 Borrmann T...... 042 Böttcher K...... 285 Cui L...... 241 Baranyai D...... 024 Culmsee C...... 088, 091, 225, 288, 290, 326 Barg M...... 467 Bouche E...... 221 Barknowitz G...... 025 Bourrier E...... 099 Barlow S...... 026 Braeuning A...... 055, 401 D Barron D...... 327 Brand E...... 136, 154, 342, 354, 366 Barros Pires V...... 027 Brand S...... 477, 056 Dahmen U...... 238 Barth H...... 028, 102, 184, 229 Brand S. - M...... 136, 154, 342, 354, 366 Dähnert I...... 039 Bastos R...... 261 Brandes R. P...... 185 Dai G...... 210, 251 Batke M ...... 426 Brandmayr J...... 304 Daiber A...... 050, 427, 469 Bätz J...... 029 Brandt D...... 209 Dao V. T. - V...... 005, 419 Bauch C...... 030, 031, 098 Brandt U...... 234 Dartsch D. C...... 128 Bauer F...... 017 Brauch H...... 270 Dascal N...... 242 Bauer L...... 193 Bräuer R...... 134 Dasenbrock C...... 111 Baum M...... 481 Braun A...... 180 De Toni E. N...... 228 Baumann J...... 403 Braun M...... 271 Dearman R. J...... 031 Bäumer W...... 351 Brauneis M. D...... 057 Debiak M...... 081 Bayer T...... 171 Bräunig J. H...... 058 Dedman J. R...... 281 Becirovic E...... 199 Brecht B...... 392 Dedon P. C...... 241 Beck A...... 035, 476 Breit A...... 059 Degen G. H...... 114, 197, 268, 269 Beck K. - F...... 053, 097 Breitenkamp A. F. S...... 060 Deigendesch U...... 376 Beck M...... 053, 097 Brenneis C...... 402 Deiss K...... 082 Becker E...... 197 Brenneisen A...... 081 Del Turco D...... 185 Becker L...... 068 Breves G...... 274 Dembla S...... 083 Becker W...... 407 Brigelius-Flohé R...... 025 Deng S...... 470 Beckert C...... 032 Brigo A...... 015 Dengjel J...... 480 Beckert U...... 033 Brill S...... 381, 458 Denzinger S...... 084 Bedke J...... 384 Bringmann G...... 008 Dettinger K...... 320 Beer-Hammer S...... 279 Brockmeyer H...... 473 Devanathan V...... 085 Beermann S...... 034, 071 Bronte V...... 480 Devor M...... 438 Beetz N...... 260 Bros M...... 017 Dhayade S...... 086 Begunk R...... 174 Brosda J...... 061 Dhein S...... 039, 188, 193, 359 Behm C...... 114 Bruckmüller H...... 062, 456 Diabaté S...... 010 Bruhn O...... 063 S111
Dibué M. A. H...... 087 Fischer J. W. ... 106, 107, 116, 127, 210, 251, 265, 331, Griessinger C...... 086 Diedrich A...... 421 332, 389, 419, 434 Griewel E...... 128 Diegel M...... 008 Fischer M...... 111 Grittner D...... 224 Diel P...... 197, 291 Fleck S. C...... 112 Groh I...... 129 Diemert S...... 088 Fleig A...... 474 Grohm J...... 091 Diener M...... 431 Flick B...... 200 Gröne H. - J...... 468 Dierolf D...... 089, 353 Flik G...... 252 Groneberg D...... 130, 264 Dietrich A...... 090 Flockerzi V. …...... 009, 019, 035, 242, 350, 454, 476 Gros N...... 346 Dietrich C...... 103 Flockerzie K...... 222 Grösch S...... 355 Diewock T...... 063 Florian S...... 025, 383 Groschner K...... 361 Dinh N. - D...... 403 Flößer A...... 113 Gross S...... 108 Dionisi F...... 327 Fokkens P...... 171 Grosse R...... 131, 132, 209 Dirsch V. M...... 017 Föllmann W...... 114 Grosser G...... 133 Dobrev D...... 003, 443, 472 Fomin N...... 424 Grosshennig A...... 421 Dodel R...... 225 Forst A. - L...... 413 Grossmann C...... 462 Doenst T...... 120, 452 Förster I...... 204 Gröters S...... 381 Doleschal B...... 361 Förstermann U...... 017, 166, 363, 404, 427, 466, 469 Groth H...... 303 Dolga A...... 091, 225 Foth H...... 461 Grötzinger K...... 359 Doll C...... 175, 297 Fox J. G...... 241 Grube M...... 169, 430 Doller A...... 106, 107 Fralish G...... 072 Grund S...... 134 Domes K...... 232, 295, 304 Framke T...... 421 Grune B...... 218, 378 Döring B...... 021 Frank H...... 383 Gruner C...... 478 Dorn S...... 391, 460 Frank N...... 262 Grünweller A...... 007 Dörr J...... 092 Frauenstein K...... 115 Grzeda E...... 239 Doti N...... 091, 225, 326 Fred K...... 199 Gudermann T. .... 059, 078, 090, 165, 248, 262, 348, 413 Drüppel V...... 366 Freese C...... 148 Gundert-Remy U...... 015, 135, 254, 294 Duning K...... 136 Freichel M...... 009, 019, 246, 454 Günther M...... 228 Durner J...... 093, 320, 417, 418 Frensch I...... 470 Gupta P...... 022 Frenzel E...... 287 Guske K...... 136, 154, 354 Freudenberger T...... 116 Guth K...... 101, 137 E Freyberger A...... 117 Friebe A...... 130, 163 Eberhardt M...... 371 Frimat J. - P...... 403 H Eberhardt W...... 106, 107 Frisch J...... 480 Eberle M...... 355 Frischauf I...... 361 Haack M...... 025 Ebert F...... 094 Fritsche E...... 115, 332 Haarmann-Stemmann T...... 115 Eckert A...... 095 Fritz G. ….... 054, 067, 075, 076, 077, 172, 230, 271, 447 Haas B...... 247, 256 Eckert G. P...... 096 Frohnweiler K...... 242 Haas K...... 395 Eckert S. H...... 096, 226 Frohwein T...... 118 Habermeier A...... 126, 427 Eckmann J...... 096 Frölich N...... 029 Haenel M. W...... 289 Ehlers A...... 205 Fromm M. F...... 119, 299, 370 Haenisch B...... 258 Eisel F...... 053, 097 Frotscher M...... 221 Haenisch S...... 062, 063, 138, 456 Eisele K...... 102 Frühwald J...... 249 Hafner S...... 395 Eisenbrand G...... 449, 481 Fu X...... 310 Hagedorn I...... 085 El-Armouche A...... 003, 215 Fuchs B...... 090 Hagemann S...... 334 Ellen D...... 059 Fuchs N...... 332 Hagn M...... 139 Elsaesser K...... 288 Fuhr U...... 335 Hahn S...... 194 Eltze T...... 030, 031, 098 Funk K...... 133 Halekotte J...... 456 Emami-Nemini A...... 099 Fürst R...... 211 Halwachs S...... 448 Emons J...... 215 Fux E...... 445 Hamann M...... 140 Endo S...... 158 Hammelmann V...... 073 Engel J...... 384 Hammer E...... 213 Engelhardt B...... 303 G Hampl V...... 262 Engelhardt S...... 006, 123, 180, 345, 346, 455 Han P. - L...... 058 Engeli S...... 100 Galuska C. E...... 133 Handler N...... 017 Engst W...... 025, 153, 383 Galuska C. G...... 021 Handschick K...... 141 Enzmann H...... 247 Ganchev B...... 270 Hanhsen B...... 075, 076 Epe B...... 190, 191 Gao J. - L...... 369 Hansen U...... 045 Erk T...... 327 Garbers C...... 265 Hardelauf H...... 403 Erker T...... 017 Garbe-Schönberg D...... 333 Hareng L...... 218 Erkes A...... 303 Garcia Garrido M...... 199, 292 Harteneck C...... 142, 390 Ermler S...... 113 Gauer S...... 384 Hartmann M. F...... 021, 133 Ernst K...... 184 Geiger H...... 384 Hartmann R. K...... 007 Esch H...... 008, 358 Geisslinger G...... 185, 234, 235, 355, 402 Hartwig A...... 143 Eschenhagen T...... 309 Gekle M...... 462 Hartwig C...... 071, 144 Escher S...... 426 Genth H...... 172 Hasan N...... 249 Esposito I...... 180 Gergs U...... 051, 188, 337, 338, 462 Hasenfuß G...... 309 Esselen M...... 129, 405 Gerhard R...... 125, 287 Häsler R...... 151 Esser C...... 291 Gerloff K...... 204, 357 Hassan M...... 053 Eyer F...... 181, 425 Gerlofs-Nijland M. E...... 171 Hauser I...... 384 Gerwin H...... 335 Hausherr V...... 372, 403 Gessner J. E...... 279 Hauswald M...... 348 F Geyer J...... 021, 133 Havermann S...... 145, 230, 271 Giegold O...... 016, 284 Hawkins E. D...... 139 Fabian E...... 040, 101, 137, 203 Gierschik P...... 068, 257, 446 Heeren J...... 177 Fahrer J...... 102, 229 Gilsbach R...... 120, 336, 452 Heide H...... 234 Fan L...... 221 Gioioso V...... 140 Heiland A...... 146, 147 Fauler J...... 377 Girod M...... 045 Heim H. - K...... 116 Faupel F...... 333 Glahn F...... 461 Hein L...... 120, 182, 221, 260, 336, 452 Faust D...... 103 Glatt H...... 025, 259, 383 Heinemeyer G...... 043, 146, 147 Fecher-Trost C...... 092, 350 Glatt H. - R...... 153 Heinen B...... 160 Fehrmann E...... 423 Gliesche D...... 121 Heinick A...... 148, 173 Feil R...... 086, 424 Gnad T...... 122 Heinrich R...... 417, 418 Feil S...... 086 Göbel P...... 123 Helms N...... 328 Feld J...... 449, 481 Gödtel-Armbrust U...... 024, 282, 470 Hempel C...... 149 Feliszek M...... 104 Gold M...... 225 Hengstler J. G...... 289, 372, 403 Fels B...... 105 Golka K...... 289 Hennekes F...... 277 Fender A. C...... 106, 107, 317 Gollasch M...... 142 Hennen J...... 080 Feng Y...... 108 Golombek M...... 465 Hennig L...... 359 Fenske S...... 073, 253, 478 Goltz L...... 124, 273, 433 Henninger C...... 150 Ferreira I...... 261 Goth C...... 101 Henrike B...... 151 Ferreiros N...... 355 Gotic M...... 420 Hensel A...... 049 Fietz D...... 021 Goy S...... 125, 287 Hentschel K...... 152, 285 Filser J. G...... 109 Graf J...... 126 Hermanns M. I...... 186 Fink C...... 049, 110 Grandoch M...... 127, 210 Herr F...... 169 Fink H...... 061 Grant A...... 402 Herrmann K...... 153 Firtz G...... 150 Gratz M...... 174 Herrmann M...... 136, 154, 342 Fischer D...... 134 S112
Herrmann S...... 155, 371 Jergas B...... 104 Koch K...... 194 Herzig S...... 060, 300 Jeruschke S...... 142 Koch M...... 160 Hescheler J...... 087 Jeske E...... 367 Koch S...... 199, 292 Hess A...... 442 Ji R. - R...... 402 Koch T...... 065, 207 Hesse D...... 177 Jobi K...... 106 Koch W...... 111, 421 Hessel S...... 156, 205 John A...... 156 Koenig C...... 257 Heukamp L...... 020 John H...... 181 Koesling D...... 208, 340, 380 Heusser K...... 421 Joost H. - G...... 177 Köhler D...... 085 Hey V...... 453 Jordan J...... 100, 421 Kojda G...... 005, 419 Heym R...... 320 Josch S...... 230 Kojima N...... 158 Hieber M...... 345 Juergens U. R...... 313 Kolatsie M...... 142 Hieke B...... 157 Junghanns S...... 283 Kolbe T...... 334 Hildebrandt I...... 158 Jurida L...... 141 Kolesova N...... 096 Hilf N...... 480 Just I...... 125, 287, 334 Kolle S...... 018, 030, 098, 200, 201, 202, 203, 442 Hill K...... 159, 373 Just S...... 175 Kolling J...... 204 Hillenbrand M...... 424 Kolrep F...... 205 Hiller K. - A...... 387 König P...... 130 Hillmann A...... 171 K Konrad M...... 414 Hintzsche H...... 160 Köritzer J...... 037 Hinz B...... 316, 368 Kabesch M...... 322 Korth M...... 472 Hinz C...... 032, 038 Kadow S...... 291 Korting H. C...... 479 Hippe A...... 331 Kaetzel M. A...... 281 Koshkina O...... 186 Hippe H. - J...... 003, 464 Kaever V...... 032, 038 Koshova O...... 398 Hirsch-Ernst K...... 245 Käfferlein H. U...... 303 Kosowski J...... 185 Hitre E...... 480 Kahl E...... 065 Kotsiou A...... 170 Hiyasat B...... 039, 359 Kahlert K...... 182 Kozlowska H...... 239 Höcherl E...... 093 Kahnt M...... 183, 433 Kozlowski M...... 239 Hoefeld H...... 384 Kaina B...... 001, 198, 277, 428 Kracht M...... 141 Hoensch H...... 161 Kaiser E...... 184 Kraft K...... 187 Höfer N...... 197 Kälble S...... 064, 322 Kraft P...... 085 Hoffmann C...... 029, 280 Kallenborn-Gerhardt W...... 185, 234, 235 Krasel C...... 288, 306 Hoffmann F...... 162 Kallies A...... 139 Krätke R...... 206 Hoffmann K...... 266 Kalwa H...... 090 Kraus J...... 207 Hoffmann L. S...... 163 Kamp H...... 200 Krause E...... 390 Hofmann F...... 232, 295, 356 Kämpfer N...... 312 Krause S...... 478 Hofmann T...... 078, 164 Kamph H...... 445 Krawutschke C...... 208 Högg C...... 165 Kansy M...... 015 Kremerskothen J...... 136 Hohlfeld J...... 421 Karginov V...... 028 Kress M...... 422 Höller Obrigkeit D...... 479 Karvouni H...... 170 Kreßner C...... 209 Höllt V...... 065, 207 Kasper J...... 186 Kretschmer I...... 210, 251, 265 Holmes G...... 369 Kasprzak B...... 366 Kretz O...... 336, 452, 454 Holz O...... 421 Katrin T...... 361 Kretzschmann V...... 211 Holzgrabe U...... 029, 374 Katus H. A...... 464 Kriebs U...... 009, 019, 454 Homey B...... 331, 332 Kaul A...... 309 Krieg S...... 088 Honscha K. U...... 448 Kaumann A. J...... 307 Krifka S...... 387 Honscha W...... 448 Kazmaier U...... 211 Kroemer H. K. .... 046, 113, 121, 152, 169, 174, 213, 285, Horgen F. D...... 474 Kebig A...... 441 430 Horke S...... 166 Kehe K...... 023, 349 Kroetz D...... 138 Horvath A...... 471 Kelber O...... 002, 049, 110, 167, 187, 189 Kroker M...... 212 Hoser S...... 167 Keller N...... 188 Kroll C...... 184 Hostettler N...... 168 Kellner M. B...... 331 Kruck S...... 384 Hoyer P...... 142 Kelm M...... 046 Krug N...... 421 Hrkac T...... 333 Kerb R...... 270 Kruppke A. S...... 385 Hruba E...... 103 Keren-Raifman T...... 242 Krutmann J...... 115, 171, 332 Hübel N...... 311 Kessler W...... 109 Kuan S. L...... 102 Hubeny A...... 169 Khalid S...... 262 Kubatzky K...... 324 Huber M...... 258 Khan J...... 131 Kübler W...... 436 Hudu M...... 170 Khayyal M. T...... 002, 189 Kuhn M...... 108 Huhse B...... 218 Khobta A...... 190, 191 Kühne E...... 446 Hullmann M...... 171 Kietzmann M...... 351 Kuner R...... 022, 278, 422, 438 Hülsenbeck J...... 150, 172 Kilic A...... 256 Kunz P. C...... 230, 271 Humpf H. - U...... 269 Kim S. W...... 088 Kuo W. C...... 474 Huppmann R...... 475 Kimber I...... 030, 031 Kurejova M...... 422, 438 Husainie D...... 438 Kinscherf R...... 110 Kurtbay G...... 213 Husser X...... 173, 281 Kipschull S...... 163 Kurz C...... 226 Hußner J...... 174 Kirch W...... 047, 048, 124, 183, 231, 273, 283, 392, 433, Kusche-Vihrog K...... 366 Hutschenreuther A...... 314 482 Kuschka J...... 140 Hüttner J...... 157 Kirchner T...... 228 Kuttruff S...... 480 Huwiler A...... 016 Kirkpatrick C. J...... 186, 349, 352 Kynast K...... 185 Kitsera N...... 190, 191 Klebe G...... 288 I Kleemann J...... 384 L Klein A...... 192 Iaroshenko V. O...... 121 Klein D...... 109 Laggerbauer B...... 123 Ibrahim A. F...... 007 Klein J...... 386 Lamarque L...... 099 Igel S...... 270 Klein S...... 193 Lambrecht C...... 214, 343 Illes P...... 328, 329 Klein T...... 252 Lämmle S...... 215 Illing S...... 175 Kleine-Ostmann T...... 160 Lampen A...... 045, 069, 070, 156, 205, 245, 481 Iro H...... 370 Kleinert H...... 017, 050, 194, 404, 469 Lamyel F...... 312 Issinger O. - G...... 300 Klenk C...... 195 Lan M. - H...... 164, 203, 216, 381, 458, 459 Kleuser B...... 046, 351 Lanciotti S...... 207 Kliesch S...... 021 Landsiedel R. .... 018, 030, 031, 040, 098, 101, 137, 164, J Kliewer A...... 196 201, 202, 203, 216, 217, 218, 378, 381, 442, 458, Klöckner J...... 029, 374 459 Jäckh C...... 040 Klugbauer N...... 074, 240 Lang A. E...... 027 Jäger J...... 261 Kluge R...... 177 Lange-Grünweller K...... 007 Jäger R...... 130, 176, 340 Klugstedt C. T...... 389 Langer T...... 315 Jahrsdörfer B...... 139 Kluxen F. M...... 197 Langeslag M...... 422 Jakupoglu C...... 460 Kneba M...... 063 Langsch A...... 218 Janousek J...... 039 Kneilling M...... 086 Laschke M. W...... 035 Janßen N...... 441 Kneuer C...... 245 Laß J...... 475 Jaschke A...... 079, 177 Knieriem T...... 202 Lau D...... 453 Jastrow C...... 160 Knizhnik A...... 198 Laue S...... 219 Jatho A...... 178 Knoess W...... 066 Leblay M...... 099 Jedlitschky G...... 430 Knutson C...... 241 Lechner S...... 422 Jehle D...... 179 Koch A...... 421 Lederer M...... 220 Jennissen K...... 163 Koch E...... 375 Leemhuis J...... 221 Jentzsch C...... 180 S113
Leffers L...... 094 Meißner M...... 078 Nowak G...... 134 Lehmann C...... 025 Melcher R...... 327 Nowara E...... 480 Lehmann L...... 008, 244, 298, 358, 364 Melching-Kollmuss S...... 250 Nuber S...... 280 Lehmann M. - L...... 289 Melchior-Becker A...... 251 Numtip W...... 109 Leiss V...... 222 Menger M. D...... 035 Nunes F...... 105, 148, 158, 173, 281 Lemoine H...... 223, 224 Menzen M. H...... 252 Nürnberg B...... 085, 222, 279, 390 Lenders M...... 354, 366 Mergia E...... 380 Nürnberg P...... 060 Lenhardt I...... 238 Merk H. F...... 479 Nüsing R. M...... 431 Leoni A. - L...... 360 Merz K. H...... 481 Nußhag C...... 282 Lepcynzsky P...... 422 Metzler M...... 112 Letsche T...... 225 Meurs H...... 252, 365 Leu C...... 093 Meusch M...... 332 O Leuner K...... 096, 226 Meyer K...... 345 Lewin G...... 422 Meyer zu Schwabedissen H...... 121, 152, 174, 213, 285 Oberleithner H...... 366 Lex K...... 081 Michalakis S...... 199, 253, 292 Oberwinkler J...... 414 Li H...... 017, 050, 363, 404, 427, 466, 469 Michaud M...... 096 Obst A...... 042 Licht O...... 227 Middendorff R...... 454 Oehlmann J...... 483 Liebl J...... 228 Mielke H...... 254, 294 Oelze M...... 050 Lies B...... 130 Mikito T...... 014 Oertel R...... 124, 183, 283 Lillich M...... 229 Mikusev V...... 096 Oesch F...... 203 Lindner J...... 480 Milde M...... 255 Oestreicher D...... 346 Lindner S...... 448 Mitschke M. M...... 256 Oetjen E...... 309 Lindtner O...... 043 Mobley M. W...... 241 Offermanns S...... 011, 014, 347, 382, 422, 431, 468 Linnebacher M...... 316 Moepps B...... 257 Ogrissek N...... 284 Lipp P...... 019 Mohamed Ali von Laue C. M. O...... 479 Okpanyi S. N...... 187 Lippmann D...... 025 Mohr F...... 414 Olausson J...... 454 Lisowsky S...... 230 Mohr F. - W...... 193, 359 Olbert M...... 285 Lochner S...... 231 Mohr K...... 029, 374, 441 Oldenburger A...... 365 Loga F...... 232 Moisch M...... 349 Oldenhage C...... 224 Lohr C...... 233, 272 Molderings G. J...... 258 Ollenschläger K...... 286 Lohse M. J...... 012, 029, 082, 084, 099, 182, 280, 339, Mönch B...... 017 Olling A...... 125, 287 439, 444, 463 Monien B. H...... 025, 153, 259, 383 Oppermann S...... 288 Loos C...... 395, 396, 397 Mönnich J...... 218 Orth J...... 179, 324, 393 Looser R...... 445 Monroy-Ordoñez E. B...... 260 Oswald S...... 151 Lorenz J. E...... 234 Monteiro-Riviere N...... 459 Othman E. M...... 412 Lorenz K...... 082, 084, 182, 339, 439 Moormann O...... 289 Ott T...... 424 Löschmann Y...... 446 Moosmang S...... 304 Ovsiannikov D...... 289 Lother A...... 336 Morange P. E...... 342 Öxler E...... 290 Lu L...... 305 Moretti A...... 455 Lu R...... 185, 235, 402 Möricke A...... 315 P Lübker C...... 236 Mostert V...... 391, 460 Lubura M...... 237 Moura D...... 261 Padberg E...... 291 Luch A...... 218, 378 Muehlich S...... 262 Panas A...... 010 Ludwig A...... 155, 371 Mueller D...... 161 Paparizos C...... 292 Ludwig J...... 480 Mühle A...... 120 Papatheodorou P...... 293 Ludwig K...... 151 Mühlfriedel R...... 199 Papavlassopoulos H...... 333 Lukowski R...... 295, 410, 415 Müller C. E...... 042, 122, 263 Park C. - K...... 402 Lunov O...... 395, 396, 397, 398, 399,400 Müller D...... 264 Parry N. M. A...... 241 Lupp A...... 238, 321 Müller F. U. ..105, 148, 158, 173, 281, 379, 389, 416, 423 Partosch F...... 254, 294 Lüth A...... 046 Müller H...... 141 Passauer J...... 377 Lutz B...... 278 Müller J...... 265 Patel A...... 411 Lutz G...... 081 Müller N...... 061 Patrucco E...... 295 Lutz S...... 003, 178, 215, 318, 464 Müller S...... 266 Pautz A...... 017, 050, 194 Müller W...... 162, 267 Pavic G...... 107 Müller W. E...... 096, 226 Pawlowski N...... 480 M Müller-Fielitz H...... 311 Peiper S. C...... 369 Münch K...... 119 Penner R...... 474 Maarsingh H...... 252, 365 Munoz K...... 268, 269 Maas R...... 119, 299 Penski J...... 430 Mürdter T...... 270 Pera T...... 252 Machala M...... 103 Murphy P. M...... 369 Mader R...... 253 Pereira L...... 261 Muschler M...... 370 Perez-Bouza A...... 362 Magiatis P...... 170 Music S...... 420 Maheswaran V...... 398 Perocchi F...... 091 Muß N...... 346 Peter M...... 256 Maier J...... 257 Mutlu S...... 122 Maier M...... 320 Peters K...... 368 Majer M...... 415 Petersen S...... 213 Petrich A...... 296 Makarova E. A...... 051 N Malinowska B...... 239 Petzel C...... 387 Mallmann R...... 074, 240 Nagel M...... 142 Petzinger E...... 133 Mangelsdorf I...... 227, 426 Nagy G...... 422 Peuker K...... 297 Mangerich A...... 241, 243 Nagy N...... 127, 419 Pexa K...... 085 Mannebach S...... 009, 019, 242, 246, 454 Nakhla A...... 271 Pfeifer A...... 122, 163, 247, 256 Marczynski B...... 303 Nauert C...... 187 Pfeiffer E...... 112 Marko D...... 376 Nem D...... 024 Pfeiffer K...... 335 Marquardt C...... 010 Neser S...... 233, 272 Pfeilschifter J...... 016, 053, 097, 284 Martello R...... 243 Neske C...... 016 Pfeilschifter W...... 355 Martiensen B...... 291 Nestler S...... 024 Pfenning C...... 298 Martin C...... 362 Nestorovich E...... 028 Pfistermeister B...... 299 Martínez Jaramillo D...... 244, 358 Neubauer P...... 273 Pfreimer M...... 257 Marx-Stoelting P...... 245 Neumann D...... 034, 071, 144, 319 Philipp M...... 072, 413 Maskos M...... 186 Neumann J...... 051, 188, 337, 338, 462 Philipp S...... 083, 249 Mathar I...... 246 Neundorf I...... 300 Pich A...... 334 Mathäs M...... 282 Ng D. Y. W...... 102 Pichler M...... 221 Maurer D...... 480 Nicken P...... 274 Piekorz R...... 085 May M...... 172 Nieber K...... 042, 167 Pietsch J...... 124 Mayer A...... 480 Niederberger E...... 185 Pietsch M...... 300 Mayer F...... 480 Niefind K...... 300 Pinheiro H...... 261 Mayer P...... 116, 247 Niemann B...... 045 Pink M...... 301, 367, 437 Mayer T...... 090 Nies A...... 275, 384 Plassmann S...... 360 Mayer W...... 202 Niessen K. V...... 276 Platzek T...... 302 Mayr D...... 228 Nieswandt B...... 085, 180 Plesnila N...... 088, 091, 326 McFaline J. L...... 241 Nikolova T...... 277 Plöttner S...... 303 Meder B...... 464 Njoo C...... 278 Plückthun A...... 195 Mederos Y Schnitzler M...... 078, 248, 413 Nobrega J. N...... 214 Podschun R...... 333 Mehling A...... 030, 098, 101, ,201 Nölke T...... 385 Pohl C...... 349, 352 Meiser J...... 083, 249 Nörenberg W...... 149 Pöll F...... 196, 297 Meissner L...... 091 Novakovic A...... 279 Polzin A...... 046 Poomvanicha M...... 304 S114
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S...... 306 Rössler B...... 480 Schneider T...... 087 Proksch P...... 004 Rothkirch D...... 337 Schnepf R...... 370 Propping S...... 307 Rötrige A...... 342 Schnorr S...... 371 Pruss R...... 096 Rottbauer W...... 464 Schöbel N...... 372, 403 Prywes R...... 262 Rötzer K...... 292 Scholich K...... 355, 402 Pütz C...... 109 Roux T...... 099 Schöll-Weidinger M...... 253 Ruh H...... 010 Scholz A...... 019 Rühle F...... 120 Scholz D...... 256 Q Rulf K...... 338 Scholz R...... 291 Ruppert C...... 339 Scholze A...... 373 Qiu H...... 024, 282 Russwurm C...... 340 Schön I...... 461 Qiu Y...... 108 Russwurm M...... 208, 340, 424 Schöndube F...... 309 Quednau R...... 467 Ruth P...... 410, 415, 472 Schönlau J...... 223 Queisser N...... 308 Ruvo M...... 326 Schonn I...... 128 Quentin T...... 309 Rybalkin S...... 295 Schoor O...... 480 Quitterer U...... 310 Schrader F...... 288 Schrader T...... 160 S R Schrage R...... 374 Schramm E...... 375 Sabha D...... 359 Raaf J...... 300 Schreck I...... 376 Salameh A...... 039 Schreier B...... 462 Raasch W...... 311 Salb K...... 341 Rabausch B...... 251 Schreiner M...... 118 Saljè K...... 169 Schrenk D...... 233, 272, 405 Racké K...... 312, 313 Salomon A...... 154, 342 Radeke H. H...... 016, 284 Schrepper A...... 452 Sander S. E...... 214, 343 Schröder A...... 334 Radicke S...... 314, 432 Sanders S. - J...... 344 Radtke S...... 315 Schröder B...... 274 Sandner A...... 461 Schröder D...... 377 Rahmer H...... 473 Sandner P...... 286 Ramba B...... 178 Schröder K...... 185 Sarikas A...... 345, 346 Schröder M...... 016 Ramer R...... 316, 368 Sass S...... 243 Ramirez T...... 200, 445 Schroeder M...... 218, 378 Sassmann A...... 347 Schrör K...... 046, 106, 107, 113, 317 Ramirez-Hernandez T...... 030, 031, 098, 203 Satagopam V. P...... 022 Rasenberger B...... 428 Schroth W...... 270 Sauer U. G...... 218, 378 Schulte J...... 020 Rasonabe Z...... 446 Schacht D...... 104 Rassaf T...... 046 Schulte J. S...... 379, 416, 423 Schaefer L...... 053 Schulte K...... 104, 281, 380 Rastan A...... 039 Schaefer M...... 149, 159, 192, 193, 314, 373, 414, 436 Rauch B. 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M...... 348 Schulz S...... 162, 175, 196, 267, 296, 297, 321 Rauwald H. - W...... 359 Schäfer M...... 349 Ravens U...... 305, 307, 432 Schulze J...... 264 Schäfer S...... 078 Schulze Selting A...... 238 Reamon-Büttner S...... 473 Schäfer-Korting M...... 137, 479 Reeh P...... 371 Schumacher D...... 382 Schalkowsky P...... 350 Schumacher F...... 383 Reemtsma T...... 481 Schaper K...... 351 Regn M...... 180 Schumacher S...... 221 Scharmach E...... 069 Schumann B...... 461 Reher T...... 319 Scheele N...... 352 Rehn S...... 367 Schupp N...... 056, 308, 453, 477 Scheider J...... 483 Schürmann A...... 079, 177, 222, 237 Reichert A...... 226 Scheitza S...... 353 Reichl F. - X...... 093, 320, 417, 418 Schütz D...... 162 Schelleckes M...... 136, 354 Schütz I...... 312 Reifenberg G...... 469 Scheller J...... 265 Reifenberger J...... 332 Schwab M...... 270, 275, 384 Schenk K...... 178 Schwan C...... 184, 385, 452 Reimann C...... 321 Scheppach W...... 327 Reinartz M. 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W...... 301, 367, 437 Schittek B...... 086 Reuther C...... 326 Seeger T...... 276, 388 Schlechtweg A...... 358 Seeliger M...... 199, 292 Rey Moreno M. - C...... 202 Schlegel F...... 359 Rey-Moreno M...... 216 Seidel A...... 156, 383 Schleh C...... 360 Seidl M. D...... 158, 389 Richling E...... 161, 327, 449, 481 Schleifer H...... 361 Richter A...... 140, 214, 343 Seifert R. ….032, 033, 034, 038, 058, 064, 071, 144, 219, Schlepütz M...... 362 236, 286, 319, 322, 323, 451, 465 Richter K...... 475 Schlichting A...... 473 Richter M...... 236 Selinski S...... 289 Schlicker E...... 104, 239 Sellmaier V...... 455 Riedel T...... 328, 329 Schlitzer M...... 288 Rieg A. D...... 362 Selvaraj D...... 022 Schlossarek S...... 309 Sendker J...... 049 Riehle M...... 142 Schlosser A...... 221 Rieke S...... 245 Shah A. M...... 185 Schlossmann J...... 157, 341, 356 Shaltiel L...... 292 Rimmbach C...... 152 Schlufter F...... 363 Rinne A...... 044, 330 Sharma N...... 140 Schmalbach K...... 364 Shengpeng W...... 014 Ripplinger A...... 410 Schmalz G...... 387 Rochais F...... 006, 123 Shipston M. J...... 472 Schmidt A...... 023, 352, 425 Shymanets A...... 390 Röck K...... 331, 332 Schmidt C...... 224 Rodewald F...... 006 Sica M...... 391, 460 Schmidt F...... 213 Sickmann A...... 123 Roeder M...... 028 Schmidt G...... 324 Roesch F...... 126 Siegert F...... 042 Schmidt M...... 365 Siegert J...... 047, 048, 392 Röhl C...... 333 Schmidt Z...... 398 Rohrbeck A...... 334 Siegert P...... 393 Schmidtko A...... 185, 234, 235, 402 Siegl S...... 394 Rohr-Udilova N...... 411 Schmitt J. P...... 084, 463 Rokitta D...... 335 Siegmund W...... 151, 169, 430 Schmitz B...... 136, 154, 342, 354, 366 Siffert W...... 020 Romanin C...... 361 Schmitz K...... 194, 303 Römer K...... 282 Silva F...... 261 S115
Simmet T...... 395, 396, 397, 398, 399, 400 Thongkam W...... 357 Walther U...... 316 Simonetti M...... 438 Thuenemann A...... 045 Wang H...... 132 Singer S...... 262 Thunemann M...... 424 Wapelhorst B...... 021 Singh H...... 480 Tiburcy M...... 344 Wareing B...... 218 Singh Y...... 401 Tigges J...... 332 Warnken M...... 312 Sinzig J...... 060 Tiret L...... 342 Wartlick F...... 054, 447 Sisignano M...... 402 Tischler A...... 425 Waßermann L...... 448 Sisnaiske J...... 372, 403 Tluczkiewicz I...... 426 Wätjen W...... 004, 067, 075, 076, 077, 145, 230, 271 Siuda D...... 050, 404 Tobaben S...... 091 Watzek N...... 449, 481 Skipper P. L...... 481 Tobias S...... 427 Weber A...... 141 Skolnik E...... 108 Todd A...... 422 Weber H...... 223 Sleman F...... 074 Tolmachev A. O...... 121 Weber S...... 142 Smit M...... 252 Tomicic M...... 001, 428 Wegener J...... 232, 304 Sobottka H...... 149 Torday L...... 480 Weggen S...... 171 Solecki G. M...... 405 Trapani J. A...... 139 Weidele K...... 450 Sommerfeld C...... 146, 147 Trautwein C...... 480 Weighardt H...... 212 Sonnenburg A...... 118, 406 Traxdorf M...... 370 Weil T...... 102, 229 Soppa U...... 407 Trègouët D. - A...... 342 Weiler F...... 451 Sothilingam V...... 199, 292 Trenk D...... 429 Weinschenk T...... 480 Speckmaier E...... 013 Treumann S...... 217, 381, 458 Weirauch U...... 007 Speit G...... 408 Triebel I...... 430 Weiser D...... 002, 110, 167, 189 Spielmann H...... 218, 378 Trinquet E...... 099 Weiss C...... 010, 376 Spiger K...... 464 Trosko J...... 070 Weiss S...... 120, 260, 336, 452 Spillner J. W...... 362 Tselnicker I...... 242 Weissenberger S...... 453 Spiteller M...... 114 Tsikas D...... 100 Weißgerber P...... 009, 019, 454 Stab J...... 414 Tsvilovskyy V...... 009, 019, 454 Weißmann N...... 090 Stahlmann R...... 118, 254, 471 Tunaru S...... 431 Welge P...... 303 Stamm S...... 228 Turnow K...... 432 Welling A...... 455 Stanulla M...... 315 Turpeinen M...... 270 Wen Yih A. W...... 033 Stark T...... 049 Tuttas K...... 273, 392, 433 Wendler O...... 370 Steffen C...... 409 Twarock S...... 251, 434 Werk A...... 062, 456 Steger M...... 234 Werner C...... 066 Steiling H...... 327 Werschkun B...... 206 Stein C...... 301 U Werz O...... 017, 457 Steinberg P...... 057, 274 West J...... 403 Steingräber A. K...... 389 Ugele B...... 133 West R...... 176 Steinhoff B...... 187 Uhl S...... 246 Wettschureck N...... 011, 347, 382, 422 Steinle M...... 410 Uhlig S...... 325, 362, 394 Wettwer E...... 305, 432 Steinmetz M...... 309 Uhlig U...... 394 Wetzel C...... 230 Steinritz D...... 023, 349, 352, 425 Ullrich A...... 211 Wetzke M...... 322 Stempelmann K...... 367 Unfried K...... 212, 420 Wetzker R...... 390 Stempin S...... 070 Unger R. E...... 186 Wichmann H...... 309 Stephan-Schnatz K...... 318 Unterberg M...... 094 Wiegand C...... 460 Sternberg K...... 121, 213 Unterberger E...... 435 Wiege K...... 279 Stieber J...... 155 Urban N...... 149, 159, 193, 436 Wieland T...... 003, 044, 108, 318, 443, 464, 472 Stöckmann D...... 420 Urcan E...... 417, 418 Wiemann M...... 217, 458 Stohr S...... 348 Utech S...... 186 Wiench K...... 164, 216, 217, 458, 459 Stoll G...... 085 Wieneke N...... 460 Stoll M...... 120 V Wiese J...... 461 Stölting I...... 311 Wiese S...... 329 Stolze K...... 411 v.d. Maagdenberg A. M. J...... 240 Wilhelm T...... 258 Stopper H...... 160, 412 van Berlo D...... 171, 357 Williamson G...... 327 Storch U...... 248, 413 van Cott A...... 202 Wilmes T...... 074 Störger C...... 454 van Den Berg M...... 272 Windshügel B...... 282 Stösser S...... 022 van Duursen M. B. M...... 272 Windt H...... 111 Straub I...... 159, 414 van Ede K. I...... 272 Winkelmann V...... 167 Straubinger J...... 415 van Ravenzwaay B. ... 018, 031, 040, 098, 101, 137, 164, Winter P...... 483 Strauss V...... 200, 217, 381, 458 200, 201, 202, 203, 216, 217, 381, 442, 445, 458, Winter S...... 384, 462 Streich C...... 335 459 Winyard P...... 142 Strilic B...... 382 van Thriel C...... 372, 403 Wirotanseng R...... 422 Strunskus T...... 333 Vass V...... 480 Wirth A...... 011 Stumm R...... 162, 267 Vatter P...... 446 Wirth M. P...... 307 Stümpel F. T...... 416 Vega M...... 268 Wishnok J. S...... 241 Styllou M...... 320, 417, 418 Vennekens R...... 246, 454 Wissenbach U...... 019, 092, 242, 350, 454 Styllou P...... 320, 417, 418 Verma N...... 367, 437 Witte I...... 166 Suchenwirth R...... 475 Vetter S...... 055 Wittig I...... 234 Sun J...... 238 Vicuna L...... 438 Wittköpper K...... 215 Sünwoldt J...... 174 Vidal M...... 339, 439 Wittmann T...... 463 Sutton V. R...... 139 Voets T...... 246 Wizemann R...... 248 Suvorava T...... 005, 419 Vogel K...... 440 Wohlleben W...... 018, 164, 203, 216, 217, 458 Sydlik U...... 212, 420 Vogel L...... 441 Wojnowski L...... 024, 282, 470 Syrovets T...... 395, 396, 397, 398, 399, 400 Vogel S...... 018, 203,442 Wolf N. M...... 003, 108, 464 Szabo B...... 220 Voigt N...... 443 Wolf S...... 152 Völker K...... 108 Wolter S...... 451, 465 Vollmar A...... 211, 228 Woolf C...... 402, 438 T Volochnyuk D. M...... 121 Worek F...... 276, 388 Völzke H...... 285 Worzfeld T...... 468 Taghizadeh K...... 241 Wrobel T...... 223 Takawale P...... 360 von der Ohe J...... 451 von Keutz A...... 274 Wruck C. J...... 098 Takefuji M...... 011 Wu Z...... 050, 466 Tanimoto N...... 199, 292 von Knethen A...... 053 von Kügelgen I...... 266 Wudy S. A...... 021, 133 Tank J...... 421 Wuest M...... 307 Tannenbaum S. R...... 241, 481 von Lehe M...... 104 Vondracek J...... 103 Wulfsen I...... 472 Tannert A...... 192 Wunder F...... 467 Tappe-Theodor A...... 422 Voss J. - U...... 227 Vranjkovic K...... 238 Wurth F...... 076, 077 Tarlinton D. M...... 139 Würtz C...... 178, 215 Tatge H...... 125, 287 Wyrsch P...... 041 Tegeder I...... 422 W Tejedor F. J...... 407 Tekook M...... 379, 423 Wächter T...... 218, 378 X Tenzer S...... 469 Wadie W...... 189 Tersteegen A...... 467 Wagner J...... 444 Xia J...... 468 Tesseromatis C...... 170 Wahl-Schott C...... 253, 292, 478 Xia N...... 363, 404, 427, 466, 469 Tevoufouet E. E...... 087 Waide S...... 403 Thiermann H...... 023, 181, 276, 352, 388, 425 Walk T...... 445 Thomas M...... 007 Walliser C...... 068, 446 Thomas S...... 461 Walter S...... 480 S116
Y Zaporojtchenko V...... 333 Zimmermann U...... 152 Zechner U...... 404 Zimmermann W...... 344 Yan T...... 470 Zeng Y...... 241 Zimmermann W. - H...... 215, 309 Yao G...... 474 Zenie A...... 147 Zimnol A...... 477 Ye W...... 241 Zenk J...... 370 Zipper P...... 332 Zhang D...... 402 Zischka H...... 091 Zhang Z...... 474 Zolk O...... 119, 315, 370 Z Zhao S...... 221 Zollner B...... 346 Zhou X. - B...... 472 Zong X...... 073, 478 Zaagsma J...... 252 Ziegler N...... 029, 280 Zörner A...... 100 Zabel R...... 471 Ziegler V...... 446 Zoschke C...... 479 Zabel U...... 029, 280, 444 Ziemann C...... 473 Zschunke M...... 218 Zablotskii V...... 400 Ziemann U...... 355 Zucker A...... 237 Zahedi R...... 123 Zierler S...... 474 Zühlke S...... 114 Zahler S...... 211, 228 Zieseniss A...... 178 Zuidhof A. B...... 252 Zaki H...... 189 Zietz B. P...... 475 Zwickenpflug W...... 165 Zakrzeska A...... 239 Zilker T...... 181, 425 Zygmunt M...... 169 Zanger U...... 270, 275 Zimmermann J...... 476